Microbiology and Molecular Biology Reviews, December 2005, p. 608-634, Vol. 69, No. 4
1092-2172/05/$08.00+0 doi:10.1128/MMBR.69.4.608-634.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Comparative Genomic Analyses of the Bacterial Phosphotransferase System
Ravi D. Barabote and
Milton H. Saier Jr.*
Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116
We
report analyses of 202 fully sequenced genomes for homologues of known
protein constituents of the bacterial phosphoenolpyruvate-dependent
phosphotransferase system (PTS). These included 174 bacterial, 19
archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not
identified in archaea or eukaryotes, showing that the horizontal
transfer of genes encoding PTS proteins has not occurred between the
three domains of life. Of the 174 bacterial genomes (136 bacterial
species) analyzed, 30 diverse species have no PTS homologues, and 29
species have cytoplasmic PTS phosphoryl transfer protein homologues but
lack recognizable PTS permeases. These soluble homologues presumably
function in regulation. The remaining 77 species possess all PTS
proteins required for the transport and phosphorylation of at least one
sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS
proteins. These homologues were analyzed for family association, range
of protein types, domain organization, and organismal distribution.
Different strains of a single bacterial species often possess
strikingly different complements of PTS proteins. Types of PTS protein
domain fusions were analyzed, showing that certain types of domain
fusions are common, while others are rare or prohibited. Select PTS
proteins were analyzed from different phylogenetic standpoints, showing
that PTS protein phylogeny often differs from organismal phylogeny. The
results document the frequent gain and loss of PTS protein-encoding
genes and suggest that the lateral transfer of these genes within the
bacterial domain has played an important role in bacterial evolution.
Our studies provide insight into the development of complex
multicomponent enzyme systems and lead to predictions regarding the
types of protein-protein interactions that promote efficient
PTS-mediated phosphoryl transfer.
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).

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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.
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When
the PTS was discovered, a single function was recognized, namely, sugar
phosphorylation. Forty-one years later, we find that this system plays
roles in many surprising aspects of bacterial physiology. Established
primary functions of the system include sugar reception, transport, and
phosphorylation, whereas secondary functions include a variety of
ramifications for metabolic and transcriptional regulation (Table
2) (37,
39,
84,
86,
87,
99,
100; for reviews, see
references 62,
76,
79, and
84).
Genetic
evidence has indicated that in various bacteria, processes regulated by
the PTS include (i) transport of non-PTS carbon sources
(35), (ii) the net
production of carbon and energy storage sources, such as
poly-ß-hydroxybutyrate
(64,
90) and glycogen
(91), (iii) the switch
between fermentative and respiratory metabolism
(36), (iv) flagellar
motility (54), and (v)
the control of
54-dependent transcription of carbon
and nitrogen metabolic genes
(12,
46,
63,
70,
74). Some evidence
implicates the PTS in the regulation of cell division
(44,
63). Still other
regulatory functions of the PTS and their physiological consequences
are listed in Table
2.
In
Escherichia coli, paralogues of EI, HPr, and the fructose IIA
protein (IIAFru), designated nitrogen enzyme I
(EINtr), nitrogen HPr (NPr), and nitrogen IIA protein
(IIANtr), 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). EINtr
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
EINtr from EI have been described previously
(65). EINtr
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. EINtr may serve a sensory function linking
carbon and nitrogen metabolism
(69). A mutation of the
orthologous EINtr-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
EINtr 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 IIADha domain that is distantly
related to IIAMan, 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.
Proposed evolutionary origins of the different PTS families have
been discussed (81).
Briefly, the substrate-recognizing protein constituents of the PTS
(enzymes IIC) are derived from at least four independent sources. Some
of the non-PTS precursor constituents have been identified, and the
evolutionary pathways taken have been proposed
(81). Analyses suggest
that two of these independently evolving systems (Gat and Dha) are
still in transition, as they have not yet acquired the full-fledged
characteristics of PTS enzyme II complexes. The mosaic nature of PTS
enzyme II complexes has also been documented
(81).
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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 IIDMan
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 IIAGlc 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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RELATIVE DISTRIBUTION OF PTS PERMEASE TYPES
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The complete PTS permeases identified in
distinct bacterial species were tabulated according to the family to
which they belong, as classified in the TCDB
(10,
77). The greatest
representation was observed for the Glc family (30%), with the Fru
family (25%) coming in second place (Fig.
2). The order of occurrence of the members of the seven families was Glc
(30%) > Fru (25%) > Man (15%) > Lac (14%)
> Asc (9%) > Gat (4%) > Gut
(3%). The Glc, Fru, Man, and Lac systems often occur in multiple
copies, while the Asc, Gat, and Gut systems are usually found as a
single copy per organism (Fig. 2).
Furthermore, about equal proportions of organisms possess the Fru and
Glc systems (86% and 83%, respectively), followed by the Asc (47%), Lac
(44%), Man (42%), Gut (18%), and Gat (16%)
systems.

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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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DISTRIBUTION OF PTS PERMEASE TYPES IN VARIOUS BACTERIAL KINGDOMS
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The occurrence of these
permease types was analyzed according to organismal type and
PTS permease family (Table
5). Actinobacteria had about equal numbers of Glc- and Fru-type
systems, with the Asc and Man families showing less but substantial
representation. Only one or two members had representatives of the
remaining three families. Among the Firmicutes, the order was
Glc > Fru > Lac > Man > Asc >
Gat > Gut. All three classes of
Firmicutes (bacilli, clostridia, and Mollicutes) had
more glucose-type systems than any other type, but in the order
Bacillales, Lac systems were more prevalent than Fru systems,
which were much more common than the Man-type systems. In contrast, the
order Lactobacillales, as well as the clostridial class, had
far more Man systems than either Lac- or Fru-type systems, which were
present in about equal numbers. This observation complements and
expands the suggestion of Zuniga et al.
(114) that the
mannose-type systems may have played a role in the establishment of
symbiotic relationships between bacteria and a wide spectrum of
eukaryotes. Finally, the Mollicutes had only Glc-, Fru-, and
Asc-type systems, at decreasing frequencies in that order.
Proteobacteria exhibited a profile quite different from those
of the Firmicutes and Actinobacteria. They were found
to possess far more Fru-type systems than any other type. Glc-type
systems were of secondary importance, followed by Man, Asc, and Lac
systems, in that order. However, the prevalence of Fru-type systems in
Proteobacteria proved to be due solely to their high
occurrence in the
-proteobacterial subdivision, for which many
fully sequenced genomes are available. The alpha and beta subdivisions
had a preponderance of Glc-type systems, while the one
-proteobacterium with a complete PTS had only a Man-type
system.
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NUMBERS OF PTS PROTEIN-ENCODING GENES VERSUS GENOME SIZE AND PHYSIOLOGY
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Figure
3A shows the numbers of PTS protein-encoding genes plotted versus genome
size. There is no good correlation, as some of the organisms with the
smallest genomes have a good representation of PTS constituents, while
some organisms with large genomes have none at all. Instead, it appears
that the PTS protein content in a bacterium correlates best with the
mode of carbohydrate metabolism utilized by that organism. Bacteria
that rely on anaerobic sugar metabolism via glycolysis for energy
production generally have the most PTS permeases, as discussed
previously (56,
57). Thus, almost all
bacteria that possess the PTS either are capable of anaerobic sugar
utilization via glycolysis or have close bacterial relatives that are
capable of such metabolism, while many of the bacteria that lack the
PTS either are strict aerobes or lack a complete glycolytic
cycle.

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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.
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We tabulated the aerobic versus anaerobic metabolic
capabilities of the 136 bacterial species analyzed in this report. The
results showed that only 20% of the bacteria that lack genes encoding
PTS proteins and 28% of the bacteria that lack PTS transport systems
are capable of anaerobic growth (Fig.
3B). However, of the
bacteria that possess PTS transport capabilities, 37% encoding 3 to 9
PTS proteins, 86% encoding 10 to 30 PTS proteins, and 100% encoding 31
to 93 PTS proteins are capable of anaerobic growth (Fig.
3B). This can be explained
by the fact that only anaerobic glycolysis yields two molecules of PEP
per molecule of hexose metabolized. One of these PEP molecules must be
used for uptake of the next sugar via the PTS, while the other is
required for biosynthetic purposes. The availability of excess
energetic PEP molecules, the phosphoryl bonds of which have higher free
energies than those of ATP, presumably provided the basis for the
establishment and selective expansion of the
PTS.
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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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BACTERIA WITH CYTOPLASMIC PTS PROTEIN HOMOLOGUES BUT NO RECOGNIZABLE PTS TRANSPORTERS
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Twenty-nine bacteria were found to have PTS phosphoryl transfer
proteins but to lack the complete complement of enzymes necessary for
sugar transport (Table
7). As noted above, Bacteroides thetaiotaomicron possesses only a
IICGat homologue and may use this protein as a secondary
carrier. A second organism, Ureaplasma parvum, possesses only
IIBGlc (GI 13357736), HPr (GI 13358152), and HprK (GI
13357632). In this organism, the HPr and HprK proteins may function in
catabolite repression by a PTS-mediated sugar transport-independent
mechanism (48), while
IIBGlc may be a relic of a complete enzyme IIGlc,
the other components of which were lost during genome minimalization
(51).
Of the
remaining 27 organisms presented in Table
7, all are gram-negative
bacteria. They include proteobacteria, chlamydiae,
spirochetes, and a green photosynthetic bacterium. All of these
organisms encode within their genomes at least one enzyme I homologue
(I or INtr) and at least one HPr homologue (HPr or NPr)
(65). Three of these
bacteria (Pirellula sp., Geobacter sulfurreducens,
and Bradyrhizobium japonicum) have two EIs, and four of them
(Parachlamydia sp., Bartonella henselae,
Bartonella quintana, and Bradyrhizobium japonicum)
have two HPrs. Furthermore, all but two groups of these bacteria, the
chlamydial group (except for Parachlamydia) and
Pirellula sp., have HprK. Finally, these organisms may possess
zero to three IIA proteins (IIANtr and/or
IIAFru and/or IIAMan).Only
two of these bacteria (Coxiella burnetii and Legionella
pneumophila) lack an identifiable IIA protein. In
Agrobacterium tumefaciens, the EINtr protein (GI
17937868) is encoded on the linear chromosome, while the rest of the
PTS proteins are encoded on the circular chromosome.
Of the IIA
homologues, Xylella species have only a single
IIAMan protein while Leptospira species have only a
single IIANtr protein. All others have either two
IIANtr proteins, two IIAFru proteins, one each of
the IIANtr and IIAFru proteins, one each of the
IIANtr and IIAMan proteins, one
IIANtr, one IIAFru, and one IIAMan
protein, or two IIANtr and one IIAMan protein
(Table 7). The species
with one IIAFru, one IIANtr, and one
IIAMan homologue are the two Bartonella species,
while Rhodopseudomonas palustris has two IIANtr and
one IIAMan 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 INtr) 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).
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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 INtr, NPr, and IIANtr. 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.
Thirteen bacterial species possess just two
types of complete PTS permeases. In all cases, one of thesystems is either a glucose- or fructose-type system, except in
Haemophilus ducreyi, where the two systems identified are Man-
and Asc-type systems. However, Haemophilus influenzae has a
single Fru-type system. Further examination of other bacteria in the
same phylogenetic group revealed that these organisms possess Fru- and
Glc-type systems in addition to Man- and Asc-type permeases (Table
9). It is therefore
possible that H. ducreyi may have lost the Fru- and Glc-type
systems during evolution. Eleven species have both glucose and fructose
systems, usually just one of each. However, Mesoplasma florum
has seven glucose-like systems and one fructose-like system.
Borrelia garinii has two fructose-like systems and one
glucose-like system, while Chromobacterium violaceum has two
of each. Corynebacterium diphtheriae has a glucose-type system
and an L-ascorbate-type system, while Haemophilus
ducreyi has a mannose-type system and an ascorbate-type system, as
noted above (Table
8).
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
|
|---|
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.
High-G+C Gram-Positive Bacteria
Among the high-G+C gram-positive
Actinobacteria, the Mycobacteria and
Tropheryma species have no recognizable PTS protein homologues
(Table 6).
Bifidobacterium longum and Nocardia farcinica have a
single glucose-type system and a single fructose-type system,
respectively. Also, these two bacteria encode the smallest number of
PTS genes (three genes) among all of the bacteria that possess a
complete transport PTS. Of the three Corynebacterium species
studied, C. efficiens has both Glc and Fru systems, C.
diphtheriae has Glc and Asc systems, and C. glutamicum
has Glc, Fru, and Asc systems. The corynebacteria represent an example
where several species within a single genus have different complements
of PTS proteins.
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
IIBCGlc and IICGlc 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.
Low-G+C Gram-Positive Bacteria
Firmicutes (orders
Bacillales and Lactobacillales and class
Clostridia in Table
9) generally have the
energy-coupling PTS proteins enzyme I and HPr as well as HprK, and with
just two exceptions, they all have Glc, Fru, and Lac systems. The two
exceptions are Clostridium tetani, which has only a Glc
system, and Clostridium perfringens, which has Glc and Fru
systems but not a Lac system. The three clostridial species analyzed
show remarkable variation in their numbers of PTS genes, and these
numbers do not correlate well with the differences in their genome
sizes. C. tetani, with a genome size of 2.87 Mb, has 4 PTS
genes, Clostridium acetobutylicum, with a genome size of 4.13
Mb, has 30 PTS genes, and C. perfringens, with an intermediate
genome size of 3.09 Mb, has 36 PTS genes. The distributions of various
PTS permeases in these three species are remarkably different as well
(Table 9).
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).
Proteobacteria
Table 9
presents the PTS protein complements in a variety of
Proteobacteria according to their subdivision classification.
Within the alpha subdivision, most either lack PTS proteins altogether
(Table 6) or only possess
phosphoryl transfer proteins (Table
7) that presumably
function in regulation. Only two have PTS permeases. Caulobacter
crescentus has two Glc systems, while Mesorhizobium loti
has one Gut system. As discussed below, Bradyrhizobium
japonicum has a complete dihydroxyacetone enzyme II complex and
therefore can phosphorylate this triose via the
PTS.
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.
Spirochetes
Among
the spirochetes, Leptospira and Treponema species
lack PTS permeases (as discussed earlier), but Borrelia
species have several. Leptospira interrogans is an obligate
aerobe that is adapted for mammalian reservoir hosts. T.
denticola is an obligate anaerobe that is commonly found in the
oral cavity in humans, while T. pallidum is an anaerobic human
pathogen that causes syphilis. Borrelia garinii has one Glc
and two Fru systems, while Borrelia burgdorferi has these
systems plus a Lac system. Borrelia species are
microaerophilic organisms that live in the gastrointestinal tract of
ticks and are capable of infecting multiple hosts via tick bites.
B. burgdorferi carries dozens of plasmids, while B.
garinii harbors only two. Interestingly, the Lac system as well as
a IICBGlc protein (GI 11497021) in B. burgdorferi
is encoded in the plasmid cp26.
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
|
|---|
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 (IICDha; dihydroxyacetone
binding), DhaL (IIBDha; ADP binding), and DhaM
(IIADha; 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
IIADha, 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 A organisms possess one and only one DHA
PTS. These bacteria include two
-Proteobacteria,
Fusobacterium nucleatum, one high-G+C gram-positive
bacterium, and eight low-G+C gram-positive bacteria (Table
10). DhaM in these
systems consists of only a IIADha domain.
Category B
organisms have just one DHA PTS, but their DhaM proteins have the
IIADha domain fused to other PTS protein domains. These
fused proteins include IIADha-HPr fusions, found in C.
diphtheriae (GI 38234870) and L. xyli (GI 50954678), a
single full-length IIADha-HPr-EI fusion (GI 46579394),
present in D. vulgaris, and tridomain proteins with a
truncated enzyme I, IIADha-HPr-EI
, characteristic
of the E. coli DhaM protein, found in
-Proteobacteria (Table
10). Only category B
systems have the IIADha 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 IIAMan homologue (GI 17988315) that
more closely resembles E. coli IIAMan than
IIADha. Category E includes four organisms encoding DhaK,
DhaL, and DhaKL, and three of these organisms have a IIAMan
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 IIAMan 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 10, one organism,
Bradyrhizobium japonicum, has an unusual gene arrangement. The
complete DHA PTS enzyme II complex as well as enzyme I (GI 27378686)
and HPr (GI 27378685) is encoded by adjacent genes that are in an
operon, dhaLMHIXK. The B. japonicum dhaM gene encodes
only the IIADha domain (GI 27378684). dhaH and
dhaI code for HPr and enzyme I homologues, respectively, while
dhaX is a hypothetical gene. It is also unusual that
dhaK and dhaL are not adjacent genes. It is
interesting that the gene order, dhaMHI, is the same as the
domain order in the fused tridomain DhaM of E. coli and that
B. japonicum possesses no other PTS enzyme II complexes. In
all other organisms listed in category A of Table
10, the ptsH and
ptsI genes map separately from the dha
operon.
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
IIAMan-like homologue (GI 17988315), as well as several PTS
energy-coupling proteins, but no IIADha 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
|
|---|
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 IIAMan
more closely than the other DhaM proteins. While C.
perfringens has a complete mannose enzyme II complex in addition
to the orphan IIAMan-like protein, B. quintana
lacks all PTS permeases, including the mannose enzyme II
complex.

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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.
|
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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.
|
|
A few organisms have "extra" DhaLs.
These include S. meliloti (GI 16264045) (Table
11, category E),
Brucella melitensis (GI 17986682) (Table
11, category D), and both
M. gallisepticum (GI 31544231) and P. syringae (GI
28870011) (Table 11,
category B). Of these organisms, all have DhaK and two DhaLs, except
M. gallisepticum and P. syringae, which have only an
orphan DhaL. The extra DhaLs in S. meliloti and B.
melitensis cluster together (Fig.
5B, cluster 6), separate
from their paralogues (cluster 5). The M. gallisepticum
protein is by itself, and it is distantly related to all other
homologues (Fig. 5B).
Extra DhaKs are present in many low-G+C gram-positive bacteria
(Table 10, category D,
and Table 11, category F)
and one
-proteobacterium, M. loti (Table
10, category F). The
extra M. loti protein (Mlo3) (GI 13488187) is by itself
(between clusters 5 and 6) (Fig.
5A), while all extra
DhaKs from low-G+C gram-positive bacteria cluster together
(cluster 1c). We conclude that these extra DhaL and DhaK
proteins did not arise by recent gene duplication events. Instead, they
either arose early to fulfill a specific but unknown function or became
"orphans" as a result of the genetic loss of their
partner proteins.
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
|
|---|
Table
12 present