I N T E R N AT I ON A L
REVIEW OF CYTOLOGY VOLUME 81
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET...
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I N T E R N AT I ON A L
REVIEW OF CYTOLOGY VOLUME 81
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS ALEXANDER
DONALD G . MURPHY ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS YUDIN
INTERNATIONAL
Review of Cytology EDITED BY
J. F. DANIELLI
G. H. BOURNE St. George's University School of Medicine St. George's, Grenada West Indies
Danielli Associates Worcester, Massachusetts
ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME 81 I983
ACADEMIC PRESS A Subsidiary of Harcortrt Bruce Jovanovich, Publishers New York London Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto
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Contents CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
Oxidation of Carbon Monoxide by Bacteria YOUNGM . K I M
AND
GEORGE D . HEGEMAN
I . Introduction . . . . . . . . . I1 . Sources and Sinks for Atmospheric Carbon Monoxide 111 . Carbon Monoxide-Oxidizing Bacteria . . . . . . . . IV. Physiology of Carbon Monoxide Oxidation . . . . . V. Environmental Significance . . . . . . . . . . . . VI . Applications . . . . . . . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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i
2 4 II 24 25 26 28
Sensory Transduction in Bacterial Chemotaxis GERALD L . HAZELBAUER A N D SHIGEAKI HARAVAMA I . Introduction . . . . . . . . . . . . . . . . . . . . I1 . Components and Features of the Sensory System . . . 111. Conventional Receptors . . . . . . . . . . . . . . . I v. Stimuli Not Mediated by Conventional Receptors . . . V. The Excitatory Link . . . . . . . . . . . . . . . . VI . Structure of Transducers . . . . . . . . . . . . . . v11 . Adaptation . . . . . . . . . . . . . . . . . . . . . v111 . Pathways for Unconventional Excitation and Adaptation IX . Concluding Remarks . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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33 36 40 45 50 52 58 63 64 65
The Functional Significance of Leader and Trailer Sequences in Eukaryotic mRNAs F. E . BARALLE 1. Introduction . . . . . . . . I 1 . The Leader Sequence . . . . I11 . The Trailer Sequence . . . . References . . . . . . . . .
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72 93 102
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CONTENTS
The Fragile X Chromosome GRANT R . SUTHERLAND
I . Introduction . . . . . . . . . . . . I1 . What Is the Fragile X? . . . . . . . 111. Tissue Culture Conditions . . . . . . I v. Cytogenetics . . . . . . . . . . . . V. Genetics . . . . . . . . . . . . . . VI . Clinical Aspects . . . . . . . . . . VII . Karyotype-Phenotype Relationship. . VIII . Conclusions . . . . . . . . . . . . IX . Apologia . . . . . . . . . . . . . . References . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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107 108
112 118
124 127 137 138
138 139
Psoriasis versus Cancer: Adaptive versus Iatrogenic Human Proliferative Diseases SEYMOUR GELFANT
I. I1 . Ill . IV. V.
Precis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Cycle Aspects of Psoriasis . . . . . . . . . . . . . . . . . . . . Cell Cycle Aspects of Cancer . . . . . . . . . . . . . . . . . . . . Psoriatic Proliferative Responses to Therapy . . . . . . . . . . . . . . Tumor Proliferative Responses to Therapy . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
145 146 149 154 156 160
Cell Junctions in the Seminiferous Tubule and the Excurrent Duct of the Testes: Freeze-Fracture Studies TOSHIO NAGANOA N D FUMIE SUZUKI I. I1 . III. IV.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intercellular Junctions between the Sertoli Cells . . . . . . . . . . . . Cell Junctions in the Epithelial Lining in the Exourrent Duct . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
163 165 178 188 188
Geometrical Models for Cells in Tissues HISAOHONDA I . Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. The Cell Aggregate Model: Cells Can Be Represented by Points . . . . .
I11 . The Boundary Shortening Model of Cells in a Tissue IV. Cell States in Tissues: Epithelium-like or Not? . . . V. Fundamental Consideration of Tension and Shape .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .
191 192 216 233 240
vii
CONTENTS VI . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
244 246
Growth of Cultured Cells Using Collagen as Substrate JASON YANGAND S . NANDI
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 I1 . Early Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 111. Studies in the 1960s . . . . . . . . . . . . . . . . . . . . . . . . . 251 I v. Studies in the 1970s . . . . . . . . . . . . . . . . . . . . . . . . . 251 V. Three-Dimensional Culture System-Early Studies . . . . . . . . . . . 254 VI . Studies in the 1980s . . . . . . . . . . . . . . . . . . . . . . . . . 255 VII . Studies in.Our Laboratory . . . . . . . . . . . . . . . . . . . . . . 258 VI11 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES A N D SUPPLEMENTS . . . . . . . . . . . . . . .
.
287 291
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Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
F. E . BARALLE (71), Sir William Dunn School of Pathology, University of Oxford, Oxford, England SEYMOUR GELFANT (145), Departments of Dermatology and Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912 SHIGEAKI HARAYAMA (33), Laboratory of Genetics, Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tohyo, Japan GERALD L. HAZELBAUER (331, BiochemistrylBiophysics Program, Washington State University, Pullman, Washington 99164 GEORGE D. HEGEMAN (I), Microbiology Group, Biology Department, Indiana University, Bloomington, Indiana 47405 HISAOHONDA(191), Kaneho Institute for Cancer Research, Misakicho 19, Kobe 652, Japan YOUNGM. KIM'( I ) , Microbiology Group, Biology Department, Indiana University, Bloomington, Indiana 47405 TOSHIO NAGANO (1631, Department of Anatomy, School of Medicine, Chiha University, Chiba 280, Japan S. NANDI(2491, Cancer Research Laboratory and Department of Zoology, University of California, Berkeley, California 94720
GRANTR. SuTnERLAND (1071, Cytogenetics Unit, Department of Histopathology, Adelaide Children's Hospital, North Adelaide, S.A. 5006, Australia FUMIESUZUKI (1631, Department of Anatomy, School of Medicine, Chiba University, Chiba 280, Japan
JASON YANG(249), Cancer Research Laboratory, University of California, Berkeley, California 94720 'Present address: Department of Biology, Yonsei University, Seoul 120, Korea. ix
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INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME 81
This Page Intentionally Left Blank
INTERNATIONAL
REVIEW OF CYTOLOGY. VOL. 81
Oxidation of Carbon Monoxide by Bacteria YOUNGM. KIM^
AND
GEORGED. HEGEMAN
MicrobioIogy Group, Department of Biology, Indiana University, Bloomington, Indiana
1. 11.
....................
Introduction Sources and
............... Ill.
IV.
V. VI.
VII.
..................
B. Sinks.. .............................................. Carbon Monoxide-Oxidizing Bacteria ......................... A. Nonutilitarian Carbon Monoxide-Oxidizing Bacteria. ......... B. Utilitarian Carbon Monoxide-Oxidizing Bacteria. . . . . . . . . . . . . Physiology of Carbon Monoxide Oxidation. . . ............ A. Mechanism of Carbon Monoxide Oxidatio B. Carbon Monoxide as Carbon and Energy Source . . . . . . . . . . . . Environmental Significance. .......... Applications .............................................. Conclusions ................ ..................
1
2 2 3 4 5 5 11
11 21 24 25 26 28
I. Introduction Carbon monoxide (CO) has been considered to be one of the most prevalent atmospheric pollutants since the first observation of CO was made by Migeotte (1949) on the basis of a study of solar spectra. Its concentration in the unpolluted troposphere is about 0.1 ppm, less than that of hydrogen or of nitrous oxide (Ehhalt and Volz, 1975), while in urban districts its concentration can be as high as 50- 100 ppm (Robinson and Robbins, 1970). It has been estimated that natural and man-made sources produce about 12-14 X lo8 tons per annum of CO, but repeated measurements have shown a relatively constant CO concentration in the atmosphere (0.03-0.9 ppm) (Robbins et al, 1968, indicating that the rate of destruction of CO must be large and at least equal to the rate of CO production (Warneck, 1975). Indeed, the turnover of CO in the lower atmosphere is quite fast and the mean residence time has been estimated to be 0.1-1 .O year (Junge et al., 1971; Seiler, 1974; Weinstock, 1969; Weinstock and Niki, 1972). The constant level of atmospheric CO suggests that there exist sinks of sufficient magnitude to convert the CO into other compounds (Warneck, 1975), some of 'Present address: Department of Biology, Yonsei University, Seoul 120, Korea. 1
Copyright 0 1983 by Academic Press. Inc. All rights of repmduclion in any form reserved. ISBN 0- 12.36448 I -X
2
YOUNG M. KIM AND GEORGE D. HEGEMAN
which are nonbiological. However, it seems likely that much of the CO generated in soil and in the lower layers of the atmosphere is oxidized to CO, locally by biological agents, principally microbes (Inmann et al., 1971; Warneck, 1975).
11. Sources and Sinks for Atmospheric Carbon Monoxide
A. SOURCES CO is continuously being added to the atmosphere in biologically significant amounts (McConnell et af., 1971) through incomplete combustion of fossil fuels and by atmospheric reactions (Levy, 1971; Weinstock and Niki, 1972). Recently it has been shown that CO is formed by various C-3 plants (Seiler et al., 1978; Seiler and Giehl, 1977), which have been estimated to produce through a lightdependent process 0.5-1 .O X lo8 tons per annum. Isotopic experiments indicate a much higher proportion of CO comes from other natural sources, including the decomposition of porphyrins (Stevens et al., 1972). CO production has also been observed during catabolism of heme and cobalamin compounds by bacteria (Engel et al., 1972, 1973) and of flavonoids by fungi (Westlake et al., 1961), and has also been observed in animal tissue where hemoglobin is converted by microsomal heme oxygenase to equirnolar amounts of biliverdin and CO (Landaw, 1970; White, 1970). In this case, the CO is formed by the oxidation of the a-methine bridge carbon of the porphyrin ring (Tenhunen ef al., 1969). Another report has suggested that CO is a product of the peroxidative degradation of lipids (Wolff and Bidlack, 1976). Some marine invertebrates (siphonophores) also have been reported to produce considerable amounts of CO (Pickwell et al., 1964). Trace amounts of CO are also produced during growth of yeasts and bacteria on glucose (Radler et af., 1974). Furthermore, there are a number of reports of light-dependent CO production by green algae, cyanobacteria, and phototrophic bacteria (Bauer et al., 1980; Loewus and Delwiche, 1963; Troxler, 1972; 'Troxler and Dokos, 1973). Several fungi are also known to produce CO (Simpson et al., 1960; Westlake et af., 1961). On the other hand, Conrad and Seiler (1980a) reported that the CO production by soil was a chemical process rather than a metabolic process of soil microorganisms. It has been estimated that several technological sources create 6.4 X lo8 tons of CO per year (Seiler, 1974) and at any time 7.4 X lo* tons are present in the atmosphere (Robinson and Robbins, 1970). Atmospheric sources include the photochemical oxidation of CH, by .OH radical (Stevens et al., 1972;Weinstock and Niki, 1972; Wofsy er al., 1972), by 0, (Crutzen, 1974) and photolysis of formaldehyde (Calvert et af., 1972), and of CO, (Wolfgang, 1970). CO can also
OXIDATION OF CO BY BACTERIA
3
be produced and stored in the Oceans (Conrad and Seiler, 1980b; Inmann et al., 1971; Junge et al., 1971; Swinnerton et al., 1970; Wilson et al., 1970). Rainwater may transport CO from the upper atmosphere to the earth’s surface (Swinnerton et al., 1971); the solubility of CO in water is similar to that of oxygen. B. SINKS Several mechanisms for removal of CO from the atmosphere have been suggested and demonstrated, but their relative importance is still unclear. It has been suggested that photochemical destruction of CO in the stratosphere (Newell et al., 1974; Pressman and Warneck, 1970), by tropospheric chemical reactions, and in biological processes were the means for removal of CO from the atmosphere (Crutzen, 1974; Kummler et al., 1969; Levy, 1971; Seiler, 1978; Zimmerman et al., 1978). Warneck (1975) estimated that oxidation of CO by OH could remove as much as 6.8 X lo8 tons of CO per year by the reaction: CO + OH + CO, .H. It is known that the biological sink is very significant in removal of CO from the atmosphere. Inmann et al. (1971) estimated that amount of CO potentially removable by this process exceeds current CO production by several fold. Krall and Tolbert (1956) and Bidwell and Fraser (1972) reported evidence of CO metabolism by plant leaves by measuring the incorporation of 14C0 into cellular materials, but concentrations of CO used in both experiments were much higher than the ambient atmospheric level and thus their ecological significance remains unknown. CO also can be oxidized in animal tissue. Breckenridge (1953) reported that purified cytochrome oxidase from pig heart muscle and muscle extracts could oxidize CO to CO,. After a critical examination of a series of reports, Fenn (1970) concluded that humans and other animals play no role as a sink for CO. However, colonic flora in humans does consume CO (Levine et al., 1982). While eukaryotic organisms may play a role in the removal of CO, the major biological sink for CO is probably oxidation of CO by microbes which are present in the soil and surface waters (Conrad and Seiler, 1980a,b; Hegeman, 1980; Heichel, 1973; Inmann et al., 1971; Jones and Scott, 1939; Spratt and Hubbard, 1981). Ingersoll et al. (1974) showed a gradual increase in the binding activity of the soil with an increase in the CO concentration and estimated the global soil uptake of CO to be 1.4 X lo9 tons per year while Seiler (1978) and Bartholomew and Alexander (1981) calculated the average uptake as 5.0 X lo8 and 4.1 X lo8 tons per year, respectively. Tests of individual species of algae (Chappelle, 1962), fungi (Hirsch, 1965; Inmann and Ingersoll, 1971), and both aerobic (Bartholomew and Alexander, 1979; Cypionka et al., 1980; Hirsch, 1965; Kirkconnell, 1978; Kistner, 1953, 1954; Meyer etal., 1980; Meyer and Schlegel, 1977, 1978, 1979; Nozhevnikova
+
4
YOUNG M. KIM AND GEORGE D. HEGEMAN TABLE I MAJOR SOURCES A N D SINKS FOR
CARBON
MONOXIDE"
Amounts Sources and sinks
( x 108 tonslyear)
References
Sources Anthropogenic Oceans Plants Forest fires Methane oxidation Hydrocarbon oxidation Total
6.4 0.2-1.2 0.5-1 .o 0.6 4.0 0.6 12.3-13.8
Seiler (1974) Seiler and Schmidt (1975) Seiler er a/. (1978) Seiler (1974) Seiler (1978) Robinson and Robbins (1969)
Sinks Soil Plants Stratosphere Oxidation by 'OH PhotoIy sis Total
5.0 7.0-70 1.1 7.0 39.0 59.1-122. I
Liebl and Seiler (1975) Bidwell and Fraser (1 972) Seiler (1974) Seiler (1976) Ehhalt (1975)
"Estimates listed here are the most current values among those reported to date.
and Zavarzin, 1974; Ooyama and Shinohara, 1971; Sanzhieva and Zavarzin, 1971; Shinohara and Ooyama, 1972; Zavarzin and Nozhevnikova, 1975, 1977) and anaerobic bacteria (Andress, 1975; Daniels eraf., 1977; Diekert and Thauer, 1978; Fischer er al., 1931, 1932; Fuchs er al., 1974; Kluyver and Schnellen, 1947; Uffen, 1976, 1981; Yagi, 1958; Lynd er al., 1982; Genthner and Bryant, 1982), have shown that these organisms can metabolize and, in some cases, grow with CO in pure culture. A summary of the major sources and sinks for atmospheric CO is given in Table I. 111. Carbon Monoxide-Oxidizing Bacteria
Bacteria that oxidize CO may be subdivided according to whether they are able to use CO as an energy source for growth (utilitarian oxidation) or whether the oxidation process is a gratuitous one (nonutilitarian oxidation) resulting from the acceptance of CO as a pseudosubstrate for an enzyme system evolved to catalyze another process. Among bacteria that can oxidize CO, nonutilitarian representatives have been studied as extensively as utilitarian CO oxidizers, and as much is known of some mechanisms of nonspecific CO oxidation as of the utilitarian process.
OXIDATION OF CO BY BACTERIA
5
A. NONUTILITARIAN CARBON MONOXIDE-OXIDIZING BACTERIA 1. Anaerobic Bacteria Fischer et al. (1931, 1932) reported that sludge from an anaerobic sewage treatment system could effect the anaerobic conversion of CO to methane and CO,. The bacterium thought to be responsible for this reaction was subsequently isolated and studied by Barker (1936) and given the name Methanosarcina barkerii (Schnellen, 1947) after Horace A. Barker who performed much of the early work on the biochemistry and physiology of methanogenic bacteria. After Barker’s work (1936) it was reported that methanogens (Daniels et al., 1977; Kluyver and Schnellen, 1947), a sulfate-reducing bacterium (Postgate, 1970; Yagi, 1958, 1959; Yagi and Tamiya, 1962), clostridia (Diekert et al., 1979a; Diekert and Thauer, 1978, 1980; Drake et al., 1980; Fuchs et al., 1974; Thauer et al., 1974), and a rumen acidogen (Lynd et a f . , 1982; see Section III,B ,2) can oxidize CO gratuitously under anaerobic conditions. Clostridium welchii has been cited in many reports (e.g., Stephenson, 1949) as a CO-oxidizing bacterium, but there is no evidence known to us for CO oxidation by this organism. In 1975, Andress found that cysteine specifically repressed CO-oxidizing activity in growing cultures of Clostridium pasteurianum. As many anaerobic bacteria are grown in the laboratory on cysteine-containing complex media or on media supplemented with reducing agents formulated with cysteine, this finding suggests that the capacity to mediate the oxidation of CO to CO, may be more widespread among anaerobic bacteria than is presently thought (Fuchs et a f . , 1975).
2 . Aerobic Bacteria The aerobic methane oxidizers can also oxidize CO gratuitously. Hubley et a!. (1974) demonstrated that CO induced both respiration and CO, production in washed cell suspensions of the two methane-oxidizing bacteria, Methylomonas albus and Methylosinus trichosporium. Pseudonomonas methanica (Ferenci, 1974, 1975; Ferenci et al., 1975) is also known to be an aerobic nonutilitarian CO-oxidizing bacteria. B . UTILITARIANCARBON MONOXIDE-OXIDIZING BACTERIA 1. Aerobic Bacteria Despite the widespread distribution of and biological consumption of CO, early attempts to isolate microorganisms capable of growth at the expense of CO were not very rewarding. The credit for the discovery of an aerobic bacterium capable of using CO as a source of carbon and energy has generally been given to Beijerinck and van Delden (1903) even though they did not mention the words
6
YOUNG M. KIM AND GEORGE D. HEGEMAN
“carbon monoxide” in their report. They isolated from garden soil a microbe which could be grown on liquid mineral medium to which no source of carbon had been added. Since growth of their organism occurred more readily in laboratory air than in “pure” air of a greenhouse, they held that the organism used a volatile substance present in the laboratory air as carbon and energy source and named it Bacillus oligocarbophilus. The role of CO as substrate was suggested 3 years later by Kaserer (1906) who isolated a hydrogen bacterium, Carboxydomonas oligocurbophila Beij. et van Delden, bearing a close resemblance to the microbe of Beijerinck and van Delden. He proposed CO as an intermediary metabolite in the reactions: H,CO, + H, + CO + 2H,O and 2CO 0, 2H,O --.* 2H,CO,. But Kaserer’s experimental evidence was subjected to a great deal of criticism by many who did not think that he gave convincing proof of CO oxidation. Lantzsch (1922) independently isolated a coccus from a pellicle which developed on distilled water and noted some branched filaments of the same diameter as the coccus, and claimed that this might be the same organism as Beijerinck’s. The morphology was dependent upon the nature of the added carbon source. Although uptake of CO was not demonstrated when grown with 0.02% CO in the gas phase of an enrichment culture, he claimed CO might be the carbon source and renamed the organism Actinomyces oligocarbophilus. Hasernann (1927) also isolated an actinomycete similar to Beijerinck’s which eliminated CO from a bell jar containing CO and air in 12-42 days. The confining liquid rose in the bell jar and gave a positive test for CO,. In 1939, Jones and Scott presented new evidence for the existence and the ubiquity of CO-oxidizing microorganisms by showing the rapid disappearance of CO from sealed mine fire areas and claimed the presence of B. oligocurbophilus in those areas. Later Pandaw et al. ( 1960) stated that a mechanism might exist for the biological oxidation of CO at the surface of the earth and that B. oligocarbophilus which was present in the soil might be a terrestrial ancestor of other biological CO oxidizers. However, a critical analysis of these investigations carried out by Kistner (1953) led him to the conclusion that their results were unsatisfactory. Hirsch (1960) and Zavarzin and Nozhevnikova (1977) confirmed Kistner’s conclusions that the organism known as B. oligocarbophilus did not oxidize CO. The first definitive studies of a CO-oxidizing organism and its action upon CO were, in fact, done by Kistner (1953, 1954). After several unsuccessful attempts at enrichment for the CO oxidizer, he was able to suppress the growth of nonoxidizers by using an atmosphere of 70% CO, 20% 0,, and 10% N,. Based on the ratio of amounts of gases consumed, he concluded that twice as much CO was used as 0, and proposed that the process had the overall stoichiometry: 2CO + 0, + 2C0,. He reported that on the basis of morphological studies and the observation that the organism could oxidize H,, the bacterium should be classi-
+
+
7
OXIDATION OF CO BY BACTERIA
fied in the genus Hydrogenomonas. He subsequently gave it the name Hydrogenomonas carboxydovorans. After Kistner’s work it was generally thought that CO is oxidized by certain hydrogen bacteria. Nevertheless, when several hydrogen bacteria were tested for their ability to use CO it was found that the growth of hydrogen bacteria at the expense of H, is generally very sensitive to CO inhibition (Zavarzin and Nozhevnikova, 1977) and CO is a well known inhibitor of hydrogenase (e.g., Lynd et al., 1982). Hirsch and Conti (1964) isolated species of Hyphomicrobium and Caulobacter using enrichment cultures established in mineral medium and inoculated with water from many sources under a CO/O,/CO,/He (75/10/0.5/14.5 or 30/15/ 0.5/54.5) gas mixture, but they did not give a clear answer regarding whether CO could be used as a source of carbon and energy by their isolates. A year later Hirsch (1965) reported that Hyphomicrobium, Caulobacter, Sarcina, Pseudomonas. corynebacteria, and Nocardia species that were capable of growing autotrophically with CO/O,/CO, were isolated from enrichments with mineral salts solution and lawn soil under CO (30-75%), 0, (10-15%), CO, (0.5-2%), and He (balance). Three strains of coryneform bacteria grew well under strictly autotrophic conditions, i.e., CO was used as the only energy source. The gas uptake observed agreed with the reaction: 2CO 0, + 2C0,. He also noted that H . carboxydovorans and B . oligocarbophilus were distinctly different from the three coryneform strains. In 1967 Davis isolated two flagellated strains of CO-oxidizing hydrogen bacteria (strains 460 and 461) from soil using CO enrichment. Among the many hydrogen bacteria she studied, these were the only strains that could grow autotrophically using either CO or H, as a source of energy. However, no physiological experiments concerning CO metabolism were carried out. Davis et al. (1970) later assigned these bacteria to the genus Pseudomonas. To develop a microbial system utilizing CO as well as N, to cope with a possible future world-wide protein shortage, Ooyama and Shinohara (1971) and Shinohara and Ooyama (1972) isolated two types of N,-fixing CO-utilizing bacteria. The first strain, S17, could use CO as a sole carbon source and H, as an energy source. Growth did not occur when either CO was omitted from the gas mixture or when N, was replaced by Ar. The second strain, A305, could use CO as a sole source of carbon and energy. Using a mineral liquid medium, Sanzhieva and Zavarzin (197 1) isolated from air tanks another organism which grew in an atmosphere of CO. The organism was polymorphic and its cells were often seen combined in stellate aggregations. Because of these traits the authors placed the organism in the genus of free-living rosette-forming bacteria, Sefiberia. Gases were consumed according to the reaction: 2CO + 0, + 2C0,. Because of its ability to oxidize not only H, but also CO, they named it Seliberia carboxydohydrogena. Meyer et a f . (1980) later
+
8
YOUNG M. KIM AND GEORGE D. HEGEMAN TABLE I1 BAC~ERIA WHICH OXIDIZE co TO C O f Mode of oxidationb
Bacteria Aerobic Pseudomonas (Hydrogenomonas) carboxydovorans P . (Seliberia) carboxydohydrogena P . carboxydojlava
U U U
P . gazotropha Comamonas compransoris Achromobacrer carboxydus Pseudomonas spp. Azoiobacrer spp. Azomonas sp. 1, 2, and 3 Hyphomicrobium Caulobacter Sarcina Nocardia Corynebacterium Strain OM2, OM3, and OM4 Strain A305 Strain S17 Actinoplanes Agromyces Microbispora Mycobacrerium Methvlomonas albus
U U U U
U U U U U U U U U
u (3 ? ? ? ? Non-U
References
Kistner (1953, 1954); Meyer and Schlegel (1978) Sanzhieva and Zavarzin (1971) Nozhevnikova and Zavarzin (1974); Kiessling and Meyer (1982) Nozhevnikova and Zavarzin (1974) Nozhevnikova and Zavarzin (1974) Nozhevnikova and Zavarzin (1974) Davis (1967); Davis er a / . (1970) Kirkconnell ( 1978) Kirkconnell (1978) Hirsch and Conti (1964) Hirsch and Conti ( I 964) Hirsch (1965) Hirsch (1965) Hirsch (1965) Cypionka et al. (1980) Shinohara and Ooyama (1972) Ooyama and Shinohara (1971) Bartholomew and Alexander (1979) Bartholomew and Alexander (1979) Bartholomew and Alexander (1979) Bartholomew and Alexander (1979) Hublev e t a / . (1974) . . (continued)
proposed that this bacterium be transferred from the genus Seliberia to the genus Pseudomonas as Pseudomonas carboxydohydrogena on the basis of electron microscopic observations, its strictly aerobic metabolism, and other traits examined. In 1973, Nozhevnikova and Zavarzin isolated several CO-oxdizing bacteria from city soils following enrichment under a gas mixture of 20% 0, and 80% CO. During the isolation of these organisms which could grow with CO as sole source of energy, they found that it was difficult to grow the organisms except as a two-component culture or without the addition of certain vitamins. Pseudomonas gazotropha was vitamin B ,-dependent and Comamonas compransoris, the other component, needed thiamine. Inoculation of each organism into sterile filtrates of cultures of the second organism gave growth at the expense of CO. They concluded from these experiments that, in natural habitats, CO oxidation
,
9
OXIDATION OF CO BY BACTERIA TABLE I1 (Conrinued)
Bacteria Merhylosinus rrichosporium P . methanica Anaerobic Methanobacrerium rhermoaurotrophicum Rhodopseudomonas gelarinosa (in the dark) Rhodospirillum rubrum (in the dark) Rhodopseudomonas spp. (in the light) Eubacterium limosum Desulfovibrio desulfuricans Methanosarcina barkerii Methanobacteriumformicicum Butyribacrerium merhylorrophicum Methanobacrerium ruminanrium Methanobacterium arbophilicum Merhanosarcina UBS Closrridium rhermoacericum Clostridium formicoacericum Clostridium pasreurianum
Mode of oxidationb
References
Non-U Non-U
Hubley er al. (1974) Ferenci er al. (1975)
U
Daniels er al. (1977)
U
Uffen (1976)
U
Uffen (1981)
U
Hirsch (1968)
U Non-U (?) Non-U Non-U Non-U and U (variant) Non-U Non-U Non-U Non-U Non-U Non-U
Genthner and Bryant (1982) Yagi (1958, 1959) Kluyver and Schnellen (1947) Kluyver and Schnellen (1947) Lynd er al. (1982) Daniels el al. (1977) Daniels er al. (1977) Daniels er al. (1977) Diekert and Thauer (1978) Diekert and Thauer (1978) Fuchs er al. (1974); Thauer er al. ( 1974)
OOrganisms for which the evidence seems ambiguous are not included. bU, Utilitarian oxidation; non-U, nonutilitarian oxidation.
could be carried out by both members of this two-component bacterial system, the probable basis for interdependence being reciprocal vitamin feeding. On the basis of microscopic examination of these bacteria they suggested that the microorganisms that oxidize CO belong to different morphological groups and cannot be assigned to a single bacterial genus. Nozhevnikova and Zavarzin (1974) and Zavarzin and Nozhevnikova (1977) carried out a more complete study of CO-utilizing, aerobic Gram-negative bacteria. Designated as sharing the ability to grow at the expense of CO were H. carboxydovorans, P. carboxydohydrogena, Pseudomonas carboxydoflava, P . gazotropha, C . compransoris, and Achromobacter carboxydus. All were regarded as a physiological group of “carboxydobacteria” meaning that bacterial group which can grow aerobically on CO as sole carbon and energy source. All but A . carboxydus were also able to grow under an atmosphere of H,, O,, and
10
YOUNG M. KIM AND GEORGE D. HEGEMAN
CO, as hydrogen autotrophs as well. These authors also placed Kistner’s Hydrogenomonas in the genus Pseudomonas because Davis et al. (1969) rejected the genus Hydrogenomonas on the grounds that the ability to use H, as an energy source was a trait that occurs in an otherwise widely diverse group of bacteria. P . gazotropha was capable both of autotrophy by aerobic growth in the presence of H, or CO as energy sources and of methylotrophic organotrophy by growth in the presence of methanol as sole source of carbon and energy. It was later found that the serine pathway was responsible for assimilation of methanol carbon (Romanova et al., 1978b). This may be the first example of the capacity for growing by means of three types of assimilatory carbon nutrition, i.e., organotrophy, autotrophy, and methylotrophy. Meyer and Schlegel (1 977, 1978) reisolated Kistner’s Pseudomonas carboxydovorans which had been lost for a long time using a mixture of 5% 0,, 5% CO,, and 90% CO. Four different Gram-negative bacteria able to use CO as sole source of carbon and energy and to grow with N, as source of nitrogen were recently obtained by direct isolation from the soil (Kirkconnell, 1978). One of these four bacteria was assigned to the genus Azotobacrer and the other three to Azomonas. These bacteria could not grow as hydrogen bacteria, like A . carboxydus and unlike other CO-oxidizing bacteria previously examined. 2. Anaerobic Bacteria Several anaerobic bacteria also have been reported to grow at the expense of CO as sole energy and carbon sources. Hirsch (1968) reported that Rhodopseudomonas spp. could use CO as a major carbon source and the cleavage of water as a hydrogen source under anaerobic-light conditions. In other reports, Rhodopseudomonas gelutinosa (Dashekvicz and Uffen, 1979; Uffen, 1976) and Rhodospirillum rubrum (Uffen, 1981) were suggested to be able to grow as facultative methylotrophs at the expense of CO as sole source of carbon and energy in the dark, anaerobically. Keppen er al. (1976), however, threw doubt on the general ability of phototrophic bacteria to use CO, since strains of Rhodopseudomonas sulfidophila, Rhodopseudomonas palustris, and Rhodomicrobium vannielii could not use CO under either phototrophic or aerobic dark growth conditions. Although Uffen (1976) reported that M . barkerii and Methanobacterium formicicum were unable to grow on a solid medium with a CO plus H, (20 : 80) gas mixture, Daniels er al. (1977) reported that Methanobacterium thermoautorrophicum grew anaerobically with CO as energy source, but the net growth was slight and the growth rate was only 1% of that observed on HJCO,. Recently a variant of Butyribacterium methylotrophicum was isolated that can grow anaerobically with CO (Lynd et al., 1982), and another recent report describes anaerobic growth of Eubacterium limosum with CO as energy source
OXIDATION OF CO BY BACTERIA
11
(Genthner and Bryant, 1982). Both organisms convert CO to CO, and acetate, and are phenotypically somewhat similar. Table I1 lists the aerobic and anaerobic bacteria that have been reported to oxidize CO to CO,.
IV. Physiology of Carbon Monoxide Oxidation It is well known that CO is inhibitory for virtually all aerobic respiratory organisms. Even in aerobic carboxydobacteria it has been reported that high concentrations of CO reduce the growth rate and cellular yield (Kim, 1981; Nozhevnikova, 1974; Saval’eva and Nozhevnikova, 1972; Zavarzin and Nozhevnikova, 1977), indicating that CO tolerance is a necessary part of the ability to use CO at higher concentrations. It is also known that CO concentrations higher than 30% in the gas phase are inhibitory to the anaerobic growth of M . thermoautotrophicum (Daniels et al., 1977) and that Rhodospirillum rubrum prefers CO concentrations lower than 100%in the gas atmosphere (Uffen, 1981). On the other hand, R. gelatinosa (Uffen, 1976, 1981) is reported to grow even with 100% CO as energy source under anaerobic, dark conditions. A. MECHANISM OF CARBON MONOXIDE OXIDATION
It has generally been considered that CO-oxidizing systems in nonutilitarian bacteria are constitutive whereas those in utilitarian bacteria are inducible, except in P. carboxydoflava (Kiessling and Meyer, 1982), M . thermoautotrophicum (Daniels et al., 1977), a variant of B. methylotrophicum (Lynd et al., 1982). E . limosum (Genthner and Bryant, 1982), and in phototrophic bacteria (Hirsch, 1968). 1. Nonutilitarian Bacteria
The mechanism underlying metabolism of CO by these organisms was clarified first for the methanogenic bacteria by the studies of Kluyver and Schnellen (1947) using whole cells of M . barkerii and M . formicicum. A mixture of CO and H, was converted to CH, according to the following equation: CO + 3H2+ CH, H,O. They proposed that this fermentation process actually proceeds in two steps: CO + H,O + CO, + H, and CO, + 4H2 + CH, + 2H,O. In agreement with this it was established that M . barkerii also acts on CO in the absence of H,. In this case the reaction course could be represented as follows: 4CO + 4 H 2 0 + 4 c 0 , + 4H, and CO, + 4H, + CH, + 2H,O. These two reactions sum to yield: 4CO + 2H,O + 3c0, + CH,. M . barkerii could bring about this conversion even in an atmosphere of 100% CO. M . formicicum be-
+
12
YOUNG M. KIM AND GEORGE D. HEGEMAN
haved like M . barkeru except that a complete conversion of a CO + H, mixture could only be obtained with low concentrations of CO. Yagi (1958, 1959) reported that formate was not an intermediate during CO oxidation in Desulfovibrio desulfuricans and that the reaction might be CO + H,O + CO, H, because of the presence of hydrogenase activity in the cellfree extracts. He suggested that a CO-activating enzyme and an hydrogenase might take part in this reaction together with an electron carrier linking them, but there was no direct evidence for this suggestion. By use of HCO, - and methyl viologen, Yagi and Tamiya (1962) demonstrated that the reaction for oxidation of CO in D . desulfuricans could be reversed in cell suspensions. They also reported that the forward and the backward reactions were inhibited completely by 1 mM KCN, but none of the following reagents at the same concentration inhibited either reaction: oxine, o-phenanthroline, EDTA, NaF, N,H,, NH,OH, As,O,, or HgCl,. Clostridial CO oxidation is in many ways like that of the methanogens, i.e., it involves nonspecific enzyme activity. However, CO dehydrogenase also performs an essential function in pyruvate oxidation. CO may be an electron donor for the reduction of methyltetrahydrofolate to acetate in these cells (Drake er al., 1980; Hu er al., 1981). Fuchs et al. (1974) found that CO was oxidized in growing cultures of C. pasteurianum by the same system in vivo and in vitro since the K, and V,,,, for CO under these conditions were almost the same. Fuchs et al. (1975) suggested that the second oxygen atom in CO, must be derived from H,O in anaerobic CO oxidation by C. pasteurianum and that the enzyme mediating the oxidation of 2H+ 2e-. CO be termed “CO dehydrogenase”: CO H,O + CO, It has been reported that free formate was not a detectable intermediate during CO oxidation in C. pasteurianum (Thauer et al., 1974). The CO-oxidizing activity was acid-labile (pH 4.5) and sensitive to molecular oxygen but was relatively stable to heat. Incubation of the extracts with cyanide (10 pM) or methyl iodide (2.5 mM) resulted in a reversible loss of CO-oxidizing activity. The activity of extracts inactivated by cyanide in the absence of CO was partially restored by incubation of the extracts with CO; the inactivation by methyl iodide was reversed by exposure of treated extracts to the light of a projection lamp. From these results it was proposed that a vitamin B,, compound was probably involved in the catalysis of anaerobic CO oxidation. Other studies on the involvement of a corrinoid enzyme in anaerobic CO oxidation were reported by Diekert and Thauer (1978) and Diekert er al. (1979b). Oxidation of CO to CO, by cell suspensions of Clostridium formicoaceticum and Clostridium thermoaceticum growing on fructose and glucose, respectively, stimulated reduction of CO, to acetate and required pyruvate for conversion of CO to CO,. The catalytic mechanism of CO oxidation in C. formicoaceticum and C . thermoaceticum was “ping-pong,” suggesting that the
+
+
+
+
13
OXIDATION OF CO BY BACTERIA
CO dehydrogenase (CO-DH) could be present in both oxidized and reduced forms. The oxidized form was shown to react reversibly with cyanide, and the reduced form with alkyl halides: cyanide inactivated the enzyme only in the absence of CO while alkyl halides inactivated it only in the presence of CO. The CO-DH inactivated by alkyl halides was reactivated by photolysis. They also reported that clostridia mediating a comnoid-independent total synthesis of acetate from CO, did not possess the CO-oxidizing system. The dependence of the synthesis of CO-DH upon cobalt was then tested because previous reports (Diekert et al., 1979b; Diekert and Thauer, 1978; Thauer et al., 1974) suggested that the CO-DH in clostridia might be a corrinoid enzyme. Such a dependence was not demonstrated, but in the course of these experiments it was found that CO-DH synthesis in C. pasteurianurn, C. therrnoacericum, and C . formicoaceticum required nickel rather than cobalt and that this unusual trace metal could be presumed to be involved in the total synthesis of acetate from CO, (Diekert et al., 1979a; Diekert and Thauer, 1980). Strong evidence that CO-DH is a nickel enzyme in Clostridium came with the observation that the radioactivity of 63Ni supplied in the medium to the cells copurified uniquely with the CO-DH of C. rhermoaceticum (Drake et al., 1980). The molecular weight of,the native enzyme was estimated to be 410,000 by gel filtration. Other properties of this enzyme were almost the same as those previously reported (Diekert and Thauer, 1978), except that the purified enzyme was not appreciably affected by alkyl halides, carbon tetrachloride, and metal chelators. The authors said it was possible that methods used to culture the bacterium and to purify and assay the enzyme might have favored the enzyme’s stability and resistance to alkylation and did not eliminate the possibility that the CO-DH was a corrinoid enzyme. However, it is possible that this nickel enzyme has properties similar to those of comnoid enzymes. Drake et al. (198 1) also reported that part of a multienzyme system which catalyzes the homoacetate-synthesizing pathway of C. thermoaceticum via a transcarboxylation reaction involving pyruvate and methyltetrahydrofolate in the terminal step was able to use CO instead of pyruvate as the C, donor. One of the components in this system contained the metallonickel CO-DH. Both 14C0 and 14CH,-tetrahydrofolate entered the acetate pool (Fig. 2). Aerobic methane-oxidizing bacteria catalyze the oxidation of CO only when metabolizing other substrates.Ferenci (1974, 1975) and Ferenci et al. (1975) have studied the oxidation of CO by the bacteria P . methanica and M . trichosporium. In both P . methanica and M . trichosporium the stoichiometry of CO oxidation was consistent with oxidation catalyzed by a nonspecific mono- or 0, + NADH H + + CO, mixed-function oxygenase complex: CO NAD+ + H,O. This is a gratuitous process that actually drains the cell’s supply of reducing power and this process does not support the growth of either organism. They suggested that alcohol oxidation could provide the necessary reducing
+
+
+
14
YOUNG M. KIM AND GEORGE D. HEGEMAN
power (NADH) to the monooxygenase in vivo, although the metabolism of ethanol by P. rnethanica was found unlikely to result in substrate-levelformation of NADH. They assumed that the failure to do so in vitro could be due to the failure of reversed electron transport to take place or to the disruption, solubilization, or dilution of essential electron transfer components upon preparation of extracts. 2. Utilitarian Bacteria Kistner (1954) first reported oxidation of CO by H . carboxydovorans.Oxidation of CO proceeded independently of lactate oxidation and was not repressed by simultaneous oxidation of lactate. However, cells grown aerobically on lactate for 24 hours almost completely lost the ability to oxidize CO. This ability was not recovered upon reincubation under CO. It is perhaps significant that cells which had been grown on CO already possessed the ability to oxidize H,, although H, was oxidized less rapidly than CO by the same culture. It was also noted that H,-grown cells do not oxidize CO. From all the above results he concluded that the oxidation of CO by H . carboxydovorans depended on the presence in the cells of a catalytic system of an adaptive nature. That system had only been found in cells grown in a mineral medium under a mixture of CO and O,, i.e., the CO-oxidizing system is inducible in H . carboxydovorans. By use of cell-free extracts of P. carboxydovorans Meyer and Schlegel(l979) found that the oxidation of CO could be coupled to the anaerobic reduction of methylene blue, thereby discounting the possibility that CO was oxidized by an oxygenase reaction. The possibility that formate or hydrogen gas is an intermediate was discounted on the basis of the differential sensitivity of the activities of formate dehydrogenase and CO-DH to various physical and chemical treatments as well as the failure to trap free formate or H, in coypled optical assays. These results supported the following equation for CO oxidation in this organism: CO H,O + CO, + 2H+ 2e-. A year later, Cypionka et al. (1980) also concluded that CO oxidation by several carboxydobacteria followed the same mechanism as that for P. carboxydovorans. Kim (198 1) used a completely anaerobic enzyme reaction system for the CODH from P. carboxydohydrogena and suggested that water was the source of the second oxygen atom in CO oxidation by P. carboxydohydrogena. However, experiments employing H2I80did not provide evidence to support this assumption for unknown reason(s), as was true for Kirkconnell and Hegeman (1978). Certain problems in the experimental system including the GUMS analysis are thought to be the reason. Since electrons are not available directly from CO it is possible that CO may form a “formate-equivalent” on the enzyme surface by reacting with water as an intermediate during oxidation to CO, even though free formate is not a true
+
+
OXIDATION OF CO BY BACTERIA
15
intermediate during CO oxidation. This “formate-equivalent’’ could be a “formyl” group formed on the surface of the enzyme and be subsequently converted to CO, and to 2H 2e- which reduces the FAD cofactor on the CO-DH. The CO, so formed can subsequently be used as a carbon source via the reductive pentose cycle. The resulting electrons may be transferred to the electron transport system to produce reducing power and energy for cell growth (Kim, 1981). Figure 1 shows a hypothetical mechanism for CO oxidation in P. carboxydohydrogena. Recent evidence (Meyer, 1982) that the enzyme of P. carboxydovorans contains molybdenum, perhaps other metals, and an iron sulfur center as well as FAD suggests that this scheme is over simplified. Anaerobic M . thermoautotrophicum could utilize CO as an energy source by disproportionating CO to CO, and CH, according to the following equation: 4CO + 2H,O + CH, + 3c0, (Daniels et al., 1977). The Occurrence of growth at the expense of CO agrees with the fact that cell-free extracts of this organism contain both an active factor 420 (F,,,)-dependent hydrogenase and a CO-DH that specifically catalyzes the reduction of F4,, with CO. The enzyme activity was reversibly inactivated by low concentrations of cyanide (2 @) and was very sensitive to inactivation by oxygen. An interesting recent report (Lynd et al., 1982) describes the anaerobic metabolism of CO to acetate by the methylotrophic acidogen, B. methylotrophicum. The parental strain consumes CO during growth on various substrates but does not grow at the expense of CO. A variant was selected, however, that was able to grow on CO alone. This acquired ability was stable, and cells of the parental strain grown on methanol-acetate medium paradoxically had even higher levels of CO dehydrogenase activity than variant cells grown on CO. Four moles of CO were converted to roughly 2 moles of CO, and 1 mole of acetate. The authors put foreward a model (Fig. 2) for anaerobic CO metabolism by B. methylotrophicum similar to that adduced for C. thermouceticum (Drake et al., 1981) and by analogy, other acetogenic clostridia (Diekert and Thauer, 1978) and C. pasteuriunum (Thauer et al., 1974). Acetate may be formed in a corrinoid-dependent transcarboxylation in which pyruvate donates its carboxyl group to methyltetrahydrofolate (Schalman et al., 1973; Welty and Wood, 1977) but in the +
+
CO + H,O
FORMYL-CO-DH(FAD)
2 ~++2eFIG. I . (1981).
Proposed mechanism for CO oxidation in P. carboxydohydrogena. Taken from Kim
16
YOUNG M. KIM AND GEORGE D. HEGEMAN
4co/zjq X-
2
C H,
co,
Fic. 2. Possible mode of formation of acetic acid and COz during anaerobic growth with CO as energy source by E . limosum, B . merhylorrophicum, and oxidation of CO by acetate-forming clostridia. “X” is an unspecified carrier or enzyme. Adapted from Drake e r a / . (1981) and Lynd er a/. (1982). See, e.g., Fig. 2 in Hu era/. (1982) for more detail. The scheme given here is simplified and differs in detail from that presented by other authors.
case of the above systems CO may be used instead of pyruvate as source of the carboxyl group (Drake et a f . , 1981; Hu et a f . , 1982). According to another recent report (Genthner and Bryant, 1982) E. limosum also grows anaerobically with CO but in a rumen fluid-supplemented medium. Here, CO serves as the sole source of energy and the stoichiometry of acetate formation is like that reported for the variant of B. methylotrophicum. Accordingly, it seems reasonable to suppose that the use of CO by E. limosum may also be described by the scheme presented in Fig. 2. There is evidence that R. gefatinosa metabolizes CO to produce equimolar amounts of CO, and H, under anaerobic dark conditions: CO + H,O + CO, + H, (Uffen, 1976, 1981). It has been suggested that Rhodopseudomonassp. grow anaerobically in the light by photodissociation of CO and hemoprotein complex (Hirsch, 1968), but the mechanistic basis for this is obscure. 3. Carbon Monoxide Dehydrogenases of Carboxydobacteria Cypionka et a f . (1980) studied physiological characteristics of various strains of CO-grown carboxydobacteria: P. carboxydohydrogena, P. carboxydojlava, C . compransoris, A. carboxydus, and three other unidentified strains. According to sucrose density gradient centrifugation, the molecular weight of the COoxidizing enzymes of all strains was 230,000, except for A. carboxydus which was 170,000. It turned out that the molecular weights of the CO-oxdizing and H,-oxidizing enzymes were identical, but that the CO-oxidizing enzymes were soluble and the hydrogenases were membrane-bound in all strains examined. Extracts of the four known strains did not show any formate-oxidizing activity. a. Properties. There have been two reports of purified CO-DHs from carboxydobacteria. Meyer and Schlegel (1980) purified CO : methylene blue ox-
OXIDATION OF CO BY BACTERIA
17
idoreductase (CO : MB oxidoreductase) from CO-grown cells of P. carboxydovorans to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. Under air the enzyme was stable for at least 6 days at -20°C had a molecular weight of 230,000, gave a single protein and activity band upon polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis reveded a single band of molecular weight 107,000. The purified CO : MB oxidoreductase was brown colored and had absorption maxima at 405 and 470 nm. From this result the authors suggested that it might be an iron-sulfur protein whose absorption spectrum was not affected by the presence of CO. Neither the absorption spectrum of the native enzyme nor the fluorescence spectrum of the trichloroacetic acid-treated preparation suggested that flavin was a constituent of this enzyme. Because the CO :MB oxidoreductase was free from fonnate dehydrogenase and practically free from hydrogenase activity (2% of that of CO : MB oxidoreductase activity), they concluded that neither hydrogenase nor formate dehydrogenase was functional in catalysis of CO oxidation. Among the metal-chelating agents tested, only cyanide (100 mM) completely inhibited the oxidation of CO. Fonnate and molecular hydrogen had no effect on the enzyme activity, and maximum reaction rates were measured at pH 7.0 and 63°C; temperature dependence followed the Arrhenius equation with an activation energy of 36.8 kJ/mole (8.8 kcalhnole). The apparent K, was 53 p M for CO. Meyer (1982), however, recently reported that the CO-DH from P. carboxydovorans is a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. This enzyme contains 2 moles of noncovalently bound FAD, 4 moles each of acid-labile sulfur and iron, and 8 moles total of iron per mole of enzyme. In addition to these components, molybdenum (1 molehole), zinc (2-3 moleshole), and copper (1-3 moleshole) are present, but nickel is not. The enzyme turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air. As is true for other molybdenum dehydrogenases, this enzyme was readily inactivated by methanol. The soluble yellow CO-DH of P . carboxydohydrogenawas purified 35-fold to better than 95% homogeneity; the purified enzyme comprised about 3% of the soluble cell protein (Kim and Hegeman, 1981a). The specific activity of this enzyme was almost 100-fold greater than that of P. carboxydovorans (1.94 pmoles of CO oxidized per minute mg-I protein) (Meyer and Schlegel, 1980). The purified enzyme is stable for a long time at -70°C under air, and this stability is greater than the enzyme of P. carboxydovorans which is quite sensitive to storage at -20°C under air. The molecular weight of the purified enzyme was found to be 4 X lo5 by gel filtration. The subunit structure of the native enzyme seems to be an unusual a3P3y3 (a,14K; P, 28K; y, 85K) which
18
YOUNG M. KIM AND GEORGE D. HEGEMAN
suggests a complicated mechanism for CO oxidation. A previous report estimates the molecular weight of CO-DH in P . carboxydohydrogenato be 2.3 X lo5 (Cypionka et al., 1980) in disagreement with that found by Kim and Hegeman (1981a) and in agreement with the value of Meyer and Schlegel(1980) for P . carboxydovoruns. The difference between the two values may be due to the different methods used. Cross-linking experiments with reversible or with nonreversible reagents showed that all three kinds of subunits of the purified CO-DH of P . carboxydohydrogena are involved in the cross-linking reaction, but the cross-linking process could not be successfully reversed (Kim and Hegeman, 1981a). Sulfhydryl groups are apparently not involved in CO oxidation. The K,,,for CO of the purified CO-DH from P . carboxydohydrogenais 63 pM. The isoelectric point of the native enzyme was found to be 4.5-4.7. One mole of native enzyme contains at least 3 moles of noncovalently bound FAD as cofactor, suggesting that one of the three types of subunits binds FAD. The purified enzyme was free from formate dehydrogenase and NAD-specific hydrogenase activities but had particulate hydrogenase-like activity (non-NAD-linked hydrogenase activity) with thionin as electron acceptor. Both soluble CO-DHs purified from aerobic utilitarian CO oxidizers have hydrogenase activity (Kim and Hegeman, 198la; Meyer and Schlegel, 1980). The hydrogenase associated with the purified CO-DHs are of a new type which is soluble but cannot reduce NAD. Association of hydrogenase activity with CO-DH may have two possible explanations: (1) Hydrogenase may be involved in CO utilization through the use of H, that may be formed as an intermediate, but not evolved in free form. (2) Hydrogenase is not involved directly in CO oxidation but H, is a pseudosubstrate for the enzyme. The presence of hydrogenase activity in cell-free extracts of A. curboxydus which cannot grow as a hydrogen bacterium (Cypionka et al., 1980) implies that hydrogen may be a pseudosubstrate for CO dehydrogenase(s), although CO is not commonly regarded to be a close analog of H,. Some hydrogen bacteria require a supply of nickel to form active hydrogenase (Friedrich et al., 1981) and the CO-DH of C. thermoacericum is a metallonickel enzyme (Drake et al., 1980). However, several chelators of divalent metals did not inactivate the purified P. carboxydohydrogena enzyme and exogenously added NiCl,, and other divalent metal salts did not activate, but sometimes inactivated, the two purified CO-DHs from carboxydobacteria(Kim and Hegeman, 1981a; Meyer, 1982). By using immunoprecipitation for purification of the CO-DH from 63Ni-growncells of P . carboxydohydrogenait was found that the enzyme does not contain a significant amount of nickel, therefore is presumably not a nickel enzyme (Kim and Hegeman, 1981a). These results and those of Meyer (1982) indicate that nickel is apparently not a necessary factor for CO oxidation in all biological systems and that there is no necessary relationship between hydrogen bacteria and carboxydobacteria with respect to the involvement of this unusual trace metal.
OXIDATION OF CO BY BACTERIA
19
From the above discussion, one might conclude that oxidation of CO as an energy source by carboxydobacteria is mediated by different enzymes of the dehydrogenase type since there are some apparent structural differences between the CO-DHs of P . carboxydohydrogena and of P . carboxydovorans found in different laboratories. However, by denaturing polyacrylamide gel electrophoresis of immune precipitates, it was found that the CO-DHs from these and other carboxydobacteria are very similar in subunit structure and in antigenicity (Kim et al., 1982; 0 . Meyer, personal communication). The differences are therefore probably not large. b. Evolution. As mentioned in the previous section, the CO-DH of P . carboxydohydrogena has a structure apparently different from that of P . carboxydovorans. Taken together with the fact that CO-utilizing bacteria are assigned to different genera and species (Kirkconnell, 1978; Nozhevnikova and Zavarin, 1974; Zavarzin and Nozhevnikova, 1977), it seems probable that there are significant differences in the mechanism of CO oxidation among aerobic utilitarian CO oxidizers. Studies of several strains of aerobic carboxydobacteria, on the other hand, reveal that these bacteria share several common physiological properties important in CO oxidation during autotrophic growth with CO (Cypionka et al., 1980; Meyer and Schlegel, 1980), indicating that the CO-oxidizing system in these bacteria may indeed be quite similar. Immunological observations made by Kim er al. (1982) indicated clearly that the CO-DHs of P . carboxydohydrogena, P. carboxydovorans, and Azomonas sp. 2 were very similar in structure. These three enzymes all have three nonidentical subunits and the molecular sizes of the corresponding subunits of all three enzymes were almost the same. The CO-DHs of P . carboxydovorans and Azomonas sp. cross-reacted with antibody prepared against the purified CO-DH of P . carboxydohydrogena(Fig. 3 ) . These observations agree with the report that the CO-DHs of all of the carboxydobacteria have a common molecular weight except that of A . carboxydus (Cypionka et al., 1980) and that the CO-DHs from several carboxydobacteria cross-react with the antiserum prepared against the purified CO-DH of P . carboxydovorans (personal communication with 0. Meyer, 1981). These facts imply that the genes for these enzymes may be very similar in the different organisms. From the results presented above, Kim et al. (1982) concluded that the oxidation of CO as an energy source by various Gram-negativebacteria is mediated by similar enzymes. Taken together with the fact that bacteria from many different biological groups oxidize CO, this suggests either that the ability to use CO evolved from a common CO-utilizing ancestor at an early time before divergence occurred with (unlikely) conservation of enzyme structure, or that genetic exchange, perhaps mediated by plasmids or other wide-ranging mechanisms, has recently dispersed genes for a common ancestral CO-DH to bacteria of many different groups. The striking structural similarities among the CO-DHs of P.
20
YOUNG M. KIM AND GEORGE D. HEGEMAN
FIG. 3. Double immunodiffusion patterns for carbon monoxide dehydrogenases from different utilitarian aerobes. Immunodiffusion was performed in 1.2% agarose gel for 24 hours at 30°C followed by staining with Buffalo black. AS, 5 pI antiserum prepared against purified CO dehydrogenase of P. carboxydohydrogena (Kim and Hegeman, 1981a); 1, purified. enzyme used to prepare antibody ( P . carboxydohydrogena. 3.75 pg); 2, crude soluble fraction from CO-grown P . carboxydohydrogena (16.5 Fg); 3 . soluble fraction from P . carboxydovorans OM5; 4, soluble fraction from Azomonas sp. 2 (Kirkconnell, 1978; 27.2 pg). Extracts prepared from cells grown in the absence of CO show no reactions. From Kim (1981).
carboxydohydrogena, P . carboxydovorans, and Azomonas sp. 2 suggest that plasmid(s) may be involved. The observation by Y. Park (unpublished observations) working in the authors’ laboratory that these three organisms all carry a small plasmid (6 kb) of apparently identical size tends to support a role for a plasmid. It is interesting to note that genes conferring the ability to grow with H,
OXIDATION OF CO BY BACTERIA
21
in Nocardia opaca (Reh and Schlegel, 1975), Pseudomonas facilis (Pootjes, 1977), and Alcaligenes eurrophus (Anderson et al., 1981) are located on plasmids. Both carboxydobacteria and hydrogen bacteria share in common the unusual ability to use an inorganic trace gas as energy source for the assimilation of CO, as carbon source, and both are facultative autotrophs. Furthermore, some carboxydobacteria (but not all) can also grow as hydrogen bacteria, even though there are some differences in physiology between the true hydrogen bacteria and the carboxydobacteria during growth with H, and CO,. Davis (1967) and Davis et al. (1970) reported that the strain known as H . carboxydovorans Kistner could not oxidize either CO or H,. Zavarzin and Nozhevnikova (1975, 1977) assumed that the loss of the ability to oxidize CO in this bacterium might come from the loss of a plasmid carrying the gene(s) for CO-qxidizing activity. The data reported by Kim ef al. (1982) strongly support that assumption.
B. CARBON MONOXIDE AS CARBON AND ENERGY SOURCE 1. Carbon Assimilation Nonutilitarian CO-oxidizing bacteria cannot use CO as .carbon and energy source under autotrophic growth conditions. However, a report that Methylococcus cupsulatus contains both ribulose biphosphate carboxylase and phosphoribulokinase (Taylor, 1977) opens the possibility that methylotrophs could grow with CO as a source of carbon following its oxidation to CO, using another compound as source of electrons (reductant). The mechanism for synthesis of cellular material from CO, derived from CO in M . thermoautrophicum (Daniels er al., 1977) is yet unknown; methanogenic bacteria do not seem to possess the reductive pentose phosphate cycle as the chemolithotrophs usually do (see Fig. 2 for a possible pathway). Experiments using I4CO indicated that CO is assimilated in aerobic carboxydobacteria after it is converted to CO, (Romanova et al., 1977; Zavarzin and Nozhevnikova, 1977). Sanzhieva and Zavarzin (1971) first reported that S . carboxydohydrogena grew autotrophically at the expense of CO through the fixation of CO, with ribulose diphosphate. Since that report it has generally been accepted that the carbon dioxide produced from CO is fixed in carboxydobacteria via the reductive pentose phosphate cycle based on the kinetics of 14C0 assimilation into early labeled products and on the presence and activity of the key enzymes of the reductive pentose phosphate cycle, ribulose biphosphate carboxylase and phosphoribulokinase (Kirkconnell, 1978; Meyer and Schlegel, 1978; Nozhevnikova and Saval’eva, 1972; Romanova et al., 1977, 1978a; Romanova and Tsyshnatii, 1978; Zavarzin and Nozhevnikova, 1977). But Romanova et al. (1978a) did not exclude the possibility that there exist other reactions for incor-
22
YOUNG M. KIM AND GEORGE D. HEGEMAN
poration of CO into cell material in P. gazotropha because of the early appearance of 14C from H14C0,- in serine (glycine), especially in the absence of an energy source. Measurement of the actual stoichiometry of uptake and fixation of CO have been made by Kistner (1953, 1954), Zavarzin and Nozhevnikova (1977), Meyer and Schlegel (1978), and Kirkconnell (1978). About 4% of the CO used by several strains of carboxydobacteria, 2% of the CO used by Azomonas sp. and Azotobacter sp., and 16% of the CO used by P. carboxydovorans were incorporated into cellular material. These differences may result from quantitative rather than qualitative differences in electron transport systems among these bacteria since it is known that there are no striking qualitative differences among the electron transport systems of several carboxydobacteria examined (see next section). 2 . Electron Acceptors and Electron Transport Systems Assay of the CO dehydrogenases has usually been done using artificial electron acceptors. Cell-free extracts of D. desulfuricans (Yagi, 1958, 1959) and several methanogens (Daniels et al., 1977) used viologen dyes as electron acceptors during the oxidation of CO. Daniels et al. (1977) reported that M . thermoautotrophicum used F420 as physiological electron acceptor, i.e., CO was used as the electron donor via the F,,,-specific CO dehydrogenase. Clostridia can also use viologen dyes, FAD, FMN, and methylene blue, but cannot use NAD, NADP, or ferredoxin from C. pasteuriunum (Diekert and Thauer, 1978; Fuchs et al., 1974, 1975; Thauer et ul., 1974) as acceptors. From the fact that FAD and FMN were both reduced at equal rates, Thauer et al. (1974) proposed that both flavin nucleotides must be considered to be possible physiological electron acceptors in C. pasteurianum. Fuchs et al. (1979, however, reported that a flavin nucleotide was not likely to be the physiological electron acceptor since the reaction was not found to be specific for one of the two flavin nucleotides and since FAD and FMN usually function as prosthetic groups rather than as dissociable coenzymes (electron acceptors), Drake et al. (1980) reported that ferredoxin purified from C. thermoaceticum and C . pasteurianum and b-type cytochromes of C . thermoaceticum were rapidly reduced by CO in the presence of the CO-DH prepared from C. thermoaceticum, and both were therefore considered to be possible native electron carriers. Since methyl and benzyl viologen were reduced by CO, it was thought that ferredoxin might be a physiological electron acceptor for C. thermoaceticum . FMN and cytochrome c j (Desulfovibrio vulgaris) were also recuced while spinach ferredoxin, FAD, NAD, and NADP were not. The reduction of b-type cytochrome by CO-DH demonstrated the potential for anaerobic oxidative phosphorylation accompanying CO oxidation, i.e., CO-DH might
23
OXIDATION OF CO BY BACTERIA
play a fundamental role in the energy metabolism of the cells, but there was no further investigation of this possibility. Studies of artificial electron acceptors for CO oxidation in carboxydobacteria using purified enzymes (Kim and Hegeman, 1981a; Meyer and Schlegel, 1980) and crude extracts (Cypionka er al., 1980; Kirkconnell, 1978) revealed that methylene blue, thionin, toluylene blue, and phenazine methosulfate, but not viologen dyes, NAD, FAD, nor FMN, could function as electron acceptors. This suggested that ubiquinone might be a physiological acceptor for electrons from CO in this group of bacteria. Restoration of the CO-DH activity in UV-treated cell-free extracts of P. carboxydohydrogena using ubiquinone 10 (UQ,o) revealed that a quinone was a necessary and physiological electron acceptor in CO oxidation (Kim and Hegeman, 1981b). However, Schlegel and Meyer (1981) reported that quinones as well as ferredoxins could not serve as electron acceptors in P. carboxydovorans,which contradicts previous reports (Cypionka et al., 1980; Meyer and Schlegel, 1979, 1980). Several carboxydobacteria possess cytochromes of the b, c, a, and o types, indicating that a typical electron transport system participates in CO oxidation (Kim and Hegeman, 1981b; Kirkconnell, 1978; Lebedinskii et al., 1976; Meyer and Schlegel, 1978; Zavarzin and Nozhevnikova, 1977). Experiments using "Nadi" reagent (to test terminal oxidase function) and electron transport system inhibitors suggested that the existing electron transport system functions during CO oxidation and that electrons from CO are delivered at the level of quinone (Kim and Hegeman, 198Ib). The presence of cytochrome o in carboxydobacteria strongly supports the conclusion that cytochrome o functions as a terminal oxidase (Jurtschuk and Yang, 1980) in cells grown with CO, and that there may be a branched electron transport system in carboxydobacteria. Cytochrome o in Bacillus megarerium has a lower affinity for CO than cytochrome a3 (Broberg and Smith, 1967). A similar system in carboxydobacteria acting together with the CO-DH may be partly responsible for CO tolerance in the carboxydobacteria. Carboxydobacteria cannot obtain reducing power directly through the reduction of NAD(P) to NAD(P)H from CO with the CO-DH. Since'electrons from CO are delivered from the CO-DH at the level of quinone, it seems that reduced pyridine nucleotide must be generated by reverse electron transport, a process which is inefficient when compared with direct reduction of NAD(P)+ by substrate. This inefficiency may explain why carboxydobacteria grow slowly with CO (doubling time = 12-42 hours) (Cypionka et al., 1980; Kim, 1981; Meyer et al., 1980; Meyer and Schlegel, 1978; Sanzhieva and Zavarzin, 1971) and why the efficiency of conversion of CO carbon to cellular material is low (2 16%) (see Section IV,B,l). A possible electron transport system for P. carboxydohydrogena during growth with CO is shown in Fig. 4 (Kim, 1981). +
-
24
YOUNG M. KIM AND GEORGE D. HEGEMAN
co + -CDDH-UQ-CYT.
HL 0
b-m.
C
b r . aa,
FIG. 4. Proposed electron transport system in P. carboxydohydrogena during growth with CO. Branching is presumed to occur at the level of cytochrome c but there is no evidence for this. Adapted from Kim (1981).
V. Environmental Significance It is now clear that the ecological distribution of CO-oxidizing bacteria is quite broad. Carboxydobacteria were thought to be a potentially powerful natural tool for removing CO from the atmosphere (Nozhevnikova, 1974; Nozhevnikova and Zavarzin, 1973; Pandaw er al., 1960; Zavarzin and Nozhevnikova, 1975) and, in nature, may be a part of the microflora utilizing organic intermediates as well as H, and CO produced by anaerobic bacteria (Zavarzin and Nozhevnikova, 1977; Kiessling and Meyer, 1982). But Bartholomew and Alexander (1979) concluded that CO oxidation in soil was not the result of autotrophic metabolism by aerobic carboxydobacteria and that “cometabolic” oxidation of CO to CO, by nonutilitarian CO-oxidizing bacteria might be the major microbial mechanism for the removal of CO in nature. The relatively high K , (53 63 pM) for CO of the purified CO-DHs from carboxydobacteria (Kim and Hegeman, 198la; Meyer and Schlegel, 1980) also casts doubt on whether utilitarian CO oxidizers can use atmospheric CO for growth since the concentration of CO in free air is so low [1.3 39 nmole/liter (0.03 0.9 ppm) depending upon the site of measurement] (Robbins et d . , 1968). Conrad and Seiler (1980a), however, tested P. carboxydovorans to see whether it could utilize the low concentrations (0.7 ppm) of CO present in laboratory air and found that CO was consumed by this organism in sterile soil. From this observation they concluded that this bacterium was a genuine oligocarbophilic microorganism and that its ecological niche in nature might include the utilization of the CO present in the atmosphere. They also reported that CO was consumed by fresh soil under anaerobic as well as aerobic conditions even though the anaerobic consumption rate was lower. Anaerobic preincubation of the soil stimulated the anaerobic CO consumption and reduced the aerobic consumption. Following this examination they suggested that anaerobic microorganisms were also of significance in the consumption of atmospheric CO. Conrad er al. (1981) tested CO consumption rates of several carboxydobacteria at high (50%) and low (0.5 ppm) mixing ratios of CO in air and concluded that carboxydobacteria cannot contribute significantly to the consumption of atmospheric CO since the K , values for CO in cell suspensions of the carboxydobacteria and in cells added to sterile soil (465-1 110 ppm) were much higher than those of the natural soils (5-8 ppm). They assumed that other microorga-
-
-
-
OXIDATION OF CO BY BACTERIA
25
nisms with a high affinity for CO are responsible for the oxidation of the atmospheric CO at the soil surface and that an ecological niche for the carboxydobacteria which have low affinity for CO may be the scavenging of CO at microsites where CO occurs locally at high concentrations during catabolism of organic matter. For instance, it is known that flavonoids and porphyrins are decomposed in the soil with formation of CO (Stevens er al., 1972; Westlake et al., 1961). Rainwater sometimes contains up to 200 times the concentration of CO expected based upon the partial pressure in the atmosphere (Swinnerton et al., 1971). Phototrophic bacteria may remove significant amounts of CO formed during degradation of photosynthetic pigments in decaying vegetative materials in anaerobic sediments (Uffen, 1976). Fuchs et al. (1974) suggested that C. pasteurianum participates in the continuous removal of CO from the environment based on the ability to grow under a low concentration (1 ppm) of CO, but this possibility was rejected by Bartholornew and Alexander (1979) since the ability to oxidize CO anaerobically was abolished by pasteurizing soil (25 minutes at 70°C).
VI. Applications Study of bacterial CO oxidation is, of course, interesting in its own right, but it has recently attracted much attention because CO oxidizers may be used to reduce locally high concentrations of CO in the environment or to produce useful chemicals or single-cell protein from toxic industrial waste gas. A better understanding of the mechanism of biological CO oxidation may also permit the development of large scale nonbiological devices for industrial use involving rational catalytic reactor design based on biological models. Since carboxydobacteria have the ability to grow in gas mixtures containing H, with significant amounts of CO, it has been proposed to cultivate these organisms in unpurified gas from blast-furnaces, synthesis gas made from coal and steam, or the CO + H, mixtures from more modem processes (Hirsch er al., 1982) to produce single-cell protein (Meyer, 1980, 1981; Sanzhieva and Zavarzin, 1971; Saval’eva and Nozhevnikova, 1972). Formyl or other CO-derived intermediates in catalytic conversions are of interest in a number of industrial processes (Haggin, 1982). For instance, it has been reported that methane (“COthane”) can be produced from CO in waste gases through a two-step process using chemical catalysts (Heylin, 1981b). The process uses nickel or cobalt as a catalyst to disproportionate CO to CO, and an active carbon : catalyst complex. The active complex further reacts with water to form equimolar quantities of methane and CO,: 4CO
270-300°C + catalyst -2C02
+ 2C :catalyst
26
YOUNG M . KIM AND GEORGE D . HEGEMAN 2C:catalyst
+ 2H20 (steam)
CH4
+ COz + catalyst
(2)
The net reaction is 4CO + 2H,O + CH, + 3co,, which is the same as that for CO oxidation in M . thermoautotrophicum(Daniels et af., 1977) and M . barkerii (Kluyver and Schnellen, 1947). Interest in practical utilization of CO in waste gases to produce useful gas, together with the assumption that a formyl intermediate may occur during CO oxidation in P . carboxydohydrogena, motivates a search for methods to produce formic acid from CO which is presently flared at industrial locations. There is already one industrial process proposed to produce formic acid from CO using catalytic carboxylation of methanol (Keylin, 198la): CHIOH
+ CO + C H 3 - O - C ( O + H
CHja(O+H
(methyl formate)
+ HzO + CHjOH + HCOOH
(3)
(4)
But this process may need further work to clean the used methanol for recycling, especially if waste gases containing contaminants are used. It is well known that CO can react with several metals such as nickel (Ni), cobalt (Co), and iron (Fe) to make metal carbonyl complexes, e.g., nickel tetracarbonyl [Ni(CO),] and iron pentacarbonyl [Fe(CO),]. The metal carbonyl complex usually reacts with water to yield metal formate [M-O-C(O)-H] and eventually produces metal hydride (M-H) and CO, upon pyrolysis (Deeming and Shaw, 1969). But if one can develop a method efficiently to hydrolyze the metal :formate complex, it would be possible to produce formic acid from
co.
It has been suggested to grow R. gefatinosa with CO to produce CO, and H, and to grow several methanogenic bacteria to produce methane from coal gasification products (CO,, H,, and CO) (Wise et al., 1978). The report that it is possible to replace pyruvate with CO as C, donor during the homoacetate synthesis in C. thermoaceticum opens another possibility that CO may be used to produce acetic acid biologically. Whether any of these suggestions will be commercially competitive is problematic (Pape, 1978).
VII. Conclusions Diverse bacteria that are present in the soil or surface water can remove CO from the biosphere by oxidizing this gas to CO,. Many of these organisms do not profit from this oxidation and oxidize CO gratuitously. It is clear that nonspecific enzymes which have various metabolic functions are involved in this nonutilitarian CO oxidation, though the mechanism of this type of CO oxidation appears not to be uniform. Anaerobic clostridia, carboxydobacteria, meth-
OXIDATION OF CO BY BACTERIA
27
anogens, and sulfate-reducing bacteria oxidize CO via a dehydrogenase, and the second oxygen for CO oxidation to CO, probably comes from water. However, ' aerobic methane-oxidizing bacteria catalyze the reaction via a nonspecific methane monooxygenase, i.e., the oxidation of CO to CO, is not the physiological function of this enzyme which uses 0, as the source of second oxygen atom during CO oxidation. The discovery of nickel in the CO-DH of the homoacetate forming clostridia and of synthesis of acetyl CoA from CO by these and other anaerobic acetogens, is an interesting outcome from studies of the anaerobic COutilizing bacteria. Studies of utilitarian CO oxidation revealed a number of important points. The ability to grow at the expense of CO is not confined to a specific, phenotypically homogeneous group of organisms, but rather is widely distributed, and it may be genetically transmissible by plasmid(s) among a physiologically related group of Gram-negative and, possibly, Gram-positive bacteria. Almost all the carboxydobacteria studied so far share common physiological adaptations for CO oxidation. The CO-dependent reduction of artificial electron acceptors has been demonstrated in most strains tested, and electrons from CO are probably delivered to the electron transport system at the level of quinone; pyridine nucleotides are apparently not involved in CO oxidation. Neither formate nor hydrogen appears to be free intermediates during aerobic CO oxidation, suggesting a direct dehydrogenation of CO by CO dehydrogenase (CO : acceptor oxidoreductase) in which water presumably serves as the source of the second oxygen for CO oxidation even though there is no direct evidence for this hypothesis: CO H,O + CO, 2H + 2e-. CO has to be converted to CO, before it is incorporated into bacterial cellular material, and key enzymes of the reductive pentose phosphate cycle have been identified in all aerobic CO-utilizing bacteria examined. Cytochromes of the b, c, 0, and a types were detected in many strains tested after aerobic growth with CO as the sole source of carbon and energy, i.e., there is no qualitative modification of the general electron transport system to avoid the toxic effects of CO during aerobic oxidation of CO. It is apparent that many carboxydobacteria, but not all, can also grow as hydrogen bacteria with H, and CO,, but bacteria isolated as hydrogen oxidizers do not usually use CO. In both cases that have been examined, the purified CO-DH from aerobic carboxydobacteria has hydrogenase activity, and growth with CO induces hydrogenase synthesis. A CO-DH may also be involved in the anaerobic utilization of CO by photosynthetic bacteria in the dark and by M . thermoautotrophicum; hydrogenase may be responsible for energy generation from H, which results from oxidation of CO with H,O in photosynthetic bacteria. The ability of M . thermoautotrophicum to use CO as energy source, even though marginal, opens the possibility that other methanogens may also use CO under optimal experimental conditions. It has recently been reported that the rumen acidogen, E. limosum, and a stable variant
+
+
+
28
YOUNG M. KIM AND GEORGE D. HEGEMAN
of a very similar methylotrophic acidogen, B. methylotrophicum, grow anaerobically with CO as sole energy source.
ACKNOWLEDGMENTS Work in the author’s laboratory on the oxidation of carbon monoxide by bacteria is supported by a research grant PCM 78-12482 from the U.S.National Science Foundation. We wish to thank Dr. 0. Meyer and many other colleagues for generously sharing unpublished findings with us and for constructive criticism.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 81
Sensory Transduction in Bacterial Chemotaxis GERALDL. HAZELBAUER* AND SHIGEAKI HARAYAMAt *BiochemistrylBiophysics Program, Washington State University, Pullman, Washington, and f k b o r a t o t y of Genetics, Department of Biology, Faculty of Science, University of Tokyo. Hongo, Tokyo, Japan I.
......................................
Introduction
A. Excitation . . ..... . . .. . . .. ... .. . . . . . . . . ..........
............... ...................
111. Conventional R
33 33 34 35 36 31 38 39 40
41 42 45 V. The Excitatory Link. VI. Structure of Transduc A. Multiple Methylation and CheB-&pendent Modification.. . . . . B. Homologies and Analogies among Transducer Genes and Their Products . . . . . . . . . . . . ........... VII. Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Control of Methylation ........... B. Adaptation in the Absence of Methylation. . . . . . . . . . . . . . . . . . C. A Functional Role for the CheB-Dependent Modification? . . . . VIII. Pathways for Unconventional Excitation and Adaptation . . . . . . . . . . IX. Concluding Remarks . . . . . . . . . .. . . .. . References .........................................
50 52
52
56 58 58 60 61 63
64 65
I. Introduction A. THESCOPEOF THISREVIEW Just over a dozen years ago, Julius Adler published a seminal paper demonstrating that chemotactic sensitivities of Escherichia cofi were mediated by specific chemoreceptors (Adler, 1969). The importance of this finding was that it identified a receptor-mediated sensory-response system which could be studied using the powerful approaches of molecular genetics and biochemistry. There are now over a dozen laboratories actively involved in the elucidation of the mecha33
Copyright Q 1983 by Academic Ress. Inc. All rights of repmduc:ion in any form reserved. ISBN 0- 12-36448I -X
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GERALD L. HAZELBAUER AND SHIGEAKI HARAYAMA
nisms of bacterial chemotaxis, primarily in the related enteric species, E. coli and Salmonella typhimurium, and the amount of information being generated makes it difficult to summarize the current understanding of all aspects of the phenomenon. The editors of this series suggested that we focus on “signal transduction in bacterial chemotaxis” and thus we have considered topics related to that subject. We have tried to summarize most of the information available about chemoreceptors and transducers since few molecular components in any receptor system are as well characterized as these proteins. We have also considered in detail the functions of excitation and adaptation which are mediated by receptors and transducers. This emphasis required that we say little about the motility system or about the products of the che genes, which are central to the control of flagellar function and adaptation. At present, most of the information about the che genes derives from genetic studies and that body of information have been recently summarized by J. S. Parkinson (1981). We refer the reader to earlier reviews which consider bacterial flagella and motility in detail (Berg, 1975; Iino, 1977; Silverman and Simon, 1977a; Macnab, 1978, 1980; Silverman, 1980) and recent reviews which provide different emphases and viewpoints (Hazelbauer, 1980; Koshland, 1980, 1981; Ordal, 1980; Taylor and Laszlo, 1981). Chemoreceptors were considered in detail several years ago (Hazelbauer and Parkinson, 1977) while transducers and methylation have been emphasized previously in two excellent reviews (Springer et al., 1979; Boyd and Simon, 1982). B. BACTERIA AS SENSORY CELLS Motile bacteria are sensory cells. Like some other sensory cells, motile Escherichia coli alternates in a random manner between two states, S and T, as diagrammed in Fig. 1. A cell spends 80-90% of the time in the S state. An average duration of residency in an S state is 1-2 seconds and in a T state 0.1-0.2 seconds. A temporal change in the chemical environment of the cell results in a rapid response in the form of an alteration in its behavioral pattern. A favorable change (increase in concentration of an attractant, decrease in a repellent) produces exclusively S state behavior and an unfavorable change results in a greatly increased proportion of time in the T state. The rapid response to a temporal gradient is termed excitation. Like many sensory cells, E. coli adapts to stimuli. Thus the response, a shifted balance between the S and T states, occurs for only a limited period of time even though the altered chemical environment persists. After a time ranging from several seconds to several minutes, depending upon the particular compound and the magnitude of the concentration change, the initial pattern of switching between S and T states is reestablished and the cells have adapted to the original stimulus. The phenomenon of adaptation implies that the sensory cell responds to changes in concentration of a compound rather than to the absolute concentration.
35
BACTERIAL CHEMOTAXIS
T
uu
UI /
excitation
+
IU u uuu u
\ adaptation t adapted state
cw
FIG.1. Schematic representation of chemotactic behavior. (A) Pattern of swims and tumbles. A cell alternates between swims (S)and tumbles (T) which result from counterclockwise (CCW) and clockwise (CW) rotation, respectively, of the flagellar motor. Addition of attractant (+att) results in immediate suppression of tumbles. After adaptation there is a short “overshoot” period during which periods of CW rotation are more frequent than before stimulation. Removal of attractant (-att) causes an increased frequency of tumbles. Adaptation to this negative stimulus occurs about 10-fold more rapidly than adaptation to the equivalent positive stimulus. (B)Changes in methylation during tactic behavior. The level of carboxyl methylation of specific glutamyl residues of the relevant transducer molecules increases during adaptation to positive stimuli and decreases during adaptation to negative stimuli.
C. BACTERIALMOTILITY The concerted functioning of the bacterial motility and sensory systems permits cells to make net progress in spatial gradients of chemicals. An E. coli cell in an isotropic environment swims in a straight line for a few seconds, then undergoes an episode of uncoordination called a tumble, after which swimming is resumed in a new, randomly chosen direction (Berg and Brown, 1972). Swims and tumbles are the S and T states described in the preceding paragraph. Continual alternation of swims and tumbles causes the cell to trace a three-dimensional random walk. The sensory system directs a cell to favorable chemical environments by biasing the random walk with longer path lengths for swims that happen to be in the direction of increasing attractant or decreasing repellent concentration (Berg and Brown, 1972; Macnab and Koshland, 1972; Brown and Berg, 1974). The phenomenon is the same as illustrated in Fig. 1. Temporal increases in attractant concentration experienced by a cell swimming in a gradient generated by diffusion from a source of the attractant are relatively small, so that the period of tumble inhibition creates swim times approximately twice the usual duration (Berg and Brown, 1972). This bias is sufficient to allow extensive accumulation of a population at the source of a diffusion gradient (Adler, 1973).
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GERALD L. HAZELBAUER AND SHIGEAKI HARAYAMA
The origin of swims and tumbles can be understood in a context of the unusual mechanism of bacterial motility. Bacterial flagella are related to eukaryotic flagella and cilia only in name and a common function as motor organelles. Bacterial flagellar filaments are left-handed helical polymers of protein subunits (often a single type of monomer) approximately the diameter of a tubulin fiber (Iino, 1977). In enteric bacteria, the filaments are 5-10 pm long, attached at randomly distributed points on the surface of cells 1-2 pm in length. The filaments have no enzymatic activity, nor do they contract. Rather, they function in motility as helical propellers, turned by a rotary motor embedded in the cell envelope (Silverman and Simon, 1974; Larsen et al., 1974b; Berg, 1974). The rotary motor consists of a shaft and a series of discs attached to the cytoplasmic membrane and cell wall (DePhamphilis and Adler, 1971a-c). Little is understood about the mechanisms by which the rotary motor functions but it is clear that the energy source is the proton motive force across the cytoplasmic membrane (Larsen et al., 1974a; Manson et al., 1977, 1980; Skulachev, 1977; Matsuura et al., 1977; Shioi el al., 1978; Glagolev and Skulachev, 1978; Khan and Macnab, 1980). Counterclockwise rotation of left-handed hellices exerts a pushing force on the cell. A universal joint, called a hook, attaches each filament to a rotary motor shaft and thus each of the 6-8 flagella on a cell can bend back at the hook to form a bundle of filaments which produces a concerted and coordinated pushing force on the cell, causing it to swim forward (Macnab, 1977). Counterclockwise rotation of closely aligned left-handed helices can occur without tangling because the sense of rotation is the same as the turn of the helix, allowing individual helices to slide past one another. In contrast, clockwise rotation of left-handed helices causes the flagellar bundle to dissociate. The flagella exert pulling forces, which are necessarily uncoordinated since each flagellum is attached to the cell at a separate point on the surface. In addition, clockwise rotation produces a torque of the opposite sense from the helix of the filament, causing deformation of the polymer, even forming localized areas of right-handed helix. The sum of all these effects is a tumble (Macnab and Omston, 1977). Thus the balance between swims and tumbles is the balance between counterclockwiseand clockwise rotation of the flagellar rotary motors. The sensory system biases the random walk by affecting the relative probabilities of the two directions of rotation of the rotary motor. 11. Components and Features of the Sensory System
The power of molecular genetics has made possible the identification of most, if not all, of the molecular components involved in bacterial behavior. In this section, we will outline the present understanding of the conventional pathways
BACTERIAL CHEMOTAXIS
37
of excitation and adaptation and then consider specific aspects and variations in more detail in later sections. A. EXCITATION Excitation is the shift in the normal balance between the two directions of flagellar rotation which occurs in response to a change in concentration of a chemical in the solution surrounding a cell. The concentration of attractants in the environment of a cell is monitored by specific receptor proteins which have recognition sites exposed to the exterior of the cell. Changes in the proportion of occupied receptor sites result in an effect on the rotary motor in approximately 200 msec (Segall et al., 1982). The recognition sites for serine and aspartate, the two most powerful attractants for enteric bacteria, are located on integral cytoplasmic membrane proteins, coded for by the genes rsr and tar, respectively (Hedblom and Adler, 1980; Wang and Koshland, 1980). The recognition sites for galactose and glucose (Hazelbauer and Adler, 197l), maltose (Hazelbauer, 1975), and ribose (Aksamit and Koshland, 1974) are contained on three separate peripheral membrane proteins. It seems likely that occupancy of a serine or aspartate site causes a conformational change in the transmembrane receptor that in turn generates a perturbation which serves as an excitatory signal that in turn affects the “gear shift” of the flagellar rotary motors. Loss of ligand from occupied transducer upon a reduction in ligand concentration (a negative stimulus) results in excitation of the opposite polarity, i.e., increased frequency of tumbles, to that observed upon concentration increases. Thus loss of ligand must induce a conformational change which generates an excitatory signal of the opposite polarity to that generated by a positive stimulus. Ligand-occupied molecules of maltose receptor interact with the Tar protein (Koiwai and Hayashi, 1979; Richarme, 1982), presumably generating an excitatory change similar to that generated by direct binding of aspartate. Excitation by galactose or ribose requires the product of frg (Kondoh et al., 1979; Hazelbauer and Harayama, 1979; Harayama er al., 1979), a cytoplasmic membrane protein analogous in many ways to the Tsr and Tar proteins (Hazelbauer er al., 1981; Harayama et al., 1982). It is likely that the ribose and galactose receptors interact with the Trg protein in the same manner as the maltose receptor interacts with the Tar protein. Because of their role in converting external stimuli in the form of temporal gradients into internal excitatory signals, the Tsr, Tar, and Trg proteins are often referred to as transducers. There is no information available about the character of the excitatory alteration undergone by transducers nor do we understand the nature of the excitatory linkage between transducer and flagellar motor. Analysis of a membrane fraction enriched in areas surrounding the motors revealed no enrichment of transducer proteins, implying that the link is not direct physical interaction of transducer and
38
GERALD L. HAZELBAUER AND SHIGEAKI HARAYAMA
motor (Engstrom and Hpelbauer, 1982). Instead it appears that occupied transducers generate an excitatory signal which travels through the cell from transducer to motor. The most attractive candidates for the signal include small, diffusible molecules or alterations in the permeability properties of the cytoplasmic membrane which could produce fluxes of specific ions across that membrane (see Section V). Whatever the nature of the excitatory signal, excitation can persist for several minutes after a maximal temporal stimulus, implying that the excitatory perturbation must be maintained over that time scale. Thus it is unlikely that the signal would be analogous to the rapid and transient membrane depolarizations which constitute action potentials in eukaryotic nerve cells. There are six cytoplasmic proteins, produced from the six che genes, which are critical for maintaining and controlling the normal balance between the two directions of flagellar rotation (Parkinson, 1978; DeFranco er al., 1979; Parkinson and Houts, 1982). Mutants missing the CheR, CheB, CheY, or CheZ proteins (Parkinson, 1981) can still be excited by several stimuli (although they are abnormal in adaptation) and mutants missing the CheA or Chew proteins can be induced to tumble by a strong acetate stimulus (Parkinson and Houts, 1982). Thus none of those proteins is an absolutely required component of the excitatory pathway. The excitatory signal is ultimately received by the flagellar motor, which responds by a shift to exclusively counterclockwise or predominantly clockwise rotation. A number of motor components are involved in determining the balance between the directions of flagellar rotation as evidenced by the observation that specific mutations in the genes coding for those components can alter the balance, creating tactically defective cells (Parkinson, 198l ; Parkinson and Houts, 1982). Particularly striking is a mutation in theflaAll gene of S. typhirnurium which shifts the rotation balance so extremely to clockwise that the flagellar filament is transformed into a right-handed helix (Khan er al., 1978). In cells containing the mutation, stimuli inducing counterclockwise rotation of the motor cause tumbling and stimuli inducing clockwise rotation cause swimming. Thus responses are inverted and the cells move away from attractants and toward repellents (Rubik and Koshland, 1978).
B. ADAPTATION Adaptation is the reestablishment, after excitation, of the normal balance between the two directions of flagellar rotation even though the altered chemical environment persists. Adaptation is strongly correlated with a covalent modification of the population of transducer molecules through which excitation passed. The simplest model suggests that the excitatory change induced by ligand or ligand-receptor binding to the transducer is effectively cancelled by the covalent modification. Detailed behavioral studies of the course of adaptation indicate that
BACTERIAL CHEMOTAXIS
39
adaptation proceeds slowly at a constant rate over the time period between excitation and reappearance of the original behavioral pattern in the adapted state (Berg and Tedesco, 1975). The time course of the covalent modification corresponds to this pattern (Springer et al., 1979). The modification is carboxyl methylation (Kort et al., 1975) of specific glutamyl residues in the transducer to form carboxyl methyl esters (Kleene et al., 1977; Van der Werf and Koshland, 1977; Stock and Koshland, 1981). A specific methyl transferase, coded for by cheR (Springer and Koshland, 1977), catalyzes the transfer of a methyl group from S-adenosylmethionineto the carboxyl group, and a specific demethylase, coded for by cheB (Stock and Koshland, 1978), catalyzes demethylation, producing methanol (Toews and Adler, 1979) and a regenerated glutamyl residue. Adaptation to positive stimuli (attractant increases, repellent decreases) is linked to increased methylation and adaptation to negative stimuli is linked to demethylation (Springer et a f . , 1977b, 1979; Silverman and Simon, 1977b). There is an asymmetry in the rates of the two types of adaptation. For a given change in receptor occupancy, methylation to the level necessary to establish the fully adapted state requires approximate 10 times as long as the same quantity of demethylation required for adaptation in the opposite direction. Protein carboxyl methylation is not limited to bacteria, but rather is widespread among many higher organisms (for reviews see Gagnon and Heisler, 1979; Paik and Kim, 1980; for recent studies see Usdin et a f . , 1982). All calf tissues examined contained some protein carboxyl methyltransferase activity and some methyl acceptor activity, with certain endocrine organs, some areas of the central nervous system, blood, and testis exhibiting high levels of activity (Kim et al., 1975; Diliberto and Axelrod, 1976). In vitro carboxyl methylation of the acetylcholine receptor (Kloog et a f . , 1980) and calmodulin (Gagnon, 1982) has been observed. There is evidence indicating that carboxyl methylation may be related to leukocyte chemotaxis (Venkatasubramiaianer al., 1979). Considerable evidence links carboxyl methylation of certain membrane protein to adaptation of Paramecium (Thomson et al., 1981). Thus it may well be that protein carboxyl methylation is often part of the mechanisms by which cells respond to chemical stimuli. C. ADAPTATIONAND "BACTERIAL MEMORY" Sensory-response systems are designed to produce the appropriate response to relevant stimuli. This usually requires that the magnitude of a response is graded in proportion to the magnitude of the stimulus. Many types of responses have an all-or-none character, including the response of the bacterial system. The cell either tumbles or not, so that the only way to grade the size of the response is by controlling its duration. This requires the processes of both excitation and adap-
40
GERALD L. HAZELBAUER AND SHIGEAKI HARAYAMA
tation. The larger the stimulus (i.e., the greater the number of occupied transducers), the longer the time required for the methylation system to neutralize the activated transducers and thus the longer the duration of the response. Any sensory-response system with an all-or-none response would be expected to have an adaptation mechanism and so it is not surprising that covalent modification of critical components is an emerging theme in the study of receptor systems. Adaptation creates a system sensitive to changes in concentration of relevant chemicals, rather than to absolute concentrations. After adaptation, a cell with occupied, methylated transducers is poised in the same state as a cell with unoccupied, unmethylated transducers. The adapted cells can respond to further occupancy of the same receptor-transducer pair (within the limits of receptor site saturation) or to occupancy of a different class of receptor sites. Receptor site occupancy is a measure of excitatory stimuli; the total level of methylation is a measure of the extent of adaptation. When these two parameters are balanced, the cell swims and tumbles in its unstimulated pattern, when they are not, the cell exhibits excessive swimming or tumbling. Since methylation is a relatively slow process, the extent of methylation at a given instant is actually a reflection of the chemical environment a short time before. In contrast the extent of receptor occupancy should reflect the instantaneous concentration of ligand. A comparison of these two parameters would allow a swimming cell to determine whether the relevant chemical environment were changing over the time period of a few seconds as the cell swam forward and to determine whether the overall change was favorable (tumble-suppressing) or unfavorable (tumble-inducing). We do not know how the comparison is accomplished, but since both parameters are directly related to the transducers, it is a reasonable hypothesis that those molecules are central to the comparison process. Thus the bacterial sensory system exhibits properties of a rudimentary memory function. The transducer proteins, in a combination of their roles in excitation and adaptation, appear to be intimately involved in the comparison of past and present.
111. Conventional Receptors
E. coli responds chemotactically to a substantial number of different small molecules and early observations indicated that the active compounds could be grouped into at least 20 chemoreceptor classes (Mesibov and Adler, 1972; Adler et al., 1973; Tso and Adler, 1974). Five chemoreceptor proteins, for the attractants galactose-glucose (Hazelbauer and Adler, 197l), maltose (Hazelbauer, 1975), ribose (Aksamit and Koshland, 1974), serine, and aspartate (Hedblom and Adler, 1980; Wang and Koshland, 1980) have been identified. Recent obser-
BACTERIAL CHEMOTAXIS
41
vations show responses to many repellents are mediated in a manner different from the conventional excitatory pathway outlined above (Repaske and Adler, 1981; Kihara and Macnab, 1981), and presently a reasonable hypothesis is that no repellent excitation occurs in the conventional manner. Many different sugars can be transported in E. coli by the phosphotransferasetransport system and each of these sugars is an attractant (Adler et al., 1973). Each of the eight sugarspecific Enzymes I1 appears to serve as a chemoreceptor (Adler and Epstein, 1974). However, recent studies imply that neither transduction of nor adaptation to these stimuli involves conventional transducers which can be carboxyl methylated (Niwano and Taylor, 1982a). Thus it is quite possible that the basic sensory-receptor system of enteric bacteria consists of only five conventional receptors and the three transducers to which those recognition sites are linked. We will discuss the conventional components first and then turn to the unconventional systems. A. AMINOACIDRECEFTORS Serine and aspartate are the two strongest attractants for enteric bacteria (Mesibov and Adler, 1972). The genetic organization of the bacteria indicates clearly that the entire motility-taxis system is integrated as a single unit since all genes are under a common cascade of genetic control (Iino, 1977; Silverman, 1980). The only two receptor protein genes included in this integrated system are tsr and tar (Clarke and Koshland, 1979) which code for the. serine and aspartate receptors, respectively (Hedblom and Adler, 1980; Wang and Koshland, 1980). A strong argument can be made that the motility-taxis system in these species developed (presumably in a common ancestor) in response to selective pressures conferring advantages on cells which could respond to gradients of these two specific amino acids. These two receptor sites are also the only ones to be integrated on the polypeptide chain of transducer proteins. Since the binding sites for ligand or ligand-receptor complex should be on the external face of the cytoplasmic membrane and methylation is controlled by enzymes in the interior of the cell, it is assumed that transducer proteins are transmembrane. The proteins have a molecular weight of about 60,000 and thus are of a sufficient size to have a receptor domain on the outer surface, a transmembrane domain, and interior domains for excitation and methylation. Crosslinking studies indicate that the Tsr and Tar proteins are complexed as tetramers in the membrane (Chelsky and Dahlquist, 1980b). Whether these are exclusively homotetramers of one type of protein, which are in fact found in mutants missing one or the other protein, or can also be heterotetramers is not known. The structure of the serine and aspartate receptors will be considered in more detail in the context of their role as methyl-accepting proteins. There appears to be a high affinity (Kd = 5 pM) and a low affinity (Kd = 300
,
42
GERALD L. HAZELBAUER AND SHIGEAKI HARAYAMA
pM) receptor site for serine which can be distinguished behaviorily (Springer et al., 1977b) and mutationally (Hedblom and Adler, 1980). The high affinity is clearly contained on the Tsr polypeptide (Hedblom and Adler, 1980; Wang and Koshland, 1980) and the available evidence is consistent with that same protein carrying the low-affinity site (Hedblom and Adler, 1980).
B. SUGARRECEPTORS (TABLE1) The recognition sites for galactose and glucose, ribose, and maltose are carried on three peripheral membrane proteins which perform two separate functions in the cell, chemoreception and transport of their respective ligands (Hazelbauer and Parkinson, 1977). These sugar-binding proteins are extreme examples of peripheral membrane proteins since in broken cells or cells with only the outer membrane disrupted, none of the molecules is found in specific association with the cytoplasmic membrane but rather in the soluble fraction (Heppel, 1971). An intact outer membrane, permeable to small molecules but a bamer to the passage of proteins, is necessary to keep the binding proteins in the periplasmic space bounded by the cytoplasmic and outer membranes. Yet characterization of mutants missing the respective binding proteins shows that they are essential for the membrane-associated functions of tactic excitation (Hazelbauer and Adler, 1971; Aksamit and Koshland, 1974; Hazelbauer, 1975) and transport (Boos, 1972; Kellermann and Szmelcman, 1974). Presumably association of binding proteins with membrane components is relatively transient and weak, and thus the dilution of the periplasm, where binding protein concentrations are in the range of 10-100 p M (Hazelbauer, 1979), releases any molecules bound to specific membrane sites. Studies using the maltose-binding protein indicate that ligand-occupied but not ligand-free protein binds to solubilized (Koiwai and Hayashi, 1979) or membrane-integrated (Richarme, 1982) Tar protein, the transducer of maltose stimuli. These observations suggest a simple model for the first step of transduction of excitatory stimuli from the sugar receptors. Binding of ligand to a binding protein has been shown to induce conformational changes (McGowan et al., 1974; Szmelcman et al., 1976; Zukin er al., 1977a,b, 1979; Zukin, 1979). If the transducer recognizes a site present only on occupied receptor, then a ligandreceptor interaction would immediately lead to an occupied receptor-transducer interaction, resulting in excitation as outlined above. Defects in mutant binding proteins define three functionally separable domains on a binding protein, the binding site, a site for interaction with a chemotactic transducer, and a site €or interaction with additional components of the particular transport system. A few mutations affecting the maltose-binding protein eliminate transport of the ligand without serious effect on ligand binding or tactic response (Hazelbauer, 1975). One galactose-binding protein mutation inactivates chemoreceptorfunction without affecting binding or transport (Hazelbauer and Adler, 1971; Ordal and Adler,
TABLE I CONVENTIONAL CHEMORECEPTORS IN ENTERIC BACTERIA Concentration (fl) for half-maximal effect E . coli
S . typhimurium
Receptor Protein la. Tsr protein (highaffiity serine site)
lb. Tsr protein (lowaffinity serine site) 2 . Tar protein
3. Galactose-binding protein
4.
Ribose-binding protein
5. Maltose-binding protein
Gene tsr
tsr
far
mgls
rbsE
mdE
Ligands Serine a-Aminoisobutyric acid Alanine Glycine Serine Aspartate a-Methyl aspartate Glutamate Glucose Galactose Glycerol-P galactoside Fucose Ribose Allose Maltose
Binding in vitro 50
> 2000
Cellular behavior 36 3000d
Binding in witro
5r (*)C
4~ 5000=
(*)