Department of Microbiology and Immunology,
Uniformed Services University of the Health Sciences, Bethesda,
Maryland 20814-4799,1 and Department of
Plant and Microbial Sciences, University of Canterbury,
Christchurch, New Zealand2
The assembly of the phosphoglyceride-linked form of enterobacterial
common antigen (ECAPG) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of o349 of the Escherichia coli
K-12 wec gene cluster revealed that this locus encodes a
Wzz protein that specifically modulates the chain length of
ECAPG polysaccharides, and we have designated this locus
wzzECA. The WzzECA-mediated
modulation of ECAPG polysaccharide chains is the first
demonstrated example of Wzz regulation involving a polysaccharide that
is not linked to the core-lipid A structure of lipopolysaccharide.
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TEXT |
Enterobacterial common antigen (ECA)
is a unique cell-surface glycolipid that is present in all
gram-negative enteric bacteria (14, 17, 21, 30). The
carbohydrate portion of ECA consists of a linear heteropolysaccharide
chain comprising the trisaccharide repeat unit
3)-
-D-Fuc4NAc-(14)-
-D-ManNAcA-(14)--D-GlcNAc-(1, where GlcNAc is N-acetyl-D-glucosamine, ManNAcA
is N-acetyl-D-mannosaminuronic acid, and Fuc4NAc
is 4-acetamido-4,6-dideoxy-D-galactose (16, 18).
The major form of ECA, ECAPG, consists of polysaccharide chains of various lengths that are covalently linked to
phosphoglyceride via phosphodiester linkage involving the
GlcNAc residue of the first repeat unit (15,
28). The occurrence of two other minor forms of ECA,
ECALPS and ECACYC, has also been demonstrated.
ECALPS is characterized by the covalent linkage of ECA
polysaccharide chains to the core region of lipopolysaccharide (LPS)
(12, 14, 31, 34), whereas ECACYC is a
water-soluble cyclic form of ECA containing four to six trisaccharide
repeat units (9, 14, 33). Although ECAPG is
present in all members of the Enterobacteriaceae, the
occurrence of ECALPS and ECACYC appears to be
restricted to certain gram-negative enteric bacteria (12, 14, 31,
34).
The known genetic determinants of ECA are located in the wec
(formerly rfe/rff) gene cluster located at min 85 of the
Escherichia coli chromosome (14, 22, 30). (The
genetic nomenclature used here is in accordance with that proposed by
Reeves et al. [27] for genes involved in the synthesis
of bacterial polysaccharides.) The nucleotide sequence of this gene
cluster includes 12 open reading frames (Fig.
1B). Biochemical and genetic studies have identified the function of 7 of these genes (30); however,
the functions of o349, wecD, o416,
o359, and o450 have not been unequivocally demonstrated. Many of the early steps involved in ECA synthesis have
been firmly established (Fig. 1A). The ECA trisaccharide repeat unit is
assembled as an undecaprenylpyrophosphate (Und-PP)-linked intermediate
(lipid III) by the same general mechanism established for the assembly
of the repeat units of Wzy-dependent O-antigens (for a review, see
reference 35). Although details concerning subsequent steps of ECA assembly have not yet been determined, the
available information suggests that these steps occur by the same
general mechanism established for the assembly of Wzy-dependent O
antigens. In this regard, previous studies led to the conclusion that
wecF (o716) of the wec gene cluster
encoded the Fuc4NAc transferase involved in ECA synthesis (5,
8). However, recent examinations of the nucleotide sequence of
this region revealed that it actually contains two putative open
reading frames, o359 and o450 (1a). The results of preliminary investigations support the conclusion that
o359 encodes WecF, and the considerable homology between the
predicted o450 gene product and several Wzy proteins
(24) suggests that o450 of the wec
gene cluster encodes the Wzy protein involved in ECA polysaccharide
chain elongation. Thus, it is likely that lipid III is also synthesized
on the cytoplasmic face of the inner membrane and subsequently
translocated to the periplasmic face of the inner membrane, where
assembly of ECA polysaccharide chains occurs by a Wzy-dependent
"block polymerization" mechanism.
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FIG. 1.
The ECA biosynthetic pathway and genes of the
wec gene cluster. (A) Current status of our knowledge
concerning the enzymatic reactions involved in the biosynthesis and
assembly of ECA. The genetic determinants of the various enzymes are
indicated next to the reaction catalyzed by each of the respective
enzymes. Abbreviations are as follows: Und-P,
undecaprenylmonophosphate; Und-PP, undecaprenylpyrophosphate; Glc,
glucose; GlcNAc, N-acetyl-D-glucosamine; ManNAc,
N-acetyl-D-mannosamine; ManNAcA,
N-acetyl-D-mannosaminuronic acid; Glc-1-P,
glucose 1-phosphate; FucNH2,
4-amino-4,6-dideoxy-D-glucose; Fuc4NAc,
4-acetamido-4,6-dideoxy-D-glucose; CoASH, coenzyme A;
-KG, -ketoglutaric acid; PPi, inorganic pyrophosphate;
ECAPG, phosphoglyceride-linked ECA polysaccharide chains.
The structures of lipids I, II, and III are defined in the inset. (B)
The wec gene cluster. Arrowheads designate the direction of
transcription. The 14-kb scale below the gene cluster is provided for
reference.
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The chain length of Wzy-dependent O antigens (35, 36), as
well as some lipid A-core-linked capsular polysaccharides (10, 11), is regulated by Wzz (Rol, Cld). A number of Wzz homologues have also been identified in a variety of bacteria that are involved in
the assembly of bacterial cell surface polysaccharides that are not
linked to core-lipid A (36), but none of these homologues has been demonstrated to function in the regulation of polysaccharide chain length. Wzz-mediated regulation results in a modal or multimodal distribution of polysaccharide chain lengths when preparations are
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, a nonmodal random distribution of chain lengths is observed in wzz mutants; however, the mechanism
of Wzz-mediated modulation of polysaccharide chain length has not been
determined. The degree of polymerization of ECA polysaccharide chains
is also modulated, and a single modal distribution of chain lengths has
been observed following SDS-PAGE analyses of ECAPG preparations (1, 2, 23). Although the genetic determinant of
ECA polysaccharide chain length regulation has not been identified, significant homology between the putative product of o349 of
the wec gene cluster and various Wzz homologues has been
noted (3, 4, 6, 25). The hydropathy profiles of all of these
proteins
including that of o349are strikingly similar,
and they all possess a large hydrophilic domain that is flanked by
apparent transmembrane domains at both the amino and carboxy termini
(25). We demonstrate here that o349 of the
wec gene cluster of E. coli K-12 does indeed encode a Wzz that functions to modulate ECA polysaccharide chain lengths. In addition, the WzzECA-mediated modulation of
ECAPG polysaccharide chains is the first demonstrated
example of Wzz regulation involving a polysaccharide that is not linked
to the core-lipid A region of lipopolysaccharide.
WzzECA is encoded by o349 of the
wec gene cluster.
The assembly of ECA polysaccharide
chains in E. coli AB1133 (23) results in a
population of polysaccharide chains whose lengths range from
approximately 1 to 14 repeat units with an apparent modal value of 5 to
7 (Fig. 2A, lane 1). A chromosomal o349::mini-TnlacZ insertion mutant was
constructed by allelic exchange in order to evaluate the role of this
gene in the regulation of ECA polysaccharide chain length. Briefly, the
transposon TnlacZ (Kmr) was used to generate
TnlacZ insertions in the coding region of o349 by
the general strategy of Pradel et al. (26). Plasmid pCA53
(Tcr Ampr), a pBR322-based construct containing
wecA and o349 on a 2.54-kb ClaI
insertion fragment (Fig. 3)
(22), was introduced by transformation into E. coli CC170 (19), which carries a chromosomal copy of TnlacZ. Cells in which transposition of TnlacZ to
the plasmid had occurred were selected by subsequent growth of the
transformants on Luria-Bertani (LB) agar plates containing tetracycline
and ampicillin as well as high levels of kanamycin (300 µg/ml). A pool of plasmid DNA prepared from these cells was used to transform strain CC118 (Lac
) (19), and
kanamycin-resistant transformants were selected on LB plates containing
kanamycin as well as
5-bromo-4-chloro-3-indoyl--D-galactopyranoside (X-Gal)
to differentiate between cells containing potentially in-frame fusions
(blue colonies) from those containing out-of-frame insertions (white
colonies). Plasmids were isolated from individual transformants, and
those containing TnlacZ insertions in o349 were
identified by restriction mapping. One of these plasmids, pJK2885,
contained a TnlacZ insertion in an orientation opposite that
of the direction of transcription of the o349 gene. The
insertion in pJK2885 was stabilized by removal of the transposase by
digestion with XhoI followed by religation to yield plasmid
pJK2887 (o349::mini-TnlacZ, Kmr).
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FIG. 2.
Effect of mutations in o349 of the
wec gene cluster and waaL on the modality of ECA
polysaccharide chain length. Exponentially growing cultures were
incubated with [3H]GlcNAc for 30 min. The incorporation
of radiolabeled GlcNAc into ECA was subsequently analyzed by SDS-PAGE
and fluorography. (A) Lane 1, strain AB1133 (wild type); lane 2, strain
CS2942 (o349::mini-TnlacZ). (B) Strain
PR4100 (o349::mini-TnlacZ + pRL123
[wecB+ wecC+ rmlB+]).
(C) Lane 1, strain PR4129
(o349::mini-TnlacZ + pRL139
[o349+]); lane 2, strain PR4130
(o349::mini-TnlacZ + pRL123
[wecB+ wecC+ rmlB+] + pRL139 [o349+]). (D) Lane 1, strain AB1133
(wild type); lane 2, strain CS2334
(waaL::TnphoA).
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FIG. 3.
Physical map of the 5' half of the wec gene
cluster. The site and orientation of the mini-TnlacZ
insertion in o349 are indicated. The cross-hatched rectangle
for rmlA denotes a partial open reading frame. The
chromosomal regions contained in plasmids pCA53, pRL123, and pRL139 are
indicated by the respective open rectangles.
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Introduction of the o349::mini-TnlacZ
insertion into the E. coli K-12 chromosome by allelic
exchange was accomplished by transformation of strain VJS803
(recBC sbcB) (32) with plasmid pJK2887.
Kanamycin-resistant transformants were selected and subsequently
screened for sensitivity to ampicillin. The insertion in one of these
transformants, strain CS2898 (Kmr Amps), was
then transduced into strain PR21548
(wecA::Tn10) (23) by using
bacteriophage P1. Kanamycin-resistant transductants were selected, and
subsequent screening for sensitivity to tetracycline revealed that all
of the kanamycin-resistant transductants were sensitive to
tetracycline. The recA56 allele of JC10240 (recA56 srl-300::Tn10) (7) was introduced
into one of the transductants, CS2900, by conjugation in order to
prevent recombination between genes harbored on plasmids and their
chromosomal counterparts during subsequent complementation studies.
Kanamycin- and tetracycline-resistant transconjugants were selected and
screened for sensitivity to UV light. One such UV-sensitive
transconjugant, CS2942, was used in all subsequent studies.
The precise site of the mini-TnlacZ insertion in the
chromosome of strain CS2942 was determined by analysis of the
nucleotide sequence of the PCR product obtained by using the
'lacZ forward primer (5' GTC ACG ACG TTG TA 3') and the
reverse primer (5' GCG CTC ACC CAG CAG 3'). The forward primer was
complementary to the coding sequence of 'lacZ at the left
end of TnlacZ (19), and the reverse primer was
complementary to the flanking nucleotide sequence immediately
downstream of the o349 stop codon (23). Nucleotide sequencing of the PCR product revealed that the
mini-TnlacZ was inserted into o349 between
nucleotides 11,400 and 11,401 of the published wec gene
cluster sequence (GenBank accession no. AE000454).
WzzLPS function is not required for the synthesis of
Wzy-dependent O antigens. In contrast, the null mutation in
o349 resulted in the apparent abrogation of ECA synthesis,
as indicated by the lack of incorporation of
N-acetyl-D-[1-3H]glucosamine into
ECA polysaccharide chains in strain CS2942 (o349::mini-TnlacZ) (Fig. 2A, lane 2).
In vivo incorporation of [3H]GlcNAc (6.6 Ci/mmol) into
ECA and analysis of radioactive ECA by SDS-PAGE and fluorography were
carried out as previously described (29). The fast-moving
broad bands in each lane that migrate immediately ahead of ECA and at
the bottom of the gel were not identified; however, they are believed
to be radiolabeled lipid A and phospholipid, respectively. Subsequent
experiments revealed that the lack of ECA synthesis by strain CS2942
was due to a polar effect of the insertion in o349 on
required downstream genes. The wecB and wecC
genes are located immediately downstream of o349 (Fig. 1B)
(20), and these genes are required for the synthesis of
UDP-ManNAcA, the donor of ManNAcA residues for ECA synthesis. In
addition, o349 and the wecBC genes appear to be
located in the same transcriptional unit (5). Thus, ECA
synthesis was restored in strain PR4100, which was constructed by the
introduction of plasmid pRL123 containing the wecBC genes
and the rmlB gene into strain CS2942 (Fig. 2B). It should be
noted that the rmlBECA and
rmlAECA genes are located immediately downstream
of the wecC gene, and these genes are required for synthesis
of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis
(20). Although the rmlBAECA genes
appear to be located in the same transcriptional unit as
o349 and the wecBC genes (5), plasmid
pRL123 contained only the rmlBECA gene (Fig. 3).
Thus, it seems likely that synthesis of ECA in strain PR4100 was also
dependent on the expression of the chromosomal rmlA allele
contained in the wbb (formerly rfb) gene cluster
of E. coli K-12 (27).
Restoration of ECA synthesis in strain PR4100 resulted in a random
nonmodal distribution of ECA polysaccharide chain lengths (Fig. 2B).
This distribution pattern is characteristic of the patterns observed
for O-antigen synthesis in wzz mutants (36). In
addition, the phenotype of PR4100 was not due to overexpression of the
wecBC and rmlB genes in pRL123 since introduction
of this plasmid into the wild-type parental strain, AB1133, did not
result in altered ECA chain length regulation (data not shown).
Wild-type modal chain length distribution was rescued in strain PR4130
(wzzECA::mini-TnlacZ) that
contained plasmid pRL123 (wecBC rmlB) as well as plasmid pRL139 (o349) (Fig. 2C, lane 2). These results clearly
support the conclusion that o349 of the wec gene
cluster functions as a regulator of ECA polysaccharide chain length;
accordingly, we have designated this open reading frame
wzzECA.
A modal distribution of ECA polysaccharide chains was also observed in
strain PR4129, which was constructed by the introduction of only
plasmid pRL139 (wzzECA) into strain CS2942 (Fig.
2C, lane 1). However, the level of [3H]GlcNAc
incorporation into ECA chains was significantly reduced in strain
PR4129 in comparison to that observed in strain PR4130, which contained
plasmid pRL139 (wzzECA) as well as plasmid
pRL123 (wecBC rmlB). These observations suggest that
wzzECA::mini-TnlacZ insertion does not completely abolish expression of genes located immediately downstream. Indeed, the apparent absence of radiolabeled chains in strain CS2942 most likely reflects a markedly reduced level
of ECA synthesis that was not detected by SDS-PAGE due to the dispersal
of the low level of incorporated radioactivity into a randomly
distributed population of ECA chains (Fig. 2A, lane 2). However,
complementation of the defect in wzzECA
facilitated the detection of the radiolabeled chains by effectively
concentrating the radioactivity into a smaller modal population of
polymers. It is also important to note that overexpression of
wzzECA in the wild-type parental strain had no
effect on either the modality of ECA chains or the amount of
incorporation of [3H]GlcNAc into ECA (data not shown).
With the exception of the data shown for strains CS2942 (Fig. 2A, lane
2) and PR4129 (Fig. 2C, lane 1), the apparent differences in the amount
of radiolabel incorporated into ECA chains primarily reflect variations
in the amount of radiolabeled sample analyzed by SDS-PAGE and the
length of time the gels were exposed to X-ray film during fluorography.
WzzECA regulates the chain length of ECAPG
polysaccharides.
ECALPS occurs only in gram-negative
enteric bacteria that are able to synthesize a complete R1, R4, or K-12
LPS core structure but that are unable to synthesize O side chains
(14, 31, 34). In contrast, all members of the
Enterobacteriaceae synthesize ECAPG, and
ECAPG is the major form of ECA in those organisms capable of synthesizing both ECAPG and ECALPS. Thus, it
has been estimated that less than 5% of E. coli K-12
core-lipid A structures are substituted with ECA polysaccharide chains
(14). Furthermore, a modal distribution of ECA
polysaccharide chains has been observed in extracts obtained from
organisms, such as Salmonella enterica serovar Typhimurium,
that do not synthesize ECALPS (1).
Taken together, these observations indicate that WzzECA is
capable of modulating the chain length of ECAPG
polysaccharides. These observations also suggest that the data
presented in this study primarily reflect the regulation of
ECAPG. In order to demonstrate this, the pattern of
incorporation of [3H]GlcNAc into ECA polysaccharide
chains was examined in strain CS2334
(waaL::TnphoA). The transfer of ECA
chains from lipid-carrier to core-lipid A receptor requires the same
O-translocase (O-ligase) required for translocation of O antigen from
lipid-carrier to core-lipid A (14, 30, 31). This enzyme is
encoded by the waaL gene, and mutations in this gene that
preclude synthesis of a functional WaaL are unable to synthesize
ECALPS (31). Radiolabeled GlcNAc was efficiently
incorporated into ECA polysaccharide chains in strain CS2334 (Fig. 2D,
lane 2), and these chains were distributed in the same modal pattern as
were the ECA polysaccharide chains synthesized by wild-type cells (Fig.
2D, lane 1). These data support the conclusion that WzzECA
does indeed modulate the chain length of ECAPG
polysaccharide chains. Furthermore, the data also demonstrate that WaaL
is not required for the synthesis of ECAPG. In this regard,
it is not known whether the chain length of ECALPS-linked polysaccharide chains is modulated by WzzECA in a manner
similar to that observed for ECAPG polysaccharide chains,
and experimental approaches to this problem have thus far been hampered
by the inability to isolate mutants that are specifically defective in the synthesis of ECAPG. Similarly, water-soluble
ECACYC has been reported to contain four to six
trisaccharide repeat units; however, it is not known whether the number
of trisaccharide repeat units in this cyclic polymer is regulated in
some manner.
The available data support the conclusion that Wzz O-antigen
(WzzOAg) proteins are not O-antigen or species specific;
however, modal values appear to be WzzOAg-dependent
(3, 13). In contrast, the activity of WzzECA
appears to be restricted to the modulation of ECA polysaccharide chain
length since the presence of a wild-type wzzECA
gene in mutants that possess a null mutation in WzzOAg does
not complement the defect in O-antigen chain length regulation.
The biochemical mechanism involved in the modulation of polysaccharide
chain length is not known; however, two models have been proposed for
the assembly of Wzy-dependent O antigens (3, 25). Both of
these models suggest that the modulation of O-antigen chain length
involves a complex interaction between WzyOAg,
WzzOAg, WaaL, and lipid-carrier-linked O antigen; however,
they differ with regard to the relative importance of interactions
between WzyOAg and either WzzOAg or WaaL. The
results of experiments presented in this study demonstrate that the
WzzECA-mediated modulation of ECAPG
polysaccharide chain length is not dependent on WaaL. The lack of a
dependence on WaaL for the WzzECA-mediated modulation of
ECAPG polysaccharide chain length may indicate that this
regulation is primarily dependent on an interaction between
WzzECA and WzyECA. Alternatively, modulation of
ECAPG polysaccharide chain length may involve specific
interactions between WzzECA, WzyECA, and an as
yet unrecognized protein that is the functional counterpart of WaaL. In
this regard, the mechanism involved in the synthesis of the linkage
between ECA polysaccharide chains and the phospholipid aglycone of
ECAPG has not yet been determined. It seems likely that
this reaction involves either the transfer of polysaccharide or
polysaccharide-1-phosphate from
polysaccharide-pyrophosphorylundecaprenol to phosphatidic acid or
diacylglycerol, respectively. In either case, it is possible that the
enzyme that catalyzes this reaction functions in agreement with the
proposed role of WaaL in the regulation of O-antigen chain length as
suggested by Morona et al. (25).
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