An alternative to the accepted phylogeny of purple bacteria based on 16S rRNA: analyses of the amino acid sequences of cytochromes C2 and C556 from Rhodobacter (Rhodovulum) sulfidophilus.

It is becoming increasingly apparent from complete genome sequences that 16S rRNA data, as currently interpreted, does not provide an unambiguous picture of bacterial phylogeny. In contrast, we have found that analysis of insertions and deletions in the amino acid sequences of cytochrome c2 has some advantages in establishing relationships and that this approach may have broad utility in acquiring a better understanding of bacterial relationships. The amino acid sequences of cytochromes c2 and c556 have been determined in whole or in part from four strains of Rhodobacter sulfidophilus. The cytochrome c2 contains three- and eight-residue insertions as well as a single-residue deletion in common with the large cytochromes c2 but in contrast to the small cytochromes c2 and mitochondrial cytochromes. In addition, the Rb. sulfidophilus protein shares a rare six- to seven-residue insertion with other Rhodobacter cytochromes c2. The cytochrome c556 is a low-spin class II cytochrome c homologous to the greater family of cytochromes c', which are usually high-spin. The similarity of cytochrome c556 to other species of class II cytochromes is consistent with the relationships deduced from comparisons of cytochromes c2. Thus, our results do not support placement of Rb. sulfidophilus in a separate genus, Rhodovulum, which was proposed primarily on the basis of 16S rRNA sequences. Instead, the Rhodobacter cytochromes c2 are distinct from those of other genera and species of purple bacteria and show a different pattern of relationships among species than reported for 16S rRNA.

Amino acid sequences of two high-potential iron-sulfur proteins (HiPIPs) from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum.

The amino acid sequences of two very different high-potential iron-sulfur protein (HiPIP) isozymes have been determined from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum. Iso-1 HiPIP, which is monomeric and contains 57 amino acid residues, is most similar to the Thiobacillus ferrooxidans iron-oxidizing enzyme (45% identity and a 6-residue deletion). On the other hand, iso-2 HiPIP, which is isolated as an oligomer, contains a peptide chain with 54 amino acid residues. It is the smallest reported to date and is only 31% identical to iso-1 HiPIP. A massive deletion of 17 residues is found at the N-terminus, such that only 2 residues remain prior to the first cysteine. Iso-2 HiPIP also has a 12-residue insertion and a 5-residue deletion. Prior to this study, there were only 2 absolutely conserved residues (Tyr 19 and Gly 75, Chromatium numbering) in addition to the 4 iron-sulfur cluster binding cysteine residues among the 13 HiPIPs sequenced to date. We found that Tyr 19 is absent in iso-2 HiPIP along with the entire N-terminal loop. Moreover, Gly 75 is substituted in both R. salinarum HiPIPs. These characteristics make the R. salinarum HiPIPs, and especially iso-2, the most divergent yet characterized.

The distance between bacterial species in sequence space.

Despite the revolution caused by information from macromolecular sequences, the basis of bacterial classification remains the genus and the species. How do these terms relate to the variety of bacteria that exist on earth? In this paper, the inter- and intraspecies differences in amino acid sequence of several bacterial electron transport proteins, cytochromes c, and blue copper proteins are compared. For the soil and water organisms studied, bacterial species can be classed as "tight" when there is little intraspecies variation, or "loose" when this variation is large. For this set of proteins and organisms, interspecies variation is much larger than that within a species. Examples of "tight" species are Pseudomonas aeruginosa and Rhodobacter sphaeroides, while Pseudomonas stutzeri and Rhodopseudomonas palustris are loose species. The results are discussed in the context of the origin and age of bacterial species, and the distribution of genomes in "sequence space." The situation is probably different for commensal or pathogenic bacteria, whose population structure and evolution are linked to the properties of another organism.

Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.

We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins are very similar with epsilon mM values of 6.27 and 5.73 mM-1 cm-1 for azurin I and II, respectively, at lambda max approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequences of two high-potential iron sulfur proteins (HiPIPs) from the moderately halophilic purple phototrophic bacterium Ectothiorhodospira vacuolata.

There are two equally abundant high-potential iron sulfur protein (HiPIP) isozymes present in the purple sulfur bacterium Ectothiorhodospira vacuolata. We have determined the amino acid sequences, which contain 71 and 72 residues. The two HiPIPs can be aligned without any internal insertions or deletions and are 65% identical to one another. The E. vacuolata HiPIPs are most similar to the HiPIP isozymes from Ectothiorhodospira halophila (32-36% identity) and require at least one internal gap for alignment. Other HiPIPs require greater numbers of insertions and deletions for alignment with those of E. vacuolata and E. halophila, and the percentage similarities are slightly smaller (19-40% identity). The E. vacuolata HiPIP isozymes appear to be slightly closer to other species than are the E. halophila isozymes. The E. vacuolata and E. halophila HiPIPs also show slightly greater similarity to the five species of Chromatiaceae, which have been studied, and less similarity to the non-sulfur purple species. These results are in agreement with other studies, which indicate that the two purple sulfur bacterial families, Ectothiorhodospiraceae and Chromatiaceae, are more closely related to one another than to the Rhodospirillaceae.

Amino acid sequences of cytochromes c2 and c' from the moderately halophilic purple phototrophic bacterium Rhodospirillum salexigens.

Rhodospirillum salexigens is a moderately halophilic purple phototrophic bacterium which grows optimally in 8% NaCl. The amino acid sequences of the two principal soluble cytochromes c have been determined. One of these is a cytochrome c2, similar in size to mitochondrial cytochrome c. While clearly of the same sequence class as mitochondrial cytochrome c and the proteins from several other Gram-negative bacteria, it does not show particular affinity to any already known sequence in terms of the percentage sequence identity. The other protein is a cytochrome c', but is also a divergent member of this widespread group. The lack of appreciable sequence identity to other species is probably due to a limit of divergence which has been reached for the majority of purple bacterial species. However, the numbers of insertions and deletions and their locations in cytochromes c2 and c' suggest that R salexigens may be related to Rhodospirillum molischianum. Like other electron transport proteins from halophiles, both of these cytochromes are notable for their high content of arginine as compared with lysine and both are acidic. However, they do not show any particular sequence homology to electron transport proteins that have been characterized from the extremely halophilic phototrophes of the genus Ectothiorhodospira. Thus, it appears that adaptation to halophilic habitats has independently occurred more than once in purple bacteria.

Amino acid sequence of a high redox potential ferredoxin (HiPIP) from the purple phototrophic bacterium Rhodopila globiformis, which has the highest known redox potential of its class.

Rhodopila globiformis HiPIP has a redox potential (ca. 450 mV) that is 100 mV higher than any other known iron-sulfur protein. The amino acid sequence contains 57 residues and can be aligned with that of Thiobacillus ferrooxidans without any insertions or deletions and is 51% identical. Rp. globiformis HiPIP is also similar to that of Rhodocyclus tenuis, but six- and two-residue gaps must be postulated and there is only 37% identity. Most of the amino acid residues near the iron-sulfur cluster are similar in these two species based on inspection of the three-dimensional structure of Rc. tenuis HiPIP. The reason for the higher redox potential may be a more hydrophilic environment of the Rp. globiformis HiPIP iron-sulfur cluster due to the above two deletions and to substitution of Ser 32 for Gly. Rp. globiformis is unusual in that it has a cytochrome c2 in addition to the HiPIP, and it too has a very high redox potential. These results suggest that the cytochrome c2 and HiPIP may function interchangeably and that the species normally resides in a very high potential environment, although it is not known to grow aerobically in the dark.

Amino acid sequences of cytochromes c-551 from the halophilic purple phototrophic bacteria, Ectothiorhodospira halophila and E. halochloris.

The cytochromes c-551 from Ectothiorhodospira halophila and E. halochloris contain 78 and 79 residues, respectively. The sequences can be aligned without the need to postulate any internal deletions or insertions to give 63% identity. They are apparently distantly related to the class I cytochromes c, based on the location of the heme attachment site near the N-terminus and the sixth ligand methionine near the C-terminus. Alignment with cytochromes c5 from Azotobacter and Pseudomonas, with cytochromes c6 from cyanobacteria and algae, and with cytochromes c-555 from the green phototrophic bacteria suggests that residues which occupy important positions in the three-dimensional structures of these proteins have their equivalents in the Ectothiorhodospira cytochromes c-551, but the levels of overall identity are very low, around 30%. Although the Ectothiorhodospira cytochromes c-551 are apparently distantly related to the above, they should be regarded as representative of a new subclass of type I bacterial cytochromes c. Homologs of all of the cytochromes c normally found in Pseudomonas and Azotobacter have now been found in one or more purple bacterial species. Among these, cytochrome c5 homologs are the most widely occurring in purple, green, and cyanobacteria. For the first time, all families of phototrophic bacteria plus Pseudomonas can be related to one another at the molecular level.

Nucleotide sequence of the heme subunit of flavocytochrome c from the purple phototrophic bacterium, Chromatium vinosum. A 2.6-kilobase pair DNA fragment contains two multiheme cytochromes, a flavoprotein, and a homolog of human ankyrin.

The gene for the cytochrome subunit of Chromatium vinosum flavocytochrome c (sulfide dehydrogenase) was cloned from an EcoRI digest of chromosomal DNA. The mature cytochrome subunit contains 175 amino acid residues and two heme binding sites in agreement with the previously reported amino acid sequence. There is also a signal peptide of 25 residues, which apparently directs the protein to the periplasmic space. There are two open reading frames upstream of the heme subunit gene, which encode a tetraheme cytochrome c and a homolog of human ankyrin. The gene for the flavoprotein subunit of flavocytochrome c is in frame 15 nucleotides downstream of the stop codon for the cytochrome gene. Messenger RNA was isolated from malate grown cells. The transcript is approximately 3 kilobases in size and does not hybridize with a probe containing the tetraheme cytochrome gene and part of the ankyrin homolog gene. The heme subunit and flavoprotein subunit genes thus appear to form an operon. The flavoprotein subunit has a 30-residue signal peptide. The clone ends 95 amino acids into the N-terminal sequence of the mature flavoprotein subunit (which should contain about 400 residues). The apparently periplasmic location of flavocytochrome c has important consequences for the presumed function as a sulfide dehydrogenase, because sulfur, which is the product of oxidation, is stored in the cytoplasm. Our results on the location of the enzyme are incompatible with this function.

Proteins and molecular palaeontology.

Protein taxonomy has existed as a concept at least since 1958, but despite the efforts of the past 30 years, comparative studies of protein sequence, structure and distribution have not revolutionized any areas of systematics. The most interesting results of single gene phylogenies have been the anomalies, such as insulin in hystricomorphs or cytochrome c in the rattlesnake. It is likely that protein sequence information can be obtained in sufficient quality and quantity from ancient material as to change this finding? The paper will assess possibilities and the likely limitations of chemical studies of ancient protein material.

Sequence variability in bacterial cytochromes c.

Cytochromes c are proteins that can be defined both phenotypically and by their possession of a characteristic sequence motif. Many sequences from bacterial sources are known, and new ones are being reported every year. An analysis can be made as to what fraction of new sequences are members of already known classes or subclasses, and how many map into previously uninhabited regions of sequence space.

Amino acid sequences of Euglena viridis ferredoxin and cytochromes c.

The Order Euglenida comprises many species and perhaps 40 genera, but almost all biochemical and genetic studies have been limited to a single species. Euglena gracilis, because of its ease of growth in the laboratory. Sequence studies of chloroplast and mitochondrial proteins from E. gracilis show that they have diverged widely from other eukaryotic lines. In the present paper we report the sequences of three proteins from another euglenoid, Euglena viridis, using material isolated from a natural bloom. The mitochondrial cytochrome c shows more than 90% sequence identity with that from E. gracilis, and contains the same characteristic features. The chloroplast cytochrome c6 has diverged to a greater extent and shows only 77% identity. The chloroplast ferredoxin from E. viridis is similar in sequence to those of cyanobacteria and algal chloroplasts, with sequence identities of up to 75%. Details of the purification, analysis and sequence determination experiments on the peptides have been deposited as Supplementary Publication SUP 50163 (32 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Rattlesnake cytochrome c. A re-appraisal of the reported amino acid sequence.

The amino acid sequence of rattlesnake cytochrome c was originally reported in 1965, and was one of the earlier sequences to be studied. When compared with other mitochondrial cytochromes c, the snake sequence was soon seen to be anomalous. There were several positions in which the snake protein resembled human cytochrome c, although comparable anomalies were not reported for the protein from other reptiles such as lizard and turtle. Explanations of these results have included accelerated evolution in the snake lineage, paralogy rather than orthology, and faulty determination of the sequence, and the rattlesnake is now often omitted from cytochrome c phylogenetic trees. We have re-investigated the sequence of the snake protein, and believe that the correct sequence differs in nine places from that used for evolutionary theorizing since 1965. Four of these differences are near the haem-attachment site, in a region that was only analysed for amino acid composition in the original investigation. The other five differences are towards the C-terminus of the molecule, and can be explained as being due to the wrong ordering of amino acids within peptides that had been satisfactorily purified. Despite these corrections, the rattlesnake cytochrome c sequence still more closely resembles human cytochrome c than it does that of any other protein we know. We believe that this is an example of convergent evolution, although it does appear that there has been accelerated change in the line connecting the rattlesnake to the ancestral vertebrate line. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50162 (16 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J.

Methylomonas J is an obligate methylotroph although it is unable to grow on methane. Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase. When grown on methanol, only the other blue copper protein is produced. We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins. The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences. The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity. The proteins show 52% identity with each other. The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.

The phototrophic bacterium Rhodopseudomonas capsulata sp108 encodes an indigenous class A beta-lactamase.

The nucleotide sequence of a 2.37 kb DNA fragment derived from cloning a total DNA digest of Rhodopseudomonas capsulata sp108 was determined. The DNA codes for a beta-lactamase, a protein showing sequence similarity to the ampR protein of Enterobacter cloacae and an unidentified open reading frame. Hybridization experiments with a probe carrying DNA from within the beta-lactamase gene suggests a chromosomal location for the coding sequences in strain sp108 and in sp109, a penicillin-sensitive revertant of sp108 in which the enzyme is not inducible. A protein-sequence comparison of the deduced amino acid sequence of the Rps. capsulata beta-lactamase indicates that it is a Class A enzyme and that its sequence can be aligned with those of the characterized beta-lactamases from Staphylococcus aureus, Bacillus licheniformis and the Escherichia coli plasmid (R-TEM enzyme), with only a few insertions or deletions. The corresponding DNA sequence is, however, characteristically rhodopseudomonad, suggesting that it is not a recently transposed gene.

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554.

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.

Amino acid sequences of cytochrome c-554(548) and cytochrome c' from a halophilic denitrifying bacterium of the genus Paracoccus.

The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M. 203 (= A.T.C.C. 12084, N.C.I.B. 8669) have been determined. Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly. The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris. Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence. Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c. Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K. from which copies are available on prepayment.

The amino acid sequence of the cytochrome c2 from the phototrophic bacterium Rhodopseudomonas globiformis.

The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined. By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome. The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion. We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].