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Comparative Genomic Analyses of the Bacterial Phosphotransferase System

Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116

SUMMARY

INTRODUCTION

OVERVIEW OF GENOME ANALYSES

RELATIVE DISTRIBUTION OF PTS PERMEASE TYPES

DISTRIBUTION OF PTS PERMEASE TYPES IN VARIOUS BACTERIAL KINGDOMS

NUMBERS OF PTS PROTEIN-ENCODING GENES VERSUS GENOME SIZE AND PHYSIOLOGY

ORGANISMS LACKING PTS HOMOLOGUES

BACTERIA WITH CYTOPLASMIC PTS PROTEIN HOMOLOGUES BUT NO RECOGNIZABLE PTS TRANSPORTERS

BACTERIA WITH A COMPLETE PTS PHOSPHORYL TRANSFER CHAIN AND JUST ONE OR TWO TYPES OF PTS PERMEASE

BACTERIA WITH A COMPLETE PTS PHOSPHORYL TRANSFER CHAIN AND MULTIPLE TYPES OF PTS PERMEASES

High-G+C Gram-Positive Bacteria

Low-G+C Gram-Positive Bacteria

Proteobacteria

Spirochetes

OCCURRENCE OF DIHYDROXYACETONE PTS ENZYME II COMPLEXES

PHYLOGENY OF DHA PROTEINS

PTS DOMAIN FUSION PROTEINS

PTS PROTEINS WITH EXTRA NON-PTS DOMAINS

PTS PROTEIN STRAIN DIFFERENCES IN SINGLE BACTERIAL SPECIES

CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Four decades ago, Kundig et al. reported the discovery of a novel sugar-phosphorylating system in Escherichia coli (38). The unique features of this phosphotransferase system (PTS) included the use of phosphoenolpyruvate (PEP) as the phosphoryl donor for sugar phosphorylation and the presence of three essential catalytic entities, termed enzyme I, enzyme II, and HPr (heat-stable, histidine-phosphorylatable protein). The discovery of this system provided an explanation for pleiotropic carbohydrate-negative mutants of E. coli described as early as 1949 (20).

In 1964, the three recognized activities of the PTS were presumed to correspond merely to three proteins. We now know that dozens of PTS proteins are present in the E. coli cell and that thousands of PTS protein homologues occur in other bacteria. Numerous genes encoding these proteins have been fully sequenced, and their phylogenetic relationships have been described (29, 81; Nguyen et al., unpublished data).

The bacterial PTS catalyzes the concomitant transport and phosphorylation of its sugar substrates (62, 96). It is a complex system that consists of general cytoplasmic energy-coupling proteins, enzyme I (EI) and HPr, which lack sugar specificity, and membranous enzyme II complexes, each specific for one or a few sugars. The enzyme II complexes usually consist of three proteins or protein domains, namely, IIA, IIB, and IIC. However, the enzyme II complexes of one of the PTS families, the mannose family, have one additional membrane-spanning protein or domain, called IID (72). Phosphoryl relay proceeds sequentially from PEP to EI, HPr, IIA, IIB, and finally, the incoming sugar, which is transported across the membrane via the integral membrane IIC porter (Table 1 and Fig. 1A).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Phosphoryl transfer chains of the bacterial phosphotransferase system. (A) Various phosphoryl relay chains of the PTS. (B) Proposed regulatory phosphoryl transfer chains based on the data in Table 7. (C) Putative phosphoryl chains in Bradyrhizobium japonicum.

In Escherichia coli, paralogues of EI, HPr, and the fructose IIA protein (IIA^Fru), designated nitrogen enzyme I (EI^Ntr), nitrogen HPr (NPr), and nitrogen IIA protein (IIA^Ntr), respectively, constitute a phosphoryl transfer chain (Fig. 1A) that has been shown to exhibit little enzymatic cross-reactivity with the classical sugar-transporting phosphoryl transfer chain consisting of EI, HPr, and various sugar-specific enzyme II complexes (65). This nitrogen-related phosphoryl transfer chain presumably functions only in regulation (46, 47, 63). EI^Ntr homologues have been shown to cluster phylogenetically together, distantly from all other enzyme I homologues (29).

Phylogenetic data have shown that NPr in E. coli is a distant paralogue of HPr (29, 63). Sequence characteristics that distinguish NPr from HPr as well as EI^Ntr from EI have been described previously (65). EI^Ntr consists of two domains, an N-terminal domain of 127 amino acids homologous to the N-terminal "sensory" domain of the NifA protein of Azotobacter vinelandii (2) and a C-terminal domain of 578 amino acids homologous to all currently sequenced enzyme I proteins. EI^Ntr may serve a sensory function linking carbon and nitrogen metabolism (69). A mutation of the orthologous EI^Ntr-encoding ptsP gene of A. vinelandii resulted in impaired metabolism of poly-ß-hydroxybutyrate as well as diminished respiratory protection of nitrogenase under carbon-limiting conditions (90). In addition to EI^Ntr and NPr, E. coli encodes within its genome three additional EI paralogues and four additional HPr paralogues. The functions of most of these proteins are still unknown.

The biochemical detection of novel PTS proteins in bacteria as diverse as Ancalomicrobium adetum (85), Spirochaeta aurantia(83), Acholeplasma laidlawii (28), Listeria monocytogenes (50), and several antibiotic-producing species of Streptomyces (6, 104) suggests the involvement of PTS proteins in cellular processes distinct from those currently recognized. It is worth noting that other families of transport systems, such as the family of ATP-binding cassette (ABC)-type permeases (27) and the major facilitator superfamily (55), apparently do not participate in metabolic and transcriptional regulation to the extent observed for the PTS.

Recently, a nontransporting enzyme II complex was characterized in E. coli that phosphorylates dihydroxyacetone (DHA) at the expense of PEP, using three soluble DHA-specific proteins in addition to EI and HPr (25). The three components of the DHA enzyme II complex are designated DhaK, DhaL, and DhaM. The DhaM protein of E. coli is a tridomain protein consisting of an N-terminal IIA^Dha domain that is distantly related to IIA^Man, a central HPr domain, and a C-terminally truncated EI domain. All three domains have been shown to be phosphorylated, using PEP and the classical enzyme I and HPr proteins as phosphoryl donors. The various currently recognized phosphoryl relay chains of the PTS are depicted in Fig. 1A.

A bifunctional HPr kinase/phosphorylase (HprK) that catalyzes the phosphorylation of HPr at Ser-46 at the expense of ATP (18, 53, 66) as well as the dephosphorylation of P-Ser-HPr (49) was discovered in Streptococcus pyogenes (19). It plays an important regulatory role in at least three different cellular processes: (i) sugar uptake via the PTS, (ii) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and (iii) inducer control via expulsion and exclusion mechanisms (for reviews, see references 7, 78, and 79). HprK homologues from gram-positive as well as gram-negative bacteria have been proposed to carry out different functions (7, 29, 97). Detailed phylogenetic analyses of HprK homologues from several bacteria have recently been presented (97).

The currently recognized structural and functional complexity of the PTS is impressive (Tables 1 and 2), but the available evidence suggests that its functional ramifications are only now beginning to be realized (7, 23). Only half of the PTS proteins recognized in E. coli have been functionally characterized (101), and as revealed by the present genomic analyses, almost all PTS proteins in other organisms are uncharacterized.

Based on the phylogeny of the IIC proteins, seven PTS permease families are currently recognized, namely, the (i) glucose (including glucoside) (Glc), (ii) fructose (including mannitol) (Fru), (iii) lactose (including N,N-diacetylchitobiose) (Lac), (iv) galactitol (Gat), (v) glucitol (Gut), (vi) mannose (Man), and (vii) L-ascorbate (Asc) families. Various sugar substrates transported by the few functionally characterized members of each of these families are presented in Table 3.

OVERVIEW OF GENOME ANALYSES

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The last few years have witnessed an explosion in the amount of sequence data available for analysis. Genome sequencers sometimes deposit new sequences into databases without rigorous scrutiny. Incorrect or imprecise annotations of genes and gene products not only obscure important genomic information but also lead to the proliferation of erroneous annotations of other genomes. Frequently, sequencing errors conceal important open reading frames (ORFs), and distant phylogenetic relationships are not noticed. Here we report our computational analyses of several completely sequenced genomes.

A total of 202 genomes were screened for the presence of homologues of all currently known constituents of the bacterial phosphotransferase system. These proteins include the general energy-coupling proteins EI and HPr, the IIA, IIB, IIC, and IID constituents of the enzyme II complexes, the DHA PTS proteins (DhaM, DhaL, and DhaK), and HprK. We used criteria established by Bächler (3) to detect homologues of the E. coli dihydroxyacetone PTS proteins (25). GenBank sequence identification (GI) numbers are provided for the proteins discussed in the text.

All completely sequenced genomes were obtained from NCBI (ftp://ftp.ncbi.nih.gov/genomes/). A standalone version (release 2.0.10) of BLAST (1) was obtained ftp://ftp.ncbi.nih.gov/blast/ and was employed to identify PTS homologues, using 57 functionally characterized PTS proteins obtained from the transporter classification database (TCDB; http://www.tcdb.org) as query sequences against each of the completely sequenced genomes. Over 3,000 protein homologues were retrieved and analyzed. Previous annotations were ignored, and all proteins were reexamined. Protein domains were identified and assigned using the NCBI CD-Search tool (42). Such rigorous scrutiny often revealed errors in previous annotations.

Enzyme II complexes that include IIA, IIB, and IIC (as well as IID^Man in the case of the mannose family) were considered to constitute complete PTS permeases. Enzyme IIC proteins that lack a cognate IIA and/or IIB domain/protein were considered incomplete systems. It should be noted that in the Glc family, and only the Glc family, several PTS porters have the IIB and IIC domains fused but lack their own IIA domain. Instead, these systems use the IIA^Glc protein of another system (101). Such Glc-type systems were considered complete.

Table 4 presents an overview of our PTS genome analyses. Except for phosphoenolpyruvate synthases and pyruvate:phosphate dikinases, which are distantly related to EIs of the PTS (101) and are present in both eukaryotes and archaea, and except for a single HPr kinase homologue found in an archaeon, Methanopyrus kandleri (97), no homologues of PTS proteins were identified in any eukaryote or archaeon. Within the bacterial domain, 22% of the species analyzed (30 organisms) encode no recognizable PTS protein homologues. Twenty-onepercent (29 organisms) encode cytoplasmic PTS phosphoryl transfer proteins but lack complete membrane-integrated PTS enzyme II complexes. Finally, 57% of these bacterial species (77 organisms) have at least one complete PTS enzyme II complex as well as the requisite PTS energy-coupling proteins.

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View larger version (21K): [in this window] [in a new window]   FIG. 2. Relative distribution of PTS permease families in bacterial genomes. The occurrence of the constituent permeases of the seven different PTS permease families (Glc, glucose; Fru, fructose; Lac, lactose; Gut, glucitol; Gat, galactitol; Man, mannose; Asc, ascorbate) was analyzed in the 77 bacterial species that were found to encode at least one putative complete PTS transport system. Only complete PTS permease systems were counted for this analysis; incomplete enzyme II complexes and orphan enzyme II constituents were not tabulated. Total numbers of complete PTS permeases (white bars) as well as total numbers of organisms that encode members of the different families (black bars) are presented. Values over the white bars indicate percentages of the total numbers of complete permease systems. Values over the black bars indicate percentages of the 77 organisms that have homologues of a particular family of permeases.

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View larger version (45K): [in this window] [in a new window]   FIG. 3. (A) PTS-encoding capacities of bacterial genomes. The total number of PTS protein homologues identified in an organism (bars) and the genome size, in mega-bp (dots), are plotted. Each point on the x axis indicates a different genome, with the genome size increasing from left to right, as indicated. (B) Correlation between the numbers of PTS proteins encoded in the various genomes and the oxygen requirements of the bacteria. Numbers along the x axis indicate the numbers of PTS proteins encoded in the genomes. "NT" indicates that the bacteria do not possess PTS permeases, while "T" indicates the presence of at least one PTS transport system. Bars are shaded according to metabolic capability, such as an aerobic, anaerobic, microaerophilic, facultative, or unknown oxygen requirement.

ORGANISMS LACKING PTS HOMOLOGUES

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All organisms whose genomes lack genes encoding identifiable PTS protein homologues are listed in Table 6. These include all archaea and eukaryotes examined, as noted above, as well as 30 bacterial species. Bacteria that totally lack PTS homologues include five actinobacteria of the genuses Mycobacterium (4) and Tropheryma (1), all six cyanobacteria examined, one Mollicute species (onion yellows phytoplasma 0Y-M), several proteobacteria of the alpha (five), gamma (two), delta (two), and epsilon (four) subdivisions, and five evolutionarily divergent bacteria of the genuses Aquifex, Bacteroides, Porphyromonas, Thermatoga, and Thermus. In the case of Bacteroides thetaiotaomicron, a single homologue of a galactitol IIC protein (GI 29349515) was found. This homologue cannot function via a PTS-dependent mechanism since this organism lacks all recognizable PTS phosphoryl transfer proteins. We have noted that the encoding gene is in a monocistronic operon with a good promoter, suggesting that this IIC homologue may function as a secondary carrier, as discussed previously (33, 81). Therefore, B. thetaiotaomicron, like Bacteroides fragilis, probably lacks PTS proteins altogether. Except for cyanobacteria, the -Proteobacteria, and several primitive bacteria in the "unclassified bacteria" category (Table 6), PTS homologues are found in all of the major bacterial kingdoms for which sufficient sequence data are available. Methylococcus capsulatus encodes two homologues of the Dha proteins (see below). However, it lacks all other PTS protein homologues, including EI and HPr. It is therefore unlikely that this organism has a functional phosphoryl transfer chain.

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Of the IIA homologues, Xylella species have only a single IIA^Man protein while Leptospira species have only a single IIA^Ntr protein. All others have either two IIA^Ntr proteins, two IIA^Fru proteins, one each of the IIA^Ntr and IIA^Fru proteins, one each of the IIA^Ntr and IIA^Man proteins, one IIA^Ntr, one IIA^Fru, and one IIA^Man protein, or two IIA^Ntr and one IIA^Man protein (Table 7). The species with one IIA^Fru, one IIA^Ntr, and one IIA^Man homologue are the two Bartonella species, while Rhodopseudomonas palustris has two IIA^Ntr and one IIA^Man protein. The fact that most organisms possess at least two homologous or nonhomologous IIA proteins suggests that the PTS phosphoryl transfer chain functions in a regulatory capacity that depends on specific functional interactions between these proteins. These putative interactions could, for example, be antagonistic or synergistic.

In summary, bacteria that possess PTS phosphoryl transfer proteins but lack PTS permeases always have at least one enzyme I (I or I^Ntr) and one HPr (HPr or NPr). They usually have two IIA proteins and one HprK. Only for one such organism, Treponema denticola, have the catalytic activities of the phosphoryl transfer proteins been demonstrated (23). Treponema pallidum lacks an active enzyme I homologue and instead possesses a ptsI pseudogene (23). We suggest that in most cases, these PTS phosphoryl transfer proteins act together, comprising a single unified phosphoryl transfer chain (63, 65) that acts biochemically as shown in Fig. 1B. Among these bacteria, two distinct, independently functioning phosphoryl transfer chains (65) appear to be present only in Bradyrhizobium japonicum. These two chains may catalyze phosphoryl transfer as shown in Fig. 1C. It is also interesting that among these bacteria, B. japonicum is the only one that possesses a complete putative DHA PTS (see below). We suggest that pathway 1 functions to phosphorylate DHA while pathway 2 functions to coordinate carbon and nitrogen metabolism (46, 47, 63, 65).

BACTERIA WITH A COMPLETE PTS PHOSPHORYL TRANSFER CHAIN AND JUST ONE OR TWO TYPES OF PTS PERMEASE

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Complete PTS permeases (enzyme II complexes) consist of IIA, IIB, and IIC (as well as IID in the case of the Man family) domains that may occur as separate peptides or may be fused in various combinations into a smaller number of polypeptide chains (see below). Table 8 lists the organisms that have only one or two types of complete PTS permease in addition to the PTS energy-coupling proteins. Of the 77 bacterial species that were found to encode at least one complete PTS transport system, 31 were found to encode just one or two types of PTS permease. Eighteen bacterial species possess just a single complete type of PTS permease. All of these organisms have both enzyme I and HPr, and many of the gram-negative Proteobacteria also have a partial or complete nitrogen regulatory phosphoryl transfer chain including I^Ntr, NPr, and IIA^Ntr. Like the case for E. coli (63, 65), these organisms may possess two independently functioning phosphoryl transfer chains, one for sugar transport and one for regulation (45-47). Nine species have just one or two fructose-type PTS permeases, while six possess only one or two glucose-type systems; two have just a single mannose system, and one has only one glucitol-type system. Except for Caulobacter crescentus, which has two Glc-type systems, and Mesorhizobium loti, which has a Gut-type system, all other -Proteobacteria examined either possess just the regulatory PTS proteins (Table 7) or lack PTS homologues altogether (Table 6). "Candidatus Blochmannia floridanus," an Enterobacteriaceae member, is one of the organisms that has just a single Man-type PTS. It is possible that it once possessed Fru- and/or Glu-type systems since many of the Enterobacteriaceae analyzed encode several Fru-, Glu-, and Man-type PTS permeases (Table 9). None of the organisms analyzed possesses only a single lactose-, galactitol-, or L-ascorbate-type system. However, orphan IIA and/or IIB and/or IIC homologues of these systems were often found. These may be residues of genome minimalization (51) where the other constituents of complete enzyme II complexes were lost.

None of the organisms that have just two types of PTS permeases have lactose-, galactitol-, or glucitol-type systems. In fact, even orphan constituents of these systems are lacking. However, several additional PTS orphan proteins (IIA, IIB, or IIC), particularly mannose IIA homologues, are found in some of these organisms. It is likely that some of these possess specific regulatory functions, but no such function is currently recognized. Others probably represent either relics of complete PTS permeases that have been partially lost or orphan genes acquired through horizontal transfer. Interestingly, in Deinococcus radiodurans, all of the PTS homologues were found on the plasmid MP1.

BACTERIA WITH A COMPLETE PTS PHOSPHORYL TRANSFER CHAIN AND MULTIPLE TYPES OF PTS PERMEASES

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Table 9 lists the 77 bacterial species that encode all of the proteins required for PTS-dependent sugar transport. Of these, 46 were found to encode more than two types of PTS permease. In the table, these bacteria are grouped according to organismal phylogenetic division, and sometimes by subdivision. These will be discussed according to their taxonomic groups in the order in which they are presented in Table 9. It should be noted that the occurrence of multiple paralogues of a specific family or subfamily of PTS permeases implies differing substrate specificities, affinities, or regulatory properties. In very few cases have these been studied in detail.

Streptomyces avermitilis has Glc and Fru systems, and surprisingly, of the 10 Actinobacteria examined, this is the only one to have HprK (GI 29826878). The HprK homologue from this bacterium clusters separately but is closest to the homologues from -Proteobacteria (97). This may represent a case of horizontal gene transfer. Streptomyces coelicolor has an Asc system in addition to Glc and Fru systems, but it lacks HprK. Of the Actinobacteria, Symbiobacterium thermophilum and Propionibacterium acnes have the most PTS permeases, with 7 and 10 complete systems, respectively. Both have Glc, Fru, and Lac systems, and S. thermophilum has three Man systems while P. acnes has just one each of the Gut-, Gat-, and Asc-type systems but no Man system.

S. thermophilum is a thermophilic bacterium that depends on microbial commensalism. Recently, a role for the Man-type PTS transporter in the establishment of symbiotic relationships has been implicated (114). The presence of three complete Man-type systems in this organism may contribute towards its commensalic growth. S. thermophilum shows remarkable similarity to bacilli and clostridia (low-G+C gram-positive bacteria) and is suggested to have shared a close common ancestor with the Bacillus/Clostridium group (105). It is interesting that the PTS family representation in this bacterium also resembles the PTS protein distribution in some of the bacilli and clostridia.

P. acnes is found ubiquitously on human skin and resides within sebaceous follicles. It has been implicated in various diseases as an opportunistic pathogen (9). Its genome encodes a great capacity to cope with changing oxygen tension, explaining its ubiquitous presence on human skin. The occurrence of a wider variety of PTS permeases may enable efficient uptake of a broad range of substrates and may allow the organism to opportunistically adapt to a pathogenic lifestyle.

All of these high-G+C gram-positive bacteria possess various orphan proteins or "partial" PTS permeases (data not shown). The IIBC^Glc and IIC^Glc components may be functional, using the IIA/B proteins present in one of the complete Glc systems (101). Precedence for this has been observed repeatedly, as several Glc-type systems lack their own IIA protein and use the general IIA protein of the authentic glucose system (101). However, the same phenomenon has not been documented for the other PTS permease families.

The occurrence of 12 complete PTSs in C. acetobutylicum correlates with the saccharolytic lifestyle of this soil bacterium. C. perfringens is commonly found in animal and human gastrointestinal tracts as a member of the normal microflora. Interestingly, the presence of multiple Man-type PTS permeases in this organism correlates with the abundance of Man-type permeases in some of the other members of the normal intestinal microflora, such as lactobacilli, Enterococcus faecalis, and several members of the Enterobacteriaceae. One of the two Man systems in C. acetobutylicum is encoded on the plasmid pSOL1.

Most of the other low-G+C gram-positive bacteria have additional PTS permeases, and some, such as three Listeria species and Lactobacillus plantarum, have all seven PTS enzyme II complex types. The bacteria with the most pts genes and PTS permeases are L. monocytogenes, with 91 genes and 30 complete PTS permeases plus several partial systems, and Enterococcus faecalis, with 93 genes and 25 complete permeases plus 14 partial systems, all of which have the IIC constituent (data not shown). Several of these may be functional, using components from the complete systems (101). The maximal percentage of genetic material devoted to the PTS is observed for L. monocytogenes, with 3.2% of all its genes encoding PTS proteins.

Of the small-genome Mollicutes organisms, all but two possess complete Glc- and Fru-type PTS permeases as well as an HprK homologue (with the exception of Mycoplasma hyopneumoniae), and several have an Asc system as well. The two exceptions (discussed above) are listed in Tables 6 and 7. A few reports have discussed some of the PTS proteins from the different Mycoplasma species (26, 30, 48, 59). The regulation of HprK in the Mollicutes may be different from that in other Firmicutes (26). The distribution and numbers of PTS permeases in the Mollicutes also differ from those in other Firmicutes. The various species within the Mycoplasma genus also show differences. Most surprisingly, Mycoplasma pulmonis and Mycoplasma pneumoniae have three PTS permeases each, Mycoplasma mycoides has four, M. hyopneumoniae has five, and M. florum and Mycoplasma penetrans have eight and nine, respectively. In some of these bacteria with genome sizes of 0.79 to 1.36 Mbp, a major fraction of the genetic material (over 2%) is devoted to the PTS. Their obligate parasitic lifestyle and dependence on glycolysis as the ATP-generating pathway may explain the retention of PTS genes, even under conditions that result in extensive genome reduction (21).

In the beta subdivision, all organisms possess regulatory PTS proteins (Tables 7 and 9), and a few also have PTS permeases that belong exclusively to the Glc and Fru families. The maximal number of probable complete PTS permeases is four, for Chromobacterium violaceum. C. violaceum lives in tropical and subtropical regions in soil and water but can occasionally be pathogenic in immunocompromised individuals, causing diarrhea. No other ß-proteobacterium has more than two complete PTS permeases. While Ralstonia solanacearum has a Glc- and a Fru-type system, Burkholderia species have only a single Glc-type system. The Glc system in R. solanacearum was found encoded in the plasmid pGMI-1000MP. Burkholderia pseudomallei is an opportunistic pathogen in humans that is usually found in terrestrial environments, while Burkholderia mallei is a host-adapted pathogen in animals and is not found outside the host.

In the gamma subdivision, only the endosymbiont of the tsetse fly, Wigglesworthia glossinidia, and the obligate methanotroph Methylococcus capsulatus lack PTS homologues altogether (Table 6). Buchnera species, which are endosymbionts of aphids, have both a single Glc system and a single Fru system. "Candidatus Blochmannia floridanus," an endosymbiont of the carpenter ant, has only a mannose system. All other enterobacteria have multiple PTS permeases. It is therefore clear that these endosymbionts have retained the minimal number of PTS permeases to allow utilization of a very restricted number of sugars provided by the host organism (11).

Several E. coli, Shigella, and Salmonella strains (see below) have all seven types of PTS permease systems. The different strains of E. coli probably have between 17 and 26 functional PTS permeases. These variations, observed for various E. coli/Shigella strains, suggest that the gain and loss of genetic material encoding PTS proteins have occurred frequently and repeatedly during the evolution of single species (108). Scrutiny of Table 9 will reveal that this is a common observation for many bacterial species and genera.

Other -Proteobacteria listed in Table 9 have far fewer PTS permeases. The pseudomonads, for example, have just one or two. The three species of Pseudomonas studied, all with large genome sizes of over 6 Mb, vary from nonpathogenic (P. putida) to plant pathogenic (P. syringae) to opportunistically human pathogenic (P. aeruginosa). In P. aeruginosa, one PTS permease is fructose specific while the other is N-acetylglucosamine specific (68; I. T. Paulsen, personal communication). The Pasteurellales have 1 to 8 PTS permeases, vibrios have 7 to 12 PTS permeases, and the two Xanthomonas species studied, which are found exclusively associated with their plant hosts and are not found free in the soil, have just one fructose-type system each (17, 80, 110). Among the Pasteurellales, "Mannheimia succiniciproducens" (proposed name), which is found in bovine rumen, has the largest number of PTS permeases. The rumen is the first section of the stomach in ruminants, where feed is collected for initial digestion. The presence of several PTS transporters in ruminant bacteria perhaps represents an adaptation to a nutrient-rich, oxygen-free ecological niche. Pasteurella multocida is found in the mucous lining of animal intestines, in the genitalia, and in respiratory tissues. It has five PTS permeases, each belonging to a different PTS family. Each of the five PTS permeases is perhaps expressed differentially depending on the host organ. H. ducreyi and H. influenzae are an obligate pathogen and an obligate parasite, respectively, in humans. The smaller numbers of PTS permeases in these bacteria may have resulted from their evolutionary adaptation to the homeostatic environment of the host.

Vibrios are abundant in marine and freshwater environments. The three species analyzed are pathogenic to humans. Vibrio vulnificus, which is an opportunistic pathogen causing a variety of diseases in immunocompromised patients, has Man-type PTS permeases that are lacking in the other two species. Interestingly, it also has the largest number of PTS permeases among the three Vibrio species analyzed. Vibrio parahaemolyticus, which causes gastroenteritis, and Vibrio cholerae, the causative agent of cholera, have similar complements of PTS proteins. The Asc systems, a few of the Fru PTS permeases, one of the two Man systems in V. vulnificus, and a few other orphan PTS proteins are encoded in the smaller chromosome, chromosome II, in all three species. All other PTS permeases are encoded in chromosome I.

Another spirochete species, Spirochaeta aurantia, whose genome has not yet been sequenced, has only a Fru-type system with specificity for mannitol (82). In this unusual system, the synthesis of all PTS enzymes (enzyme I, HPr, and the mannitol enzyme II) as well as that of mannitol-1-P dehydrogenase is induced 200-fold by the inclusion of mannitol in the growth medium (83).

OCCURRENCE OF DIHYDROXYACETONE PTS ENZYME II COMPLEXES

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Many bacteria possess nontransporting DHA-specific enzyme II complexes. Only for E. coli has such a system been characterized (22, 25, 94). The system consists of three proteins, named DhaK (IIC^Dha; dihydroxyacetone binding), DhaL (IIB^Dha; ADP binding), and DhaM (IIA^Dha; phosphoryl donor for ADP phosphorylation in DhaL) (4, 22, 81). The DhaM protein of E. coli is a three-domain protein consisting of an N-terminal IIA^Dha, a central HPr-like domain, and a C-terminal truncated enzyme I-like domain (25). DhaL subunits exhibit sequence characteristics that distinguish them from the homologous ATP-dependent DHA kinases (3, 4, 93). The DhaK subunit covalently binds the substrate dihydroxyacetone to a histidyl residue in the protein (22). These sequence characteristics allowed us to distinguish ATP- from PEP-dependent systems and therefore to tentatively conclude that all PTS DHA enzyme II complexes consist of split DhaK/DhaL systems, while all ATP-dependent kinases have these two functional domains fused in a single polypeptide chain (DhaKL).

Table 10 presents all bacteria for which we found homologues of the subunits of the DHA PTS enzyme II complex. No such homologues were found in cyanobacteria, chlamydia, and the epsilon division of the Proteobacteria. Twenty-seven organisms appear to have a complete DHA PTS, and of these, 15 (including two Mycoplasma species) are low-G+C gram-positive bacteria. Other organisms with complete DHA PTS enzyme II complexes include (i) -, -, and -Proteobacteria (six organisms), including Bradyrhizobium japonicum (), Mannheimia succiniciproducens (), Desulfovibrio vulgaris (), E. coli (), Shigella flexneri (), and Mesorhizobium loti (); (ii) high-G+C gram-positive bacteria (five organisms), including Streptomyces avermitilis, S. coelicolor, Corynebacterium diphtheriae, Leifsonia xyli, and Propionibacterium acnes; and (iii) other divergent organisms, including Fusobacterium nucleatum and Deinococcus radiodurans. Unlike all other DhaL proteins examined, that in D. radiodurans is split into two polypeptide chains. These chains could only be identified using nucleotide BLAST searches because these protein fragments had not been assigned protein accession numbers. All four putative dha genes in D. radiodurans (including the genes for DhaM and DhaK and the two split genes that together comprise DhaL) were adjacent on a plasmid, the MPI plasmid (41, 109). Splicing of the putative DhaL gene could have resulted from a sequencing error. Furthermore, since the functionality of its gene product has not been demonstrated, the significance of this observation has yet to be determined.

Category B organisms have just one DHA PTS, but their DhaM proteins have the IIA^Dha domain fused to other PTS protein domains. These fused proteins include IIA^Dha-HPr fusions, found in C. diphtheriae (GI 38234870) and L. xyli (GI 50954678), a single full-length IIA^Dha-HPr-EI fusion (GI 46579394), present in D. vulgaris, and tridomain proteins with a truncated enzyme I, IIA^Dha-HPr-EI, characteristic of the E. coli DhaM protein, found in -Proteobacteria (Table 10). Only category B systems have the IIA^Dha domain fused to other PTS protein domains.

Listeria species have two complete sets of DHA PTS (category C). Category D, which includes bacteria that possess a complete DHA PTS plus an extra DhaK, includes only low-G+C gram-positive bacteria. Possibly, the extra DhaK protein allows phosphorylation of an alternative substrate. It is always encoded by a gene distantly related to that encoding DhaK carried within the dha operon, which also encodes the remaining proteins of the complete DHA system.

Category E systems, with complete PEP- and ATP-dependent DHA phosphorylating systems, are from two high-G+C gram-positive bacteria. Category F, with just one -proteobacterial representative, has complete PEP- and ATP-dependent systems but also has two extra DhaKs (GI 13476064 and GI 13488187) and one extra DhaL (GI 13476063). One of the DhaK homologues (GI 13488187) of M. loti is encoded on plasmid pMLa.

Several bacteria lack a complete DHA PTS but nevertheless encode within their genomes at least one PTS Dha homologue, as revealed by the data in Table 11. Several different combinations of these proteins can be found. For example, two organisms each have only DhaM (category A) or DhaL (category B). No organism has just DhaK. Just three organisms have only ATP-dependent DhaKL (category C). The DhaKL protein (GI 17937250) of A. tumefaciens is encoded on the linear chromosome. Several bacteria (category D) have both DhaK and DhaL, and one of these organisms (Brucella melitensis) has a second copy of DhaL (GI 17986682) as well as a IIA^Man homologue (GI 17988315) that more closely resembles E. coli IIA^Man than IIA^Dha. Category E includes four organisms encoding DhaK, DhaL, and DhaKL, and three of these organisms have a IIA^Man homologue. All four genes encoding the Dha proteins in Sinorhizobium meliloti were found on the plasmid pSymB. In both B. mallei and B. pseudomallei, the DhaK and DhaL proteins are encoded on chromosome 2, while the DhaKL protein is encoded on chromosome 1 along with the other PTS homologues found in these organisms. Interestingly, Bacillus cereus, which lacks the orphan IIA^Man homologue, has a short DhaL fragment (GI 52144352). Finally, organisms in category F, all Bacillus species, have DhaKL as well as DhaK. It seems likely that many of these orphan PTS Dha proteins have resulted from genome minimalization, but the possibility that some of them serve a specific function, perhaps to allow phosphorylation of a novel substrate or to provide a regulatory function, should be kept in mind.

Of the organisms listed in Table 11, Bartonella quintana has DhaM (GI 49473737) and several PTS energy-coupling proteins, but no complete enzyme II complex (Table 7); Brucella melitensis has DhaK (GI 17986679), DhaL (GI 17986680), and a IIA^Man-like homologue (GI 17988315), as well as several PTS energy-coupling proteins, but no IIA^Dha and no PTS permeases; and Methylococcus capsulatus has DhaK (GI 53802782) and DhaL (GI 53802783), but no other PTS protein of any kind. It seems likely that none of these organisms can phosphorylate DHA with either PEP or ATP, but conceivably M. capsulatus might use ATP with DhaK and DhaL in a split DhaKL system. We tentatively suggest that some of these organisms are "in transition," losing or gaining complete systems, either by genome reduction or by horizontal transfer, respectively (51, 108).

PHYLOGENY OF DHA PROTEINS

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The Dha proteins were analyzed by generating phylogenetic trees. This was done in several ways. First, a tree was generated just for the fused DhaKL proteins. The results showed that these proteins did not cluster according to organismal group, suggesting either that horizontal transfer of these genes has occurred or that rates of evolutionary sequence divergence have been radically different for the different organisms (11) (Fig. 4). Three separate trees were generated, for the DhaK (Fig. 5A), DhaL (Fig. 5B), and DhaM (Fig. 5C) proteins, for organisms that possess homologues of these proteins (Tables 10 and 11). The analyses clearly suggest that these three proteins have evolved in parallel without shuffling of constituents between systems. However, the 16S rRNA phylogeny results for these organisms (data not shown) suggested that horizontal transfer of complete systems has occurred repeatedly and that different rates of sequence divergence in the different organisms (11) are unlikely to explain the results. The lone DhaM protein (GI 15901038) in Streptococcus pneumoniae (Table 11) clusters with the other streptococcal DhaMs (Fig. 5C), but those from Clostridium perfringens (GI 18311611) and Bartonella quintana (GI 49473737) cluster very loosely together and distantly from all other DhaMs. These two proteins resemble IIA^Man more closely than the other DhaM proteins. While C. perfringens has a complete mannose enzyme II complex in addition to the orphan IIA^Man-like protein, B. quintana lacks all PTS permeases, including the mannose enzyme II complex.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Phylogenetic tree for the putative ATP-dependent DHA kinases. The DhaKL fusion proteins from 12 organisms (Tables 10 and 11) and the known ATP-dependent dihydroxyacetone kinase from Citrobacter freundii (Cfr) were aligned using the Clustal X program, and neighbor-joining trees were generated (102). Trees were viewed using the TreeViewPPC program (113). When multiple strains of a species had been sequenced, only one orthologue was included.

View larger version (55K): [in this window] [in a new window]   FIG.5. Phylogenetic trees for DhaK (A), DhaL (B), and DhaM (C) homologues. The trees were generated as described in the legend to Fig. 4. Multiple paralogues from a single organism are distinguished by numbers at the ends of the names. For the DhaK and DhaL trees, the DhaKL fusion proteins were split into the DhaK and DhaL domains and were included in the two trees, respectively. These domains are indicated with an "F" at the ends of the names, and their clusters are designated with letters (A, B, C, and D), while the remaining homologues were grouped numerically into seven clusters. The alphabetical and numerical cluster designations were maintained in the two trees shown in panels A and B. The Clustal X program (102) was used to derive the trees. Abbreviations of organism names are listed in Tables 10 and 11.

In categories D and E in Table 11, the DhaL and DhaK proteins are present, but no DhaM homologue could be found. All of the DhaL-DhaK pairs were derived from proteobacteria. Most of these proteins cluster together (cluster 5) in both the DhaL (Fig. 5B) and DhaK (Fig. 5A) trees. They do not cluster with the ATP-dependent DhaKL kinases (clusters A to D). These observations led us to suggest that these DhaL-DhaK protein pairs have coevolved from a common ancestor and serve a unified but unknown function. As noted above, it is possible that they use ATP or another phosphoryl donor to phosphorylate DHA. Alternatively, they could act on another substrate. It is interesting that the Mlo2 DhaL protein (GI 13476063) clusters with Sme2 (GI 16264045) and Bme2 (GI 17986682) (Fig. 5B). The corresponding Mlo2 DhaK protein (GI 13476064) (Fig. 5A) is by itself because there is no corresponding orthologue in S. meliloti and B. melitensis.

PTS DOMAIN FUSION PROTEINS

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Table 12 present