In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of
Proteus mirabilis. Since
P.
mirabilis and
Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common
P.
mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of
K.
pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined
K.
pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an
N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called
waa gene cluster, although one common core biosynthetic gene (
wabO) was found outside this cluster.Gram-negative motile and frequently swarming bacteria of the genus
Proteus and the family
Enterobacteriaceae are opportunistic human pathogens (
33). Currently, the genus consists of five species (
Proteus mirabilis,
P.
penneri,
P.
vulgaris,
P.
myxofaciens, and
P.
hauseri) and three genomospecies (4, 5, and 6) (
33,
35).
P.
mirabilis is a common uropathogen that causes urinary tract infections especially in individuals with functional or anatomical abnormalities of the urinary tract (
52) and elderly persons undergoing long-term catheterization (
53) but less frequently in normal hosts (
43). Potentially serious complications arising from
P.
mirabilis infections include bladder and kidney stone formation, catheter obstruction due to the formation of encrusting biofilms, and bacteremia (reviewed in reference
2). This bacterium is found more frequently than
Escherichia coli in kidney infections (
14) and may be associated with rheumatoid arthritis (
38). Studies aimed at the identification of
P.
mirabilis virulence factors showed that flagella and fimbriae (MR/P and PMF) are required for entry into and colonization of the bladder, respectively (reviewed in reference
12). Other important virulence factors are urease, hemolysin, and iron acquisition (
12). More recently, an extracellular metalloprotease (
37) and several putative DNA binding regulatory, cell-envelope related, and plasmid-encoded proteins have been identified by signature-tagged mutagenesis (
8,
21).The lipopolysaccharide (LPS), as in other members of the family
Enterobacteriaceae, consists of three domains, an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O-antigen, and an intervening core oligosaccharide (OS) region. The O-antigen is the major surface antigen, and its serological O specificity, in contrast to that of other Gram-negative bacteria (
31), is defined by the structure of the O-PS chain and that of the core OS (
51). On the basis of immunospecificity, 60 O serogroups (
28,
36) have been recognized in
P.
mirabilis and
P.
vulgaris, and several new
Proteus O serogroups have been proposed for
P.
penneri (
27,
55). The LPS is a potential
Proteus virulence factor (
42), and recently two mutants deficient in a glycosyltransferase and with attenuated virulence have been isolated and it has been speculated that this glycosyltransferase could be involved in LPS biosynthesis (
21). LPS plays a significant role in the resistance of
P.
mirabilis to antimicrobial peptides (
32), and LPS charge alterations may influence the swarming motility of the bacterium (
3,
32). In addition, the core LPS is a charged OS which plays an important role in the biological activities of the LPS and the function of the bacterial outer membrane (
10). In
Proteus, the core OS structures of up to 34 strains of different O serogroups have been determined (
51). These structures revealed that
Proteus core OSs share a heptasaccharide fragment that includes a 3-deoxy-α-
d-
manno-oct-2-ulosonic acid (Kdo) disaccharide, an
l-
glycero-α-
d-
manno-heptose (
l,d-Hep) trisaccharide, and one residue each of
d-glucose (
d-Glc),
d-galacturonic acid (
d-GalA), and either
d-glucosamine (
d-GlcN) or
d-galactosamine (
d-GalN) (
51). This common fragment is also found in the core LPSs of
Klebsiella pneumoniae and
Serratia marcescens (
11,
41,
50). The rest of the
Proteus core OS is quite variable, and it is possible to recognize up to 37 and 11 different structures in the genus and
P.
mirabilis, respectively (
51). Some
P.
mirabilis core OS structures are characterized by the presence of unusual residues, such as, for instance, quinovosamine; an open-chain form of
N-acetylgalactosamine (GalNAc); or unusual amino acids (
51). In contrast, little is known about the genes encoding enzymes involved in core LPS biosynthesis in
P.
mirabilis, which makes detailed genetic analysis of the role of LPS in
P.
mirabilis pathogenesis difficult. Thus, we decided to identify these genes by using
P.
mirabilis strains R110 and 51/57, the whole structures of whose core LPSs are known (Fig. ).
Open in a separate windowChemical structures of the core LPSs of
P.
mirabilis strains R110 and 51/57 (
51),
K.
pneumoniae types 1 (
50) and 2 (
41), and
S.
marcescens N28b (
11).
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