Sequence analyses of cyanobacterial bicarbonate transporters and their homologues

The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA. SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs). These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product. A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin. Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters. The tree topology suggests that SbtA family members display multiple transport functions.     

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members.     

Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family

Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes.     

Evolution of the Oligopeptide Transporter Family

The oligopeptide transporter (OPT) family of peptide and iron-siderophore transporters includes members from both prokaryotes and eukaryotes but with restricted distribution in the latter domain. Eukaryotic members were found only in fungi and plants with a single slime mold homologue clustering with the fungal proteins. All functionally characterized eukaryotic peptide transporters segregate from the known iron-siderophore transporters on a phylogenetic tree. Prokaryotic members are widespread, deriving from many different phyla. Although they belong only to the iron-siderophore subdivision, genome context analyses suggest that many of them are peptide transporters. OPT family proteins have 16 or occasionally 17 transmembrane-spanning α-helical segments (TMSs). We provide statistical evidence that the 16-TMS topology arose via three sequential duplication events followed by a gene-fusion event for proteins with a seventeenth TMS. The proposed pathway is as follows: 2 TMSs → 4 TMSs → 8 TMSs → 16 TMSs → 17 TMSs. The seventeenth C-terminal TMS, which probably arose just once, is found in just one phylogenetic group of these homologues. Analyses for orthology revealed that a few phylogenetic clusters consist exclusively of orthologues but most have undergone intermixing, suggestive of horizontal transfer. It appears that in this family horizontal gene transfer was frequent among prokaryotes, rare among eukaryotes and largely absent between prokaryotes and eukaryotes as well as between plants and fungi. These observations provide guides for future structural and functional analyses of OPT family members.     

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Correspondence to: E.A. Craig     

Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.     

The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions

We have identified all homologues in the current databases of the ubiquitous protein constituents of the general secretory (Sec) pathway. These prokaryotic/eukaryotic proteins include (1) SecY/Sec61alpha, (2) SecE/Sec61gamma, (3) SecG/Sec61beta, (4) Ffh/SRP54 and (5) FtsY/SRP receptor subunit-alpha. Phylogenetic and sequence analyses lead to major conclusions concerning (1) the ubiquity of these proteins in living organisms, (2) the topological uniformity of some but not other Sec constituents, (3) the orthologous nature of almost all of them, (4) a total lack of paralogues in almost all organisms for which complete genome sequences are available, (5) the occurrence of two or even three paralogues in a few bacteria, plants, and yeast, depending on the Sec constituent, and (6) a tremendous degree of sequence divergence in bacteria compared with that in archaea or eukaryotes. The phylogenetic analyses lead to the conclusion that with a few possible exceptions, the five families of Sec constituents analyzed generally underwent sequence divergence in parallel but at different characteristic rates. The results provide evolutionary insights as well as guides for future functional studies. Because every organism with a fully sequenced genome exhibits at least one orthologue of each of these Sec proteins, we conclude that all living organisms have relied on the Sec system as their primary protein secretory/membrane insertion system. Because most prokaryotes and many eukaryotes encode within their genomes only one of each constituent, we also conclude that strong evolutionary pressure has minimized gene duplication events leading to the establishment of Sec paralogues. Finally, the sequence diversity of bacterial proteins as compared with their archaeal and eukaryotic counterparts is in agreement with the suggestion that bacteria were the evolutionary predecessors of archaea and eukaryotes.     

Topological and phylogenetic analyses of bacterial holin families and superfamilies

Holins are small “hole-forming” transmembrane proteins that mediate bacterial cell lysis during programmed cell death or following phage infection. We have identified fifty two families of established or putative holins and have included representative members of these proteins in the Transporter Classification Database (TCDB; www.tcdb.org). We have identified the organismal sources of members of these families, calculated their average protein sizes, estimated their topologies and determined their relative family sizes. Topological analyses suggest that these proteins can have 1, 2, 3 or 4 transmembrane α-helical segments (TMSs), and members of a single family are frequently, but not always, of a single topology. In one case, proteins of a family proved to have either 2 or 4 TMSs, and the latter arose by intragenic duplication of a primordial 2 TMS protein-encoding gene resembling the former. Using established statistical approaches, some of these families have been shown to be related by common descent. Seven superfamilies, including 21 of the 52 recognized families were identified. Conserved motif and Pfam analyses confirmed most superfamily assignments. These results serve to expand upon the scope of channel-forming bacterial holins.     

The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions

We have identified all homologues in the current databases of the ubiquitous protein constituents of the general secretory (Sec) pathway. These prokaryotic/eukaryotic proteins include (1) SecY/Sec61α, (2) SecE/Sec61γ, (3) SecG/Sec61β, (4) Ffh/SRP54 and (5) FtsY/SRP receptor subunit-α. Phylogenetic and sequence analyses lead to major conclusions concerning (1) the ubiquity of these proteins in living organisms, (2) the topological uniformity of some but not other Sec constituents, (3) the orthologous nature of almost all of them, (4) a total lack of paralogues in almost all organisms for which complete genome sequences are available, (5) the occurrence of two or even three paralogues in a few bacteria, plants, and yeast, depending on the Sec constituent, and (6) a tremendous degree of sequence divergence in bacteria compared with that in archaea or eukaryotes. The phylogenetic analyses lead to the conclusion that with a few possible exceptions, the five families of Sec constituents analyzed generally underwent sequence divergence in parallel but at different characteristic rates. The results provide evolutionary insights as well as guides for future functional studies. Because every organism with a fully sequenced genome exhibits at least one orthologue of each of these Sec proteins, we conclude that all living organisms have relied on the Sec system as their primary protein secretory/membrane insertion system. Because most prokaryotes and many eukaryotes encode within their genomes only one of each constituent, we also conclude that strong evolutionary pressure has minimized gene duplication events leading to the establishment of Sec paralogues. Finally, the sequence diversity of bacterial proteins as compared with their archaeal and eukaryotic counterparts is in agreement with the suggestion that bacteria were the evolutionary predecessors of archaea and eukaryotes.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system

Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families. Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria. Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members. The Arabidopsis thaliana genome encodes three of each. Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle. Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB). When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB. Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA. In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type. These results are discussed in terms of their probable structural, functional and evolutionary significance.     

Homologues of archaeal rhodopsins in plants, animals and fungi: structural and functional predications for a putative fungal chaperone protein

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR family. We have used modeling techniques to predict the structure of one of the putative yeast chaperone proteins, YRO2, based on homology with bacteriorhodopsins (BR). Availability of the structure allowed depiction of conserved residues that are likely to be of functional significance. The results lead us to predict an extracellular protein folding function and a transmembrane proton transport pathway. We suggest that protein folding is energized by a novel mechanism involving the proton motive force. We further show that MR family proteins are distantly related to a family of fungal, animal and plant proteins that include the human lysosomal cystine transporter (LCT) of man (cystinosin), mutations in which cause cystinosis. Sequence and phylogenetic analyses of both the MR family and the LCT family are reported. Proteins in both families are of the same approximate size, exhibit seven putative transmembrane alpha-helical spanners (TMSs) and show limited sequence similarity. We show that the LCT family arose by an internal gene duplication event and that TMSs 1-3 are homologous to TMSs 5-7. Although the same could not be demonstrated statistically for MR family members, homology with the LCT family suggests (but does not prove) a common evolutionary pathway. Thus, TMSs 1-3 and 5-7 in both LCT and MR family members may share a common origin, accounting for their shared structural features.     

Internal gene duplication in the evolution of prokaryotic transmembrane proteins

We investigated the evolution of transmembrane (TM) topology by detecting partial sequence repeats in TM protein sequences and analyzing them in detail. A total of 377 sequences that seem to have evolved by internal gene duplication events were found among 38,124 predicted TM protein sequences (except for single-spannings) from 87 prokaryotic genomes. Various types of internal duplication patterns were identified in these sequences. The majority of them are diploid-type (including quasi-diploid-type) duplication in which a primordial protein sequence was duplicated internally to become an extant TM protein with twice as many TM segments as the primordial one, and the remaining ones are partial duplications including triploid-type. The diploid-type repeats are recognized in many 8-tms, 10-tms and 12-tms TM protein sequences, suggesting the diploid-type duplication was a principle mechanism in the evolutionary development of these types of TM proteins. The "positive-inside" rule is satisfied in whole sequences of both 10-tms and 8-tms TM proteins and in both halves of 10-tms proteins while not necessarily in the second half of 8-tms proteins, providing fit examples of "internal divergent topology evolution" likely occurred after a diploid-type internal duplication event. From analyzing the partial duplication patterns, several evolutionary pathways were recognized for 6-tms TM proteins, i.e. from primordial 2-tms, 3-tms and 4-tms TM proteins to extant 6-tms proteins. Similarly, the duplication pattern analysis revealed plausible evolution scenarios that 7-tms TM proteins have arisen from 3-tms, 4-tms and 5-tms TM protein precursors via partial internal gene duplications.     

Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana

The Spo11 protein of yeast has been found to be covalently bound to double-strand breaks in meiosis, demonstrating a unique role of the protein in the formation of these breaks. Homologues of the SPO11 gene have been found in various eukaryotes, indicating that the machinery involved in meiotic recombination is conserved in eukaryotes. Here we report on SPO11 homologues in plants. In contrast to what is known from other eukaryotes, Arabidopsis thaliana carries in its genome at least two SPO11 homologues, AtSPO11-1 and AtSPO11-2. Both genes are not more closely related to each other than to other eukaryotic SPO11 homologues, indicating that they did not arise via a recent duplication event during higher plant evolution. For both genes three different polyadenylation sites were found. AtSPO11-1 is expressed not only in generative but also to a lesser extent in somatic tissues. We were able to detect in different organs various AtSPO11-1 cDNAs in which introns were differently spliced—a surprising phenomenon also reported for SPO11 homologues in mammals. In the case of AtSPO11-2 we found that the 3′ end of the mRNA is overlapping with a mRNA produced by a gene located in inverse orientation next to it. This points to a possible antisense regulation mechanism. Our findings hint to the intriguing possibility that, at least for plants, Spo11-like proteins might have more and possibly other biological functions than originally anticipated for yeast.     

Tracing pathways of transport protein evolution

We have conducted bioinformatic analyses of integral membrane transport proteins belonging to dozens of families. These families rarely include proteins that function in a capacity other than transport. Many transporters have arisen by intragenic duplication, triplication and quadruplication events, in which the numbers of transmembrane alpha-helical hydrophobic segments (TMSs) have increased. The elements multiplied may encode two, three, four, five, six, 10 or 12 TMSs and gave rise to proteins with four, six, seven, eight, nine, 10, 12, 20, 24 and 30 TMSs. Gene fusion, splicing, deletion and insertion events have also contributed to protein topological diversity. Amino acid substitutions have allowed membrane-embedded domains to become hydrophilic domains and vice versa. Some evidence suggests that amino acid substitutions occurring over evolutionary time may in some cases have drastically altered protein topology. The results summarized in this microreview establish the independent origins of many transporter families and allow postulation of the specific pathways taken for their appearance.     

The major facilitator superfamily (MFS) revisited

The major facilitator superfamily (MFS) is the largest known superfamily of secondary carriers found in the biosphere. It is ubiquitously distributed throughout virtually all currently recognized organismal phyla. This superfamily currently (2012) consists of 74 families, each of which is usually concerned with the transport of a certain type of substrate. Many of these families, defined phylogenetically, do not include even a single member that is functionally characterized. In this article, we probe the evolutionary origins of these transporters, providing evidence that they arose from a single 2-transmembrane segment (TMS) hairpin structure that triplicated to give a 6-TMS unit that duplicated to a 12-TMS protein, the most frequent topological type of these permeases. We globally examine MFS protein topologies, focusing on exceptional proteins that deviate from the norm. Nine distantly related families appear to have members with 14?TMSs in which the extra two are usually centrally localized between the two 6-TMS repeat units. They probably have arisen by intragenic duplication of an adjacent hairpin. This alternative topology probably arose multiple times during MFS evolution. Convincing evidence for MFS permeases with fewer than 12?TMSs was not forthcoming, leading to the suggestion that all 12?TMSs are required for optimal function. Some homologs appear to have 13, 14, 15 or 16 TMSs, and the probable locations of the extra TMSs were identified. A few MFS permeases are fused to other functional domains or are fully duplicated to give 24-TMS proteins with dual functions. Finally, the MFS families with no known function were subjected to genomic context analyses leading to functional predictions.     

The copper transporter (Ctr) family of Cu+ uptake systems

Copper (Cu(+)) transporters of the Ctr family are sequence diverse eukaryotic proteins that function by an unknown mechanism of action. We have conducted bioinformatic analyses of sequenced Ctr proteins. Multiple paralogues are found in single organisms, and these may be either closely or distantly related to each other. Protein phylogeny generally correlates with organismal source and protein size with proteins of each cluster being derived from a specific eukaryotic kingdom and exhibiting characteristic domain arrangements. Some homologues exhibit repeats of the basic 3 TMS unit. Regions of conserved hydrophobicity and amphipathicity suggest functional roles, particularly for putative TMSs 2 and 3 which possess a nearly fully conserved M X(3) M motif in putative TMS2. We propose that this motif comprises the transmembrane Cu(+)-binding site in oligomeric channels that take up Cu(+) by a passive, membrane potential-dependent mechanism.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Identification of the human methylmalonyl-CoA racemase gene based on the analysis of prokaryotic gene arrangements. Implications for decoding the human genome

In this report, we identify the human DL-methylmalonyl-CoA racemase gene by analyzing prokaryotic gene arrangements and extrapolating the information obtained to human genes by homology searches. Sequence similarity searches were used to identify two groups of homologues that were frequently arranged with prokaryotic methylmalonyl-CoA mutase genes, and that were of unknown function. Both gene groups had homologues in the human genome. Because methylmalonyl-CoA mutases are involved in the metabolism of propionyl-CoA, we inferred that conserved neighbors of methylmalonyl-CoA mutase genes and their human homologues were also involved in this process. Subsequent biochemical studies confirmed this inference by showing that the prokaryotic gene PH0272 and its human homologue both encode DL-methylmalonyl-CoA racemases. To our knowledge this is the first report in which the function of a eukaryotic gene was determined based on the analysis of prokaryotic gene arrangements. Importantly, such analyses are rapid and may be generally applicable for the identification of human genes that lack homologues of known function or that have been misidentified on the basis of sequence similarity searches.     

Phylogenetic analyses of the constituents of Type III protein secretion systems

Multicomponent Type III protein secretion systems transfer gram-negative bacterial virulence factors directly from the bacterial cytoplasm to the cytoplasm of a host eukaryotic cell in a process that may involve a single energy-coupled step. Extensive evidence supports the conclusion that the genetic apparatuses that encode these systems have been acquired independently by different gram-negative bacteria, presumably by lateral transfer. In this paper we conduct phylogenetic analyses of currently sequenced constituents of these systems and their homologues. The results reveal the relative relatedness of these systems and show that they evolved with little or no exchange of constituents between systems. This fact suggests that horizontal transmission of the genes encoding these systems always occurred as a unit without the formation of hybrid gene clusters. Moreover, homologous flagellar proteins show phylogenetic clustering that suggests that the flagellar systems and Type III protein secretory systems diverged from each other following very early duplication of a gene cluster sharing many (but not all) genes. Phylogenies of most or all of the flagellar proteins follow those of the source organisms with little or no lateral gene transfer suggesting that homologous flagellar proteins are true orthologues. We suggest that the flagellar apparatus was the evolutionary precursor of Type III protein secretion systems.