INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 23, No. 4 October 1973, p. 459-467 Prin ted in U.S.A. Copyright 0 1973 International Association of Microbiological Societies Distribution of in Bacteria: Relationship to General Physiology DAVID J. MEYER' and COLIN W. JONES

Department of Biochemistry, The University of Leicester, England

A review of occurrence in bacteria is presented which gives the taxonomic distribution of cytochromes and which relates this to general physiological characteristics. Data obtained from published research and recent experimental studies on a total of 169 species of bacteria suggested the existence of four major groupings: (i) the aerobic and facultatively anaerobic, heterotrophic gram positives (cytochrome pattern aa3.0. b.c); (ii) the aerobic and facultatively anaerobic, heterotrophic gram negatives (cytochrome pattern either al.d.o.b.c, a1.o.b.c or aa3.o.b.c); (iii) anaerobic and microaerophilic hetero- trophs (cytochrome pattern b sometimes with al /d/o), and (iv) the ch'emo- and photo-autotrophs (cytochrome pattern c plus czl /aa3/o/b). The absence or minor presence of cytochrome c in facultatively anaerobic and anaerobic heterotrophs was confirmed and was also observed in plant and animal pathogens. Cytochrome d was confined in occurrence mainly to a small taxonomic group of organisms characterized by a high degree of adaptability to unstable habitats. This group was considered for further subdivision dependent upon the conditions causing the production of cytochrome d.

As part of an investigation into the occur- ilated on organisms in the three major bacterial orders: rence of more than one spectral type of (i) the taxonomic status of the species according to cytochrome oxidase in many bacteria, a survey Bergey 's Manual of Determinative Bacteriology (13) of published data was carried out. In addition, a (i.e., the order, family, genus, species, strain, and number of species were examined experimen- response to Gram stain); (ii) the physiological type (e.g., heterotrophic, facultatively anaerobic); (iii) the tally to extend existing data. culture conditions with respect to the terminal In contrast to previous reviews (14, 39, 56, electron acceptor, the sources of carbon and nitrogen, 57, 89, 90), this study constitutes a general and the phase of growth of the culture; and (iv) the approach relating cytochrome composition to qualitative cytochrome content. (Although the sev- the physiology and taxonomy of a large enth edition of Bergey 's Manual (1957) is considerably number of bacterial species. Three general out of date, it was considered to be the most workable observations have been reported: firstly, that classification for the present study.) Where a publica- the cytochrome pattern aa3.o.b.c occurred tion failed to conclude whether ana type cytochrome was a, or aa3, the cytochrome was classified for the predominantly in gram-positive bacteria, and survey on the basis of the position of its 01 peak in the the pattern al.d.o.b. c occurred predominantly reduced minus oxidized spectrum, namely, a,, 586 to in gram-negative species (65); secondly, that 596 nm, andaa,, 598 to 608 nm. cytochrome c was very often concluded to be Experimental studies. Bacteria mainly from taxa absent from facultatively anaerobic species (42, not previously investigated were cultured at 30 C in 65); and, thirdly, that cytochromes al and d 600-ml batches of nutrient broth (Oxoid, England), 26 always occurred together (86). This investiga- g plus 3 ml of Silicone MS Antifoam Emulsion RD per tion evaluates and extends these suggestions. liter (Hopkins and Williams, England) in 21 tri-baffled flasks. Aeration was by gyratory shaking (180 rpm; oxygen dissolution rate, 144 mmol per liter per h as measured by a titrimetric procedure [26] ). MATERIALS AND METHODS Cells in either the logarithmic or stationary growth phase were harvested by centrifugation at 4 C, Analysis of published work. The following types of homogenized in distilled water, recentrifuged, and data from relevant scientific publications were assim- resuspended by homogenization in 25 mM sodium ' Present address: Department of Biochemistry, phosphate buffer (pH 7.4) to an extinction at 680 nm Dartmouth Medical School, Hanover, N.H. 03755. of 50 to 80. Cell suspensions in 8- to 10-ml portions 459 460 MEYER AND JONES INT. J. SYST. BACTERIOL. were disrupted by sonic treatment at 0 to 4 C by use occurred not only in heterotrophic, facultative of an MSE 100-W ultrasonic disintegrator at an anaerobes but also in species pathogenic for amplitude of 7.8 pm (peak to peak). In general, 3 min plants and animals, znd in several other were required to disrupt gram-negative cells, 5 min for heterotrophic species. gram positives. Whole cells and cell-wall material were Although the quantitative and, to a lesser removed by centrifugation at 7,000 X g for 10 min, extent, the qualitative cytochrome content and the respiratory membranes were sedimented by further centrifugation at 157,000 X g for 1 to 2 h. The varied with growth conditions in many cases, membranes were finally suspended in the same buffer four groups of physiologically and/or taxo- to a protein concentration of 4 to 10 mg/ml as nomically related species could be distinguished determined by a modified biuret method (41). on the basis of their total cytochrome content, Cytochrome content was determined from di- as follows. thionite-reduced minus oxidized, and dithionite- Group A. The aerobic and facultatively reduced plus CO minus dithionite-reduced difference anaerobic, het erotrophc, gram-posit ive bacteria spectra obtained at room temperature by use of a of the orders Eubacteriales and Actino- Shimadzu MPS 50-L split-beam recording spectre mycetales indeed contained predominantly the photometer. The wavelength accuracy was often checked by use of a filter of known spectrum. aa3.0.b.c pattern (see Table 1). In some cases Distilled water was used to make up the growth either cytochrome aa3 or o was absent, and in a medium, and glass-distilled, deionized water was used few cases (Bacillus megaterium [ KM] [ 141, for the preparation and examination of respiratory Micrococcus denitrificans [ 8 1] , and Bacillus membranes. Reagents were of analytical grade. subtilis [ D4731 -see Table 5) cytochrome aa3 predominated during logarithmic growth and RESULTS cytochrome o was present particularly in the stationary phase of growth. The data obtained in the present work, plus Certain exceptions to the general pattern those gathered from the literature, are sum- occurred: in the family Bacillaceae, cytochrome marized in Tables 1 to 4, which include the al and d were observed “in some cultures” of sources of data. the unusual insect pathogen B. popilliae (71), General observations. Few of the publica- and cytochrome al was concluded to be tions examined contained complete informa- present in B. licheniformis S294 (82), although tion; the presence or absence of cytochrome o in another strain of this species the expected was uncertain in nearly two-thirds of the cytochrome aa3 was present in place of al (see species due to the omission of a reduced plus Table 5). In the Micrococcaceae, M. denitrifi- CO minus reduced spectrum. Where suitable cans was unusual in its ability to produce a spectra were obtained, cytochrome o was found cytochrome cd () complex to be present in 75% of the species. when grown anaerobically with nitrate as There was, in the majority of cases, a close terminal acceptor (62). This organism was correlation between the cytochrome content subsequently transferred to a new genus, and the taxonomic family. Paracoccus Davis (28). The absence of a distinct absorption peak(s) Arthrobacter globiformis NCIB 8602 (family or shoulder corresponding to cytochrome c Corynebacteriaceae) appeared to be an excep

TABLE 1. Cytochrome pattern of aerobic and facultatively anaerobic,gram-positive heterotrophs

No. of species Cy to chrome in agree- Excep- pattern Order Family ment tions Sources of information aa 3. 0.b. c. A ctinom y cetales A ctino planacea e 10 (31,52, -a) (aa3 or o are My co bacteriaceae 6 (8,34,40,57,62,76) sometimes ab- Streptomycetaceae 1 1 (43,661 sent; c is often ab sent) Eu bacteriales Bacillaceae (14, 17,18,19,29,32,61, 63,65,71,82,83,85, 93, I l4 98) Brevibacteriaceae 2 (-9 Corynebacteriaceae (42, 69,80) Micro coccaceae 1 (17, 35,48,49,56,79,81, 85,100,105) a -, See Table 5. VOL. 23, 1973 CYTOCHROME DISTRIBUTION IN BACTERIA 46 1

TABLE 2. Cytochrome patterns of aerobic and facultatively anaerobic, gram-negative heterotrophs (group B)

~~ 7 No. of species in EX- agree- c=P- Sources of Cytochrome pattern Order Family ment tions information

Group B-1: al.d.o.b.c.; Pse udom onadales Pseudomonadaceae 20 1 (7,9,10,46,59,60, c is often absent, al 77,85,89,97) andd are often Eubacteriales A chromobacteraceae 1 0 (6 ) present only after Azotobacteraceae 2 0 (17,27,51,85) log phase Brucellaceae 4 0 (36, 106,103, -") Enterobacteriaceae 11 0 (17, 33, 37,85) R hizobiaceae 0 1 (-a)

Group B-2: a .o.b.c. Pse udom onadales Methanomonadaceae 2 0 (75, -a) Pseudomonadaceae 7 2 (17, 21, 27,72,85) Sp irillaceae 3 0 (24,25)

Group B-3: aas .o.b.c. Pseudomonadales Caul0bac teraceae 1 0 (-a) Pseudomonadaceae 4 2 (20, 21, 22,47,89) Eubacteriales R hizobiaceae 2 -0 (4,5,44) =-, See Table 5.

No. of species in Sources of Cytochrome pattern Order Family agreement Exceptions information

Either no cytochromes, Eubacteriales Bacillaceae (Clostridia) 3 0 (30) or Bac t ero ida c ea e 3 0 (104) sometimes also with Lac tobacillaceae 13 0 (11, 15,30, a,/d/o. (No c 107) observed.) Propionibacteriaceae 5 0 (88,101)

TABLE 4. Cytochrome pattern of autotrophs (group 0)

1 sizizL I 1 Cytochrome pattern Order Family agreement Exceptions Sources of information

c, to ether with Pseudomonadales ~ Athiorhodaceae 4 0 (56,58,78,91, 102) a, faa3/o/b. Chlorobacteriaceae 2 0 (39,561 (No d.) Nit0 bacteriaceae 3 0 (2,3,45,74,90,95,96)

Spirilhcea e 3 0 (23,50,73) Thio bact eriaceae 5 0 (1,70,94,99) Thio rhodaceae 1 0 (6 8)

tion that proved the rule: the cytochrome below and Table 5). Concomitant with this pattern of this gram-variable organism was, in change was the appearance, in mixed chains, of the logarithmic phase, that of a typical gram- gram-negative as well as the original gram-posi- positive, heterotrophic aerobe (aa3.0. b.c), but tive cells. This organism is of further interest after the logarithmic phase, cytochromes aa3 since it is the only organism observed to and c disappeared and were replaced by produce four spectrally distinct classes of cytochromes al and d to yield the pattern of a CO-b inding cyt o chromes. typical gram-negat ive, he terot ro phic, fa cult at ive Group B. The aerobic and facultatively anaerobe of subgroup B-2, i.e., al.d.o.b.c (see anaerobic, het erotro phic, gram-negat ive bac- 462 MEYER AND JONES INT. J. SYST. BACTERIOL. teria of the orders Eubacteriales and Pseudo- aa3 and d (7), and Chromobacterium viola- monadales are a group which have been distin- ceum, which contained cytochromes d and o guished from bacteria in group A on the basis but no al (see Table 5). The latter feature may of the regulation of their citrate synthase be attributable to the physiological production and, hence, their general intermediary of hydrogen cyanide by ths species ( 16). metabolism (53). This group could be split into The organisms producing cytochrome d merit three subgroups on the basis of cytochrome some additional consideration. They appeared pattern dependent upon the a type cyto- to be divided into the following: facultatively chrome(s) present (see Table 2). If no a type anaerobic species, which ,produced fairly large cytochromes were detected, species were classi- amounts of cytochromes al and d in the late fied according to the usual taxonomic consid- logarithmic or stationary phases of growth (6, erations. (It was assumed here that a type 16); aerobic species, which produced only small cytochromes may well have been produced by quantities of cytochromes al and d in the these species under suitable conditions of stationary phase unless cultured under severe culture.) oxygen limitation (see Table 5); and species As a result of these subdivisions, representa- which produced significant amounts of cyto- tives of only two families appeared in more chromes d and/or al mainly when grown than one group: the Pseudomonadaceae, the anaerobically with nitrate as the terminal largest family in terms of number of species and electron acceptor (67, 103, 108). It must be one whose taxonomy remains the subject of stressed, therefore, that for taxonomic use the intensive research, and the Rhizobiaceae, which cytochrome pattern of these species should be contains three genera, Agrobacten'um, Chrorno- examined under a variety of growth conditions. bacterium, and Rhizobium, which are related Group C. The anaerobic and microaerophilic mainly by the relatively crude criterion of heterotrophs of the order Eubacteriales (see morphological similarity. Table 3) were for a long time thought not to Exceptions to these groupings were generally contain cytochromes, but in the last decade of a minor nature, e.g., the absence of cytochromes (always including cytochrome b) cytochrome b in Pseudomonas testosteroni and were found to be present in several species P. acidivorans (89); the notable exceptions were (although usually associated with the inclusion P. riboflavina, which contained cytochromes of in the growth medium). Cytochrome b

TABLE 5. Qualitative cytochrome content of bacteria examined experimentally

~~ Cytochrome content of cells in logarithmic (log) or stationary (stat) Species phase of growth

~ Acinetobacter lwoffi [4B-P. D. J. Weitzman] o b ca (log) a," d" o b ca (stat) Agromyces ram osus aa, o b c (log and stat) Arthrobacter globformis [NCIB 86021 aa, o b c (log) a, d o b (stat) Bacillus lichen formis' aa, o b c (log and stat) Bacillus subtilis [D-473Ib aas b c (log) aa, 0" b c (late stat) Brevibacterium linens [ATCC 9 1741 aa, oa b c (log and stat) Caulobacter vibrioides [ATCC 1 17631 aa, o b c (log and stat) Chromobacterium violaceurn [D-3O1lb d o b ca (stat) Cellulomonas biazotea [NCIB 80771 aa, o b c (log and stat) Hydrogenomonas facilisd a1 o b c (stat) Kurthia zopfii [C-5Ib o b (log and stat) L isteria rnonocytogenes b (stat) Xanthomonas hyacinthi [NCPPB 5 191 0 b (log) a, d o b (stat) Trace amount. The culture collection of the Medical Research Council's Microbial Systematics Research Unit, University of Leicester, England. ' Strain supplied by T. Coultate. Strain provided by J. Heptinstall. VOL. 23, 1973 CYTOCHROME DISTRIBUTION IN BACTERIA 463

FIG. 1. Distribution of cytochromes in bacteria. was also found to mediate the anaerobic electron transport systems, such as occur in the reduction of fumarate (88, 104). autotrophic bacteria. The families Lactobacillaceae and Propioni- The data presented in Tables 1 to 4 were bacteriaceae prompted special interest since the summarized diagramatically in order to empha- a type cytochromes in these families were al size the taxonomic distribution of each class of and d rather than aa3 as in other gram positives. cytochrome (see Fig. 1). Note the relatively This observation suggested a relationship be- small taxonomic region in which cytochrome d tween these species and the gram-negative occurs. group B-1 (see above). This relationship could be a direct evolutionary relationship, or it may be connected to their high degree of adaptabil- DISCUSSION ity (see Discussion). This feature also strongly excludes from the Lactobacillaceae the atypical The results obtained with the recent mathe- Microbacterium thermosphactum (at present matical analysis of Shipp (84) suggest strongly reservedly classified with the Corynebacte- that two or more cytochromes may exhibit riaceae), which produces cytochromes aa3 and only a single absorption peak at room tempera- b (42). ture. These findings cast serious doubts on a The only group of bacteria in which the conclusion that het erotro phic, facultative presence of cytochromes has not been detected anaerobes and pathogens lack cytochrome c. is the genus Clostridium. Perhaps these orga- However, it would seem likely that, although nisms have been anaerobes throughout their cytochrome c may be present, it is a minor evolution. component in these bacteria and must represent Group D. The chemo- and photo-autotrophs a considerable difference in the aerobic oxida- of the order Pseudoinonadales (see Table 4) are tion pathway. a complex and diverse group characterized in The above technique may serve as a refine- particular by the omnipresence of cyto- ment to the use of cytochrome composition as chrome(s) c. The properties of these cyto- a taxonomic characteristic. chromes c appear to be quite variable (for It is interesting to speculate that the bacteria review, see references 53, 54), and it was (mainly group B- l), which commonly contain speculated that these observations could be three CO-binding cytochromes, are species explained on the basis of cytochrome c having a which are particularly adaptable in unstable greater potential for diversification and for environmental conditions, with respect not evolution as a component in a wide variety of only to the terminal electron acceptor but also 464 MEYER AND JONES INT. J. SYST. 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