Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates

General features of cps gene clusters of K. pneumoniae

Seven newly sequenced and five already sequenced cps of K. pneumoniae were analysed in this paper. The cps of K. pneumoniae NTUH-K2044, Chedid, MGH78578, NK8, NK29, NK245, VGH404, VGH484, VGH916, VGH698, A1142 and A1517 are named as cps_kpK1, cps_kpK2, cps_kpK52, cps_kpA, cps_kpB, cps_kpC, cps_kpK5, cps_kpK9, cps_kpK14, cps_kpK62, cps_kpK57 and cps_kpKpA1517, respectively. The genetic organization of the K. pneumoniae cps gene clusters are shown in Fig. 2(a)⇓. In general, the cps region of K. pneumoniae begins at galF and ends at ugd. The length of the K. pneumoniae cps gene clusters ranges from 21 to 30 kb (Table 2⇓). The number of ORFs found in the cps region of 12 K. pneumoniae cps gene clusters ranges from 16 to 25 (Table 2⇓). The transcriptional direction of all ORFs is from galF to ugd, except for kpc7 of cps_kpC (Fig. 2a⇓). The GC content of each ORF is also shown in Fig. 2(a)⇓. The GC content of K antigen gene clusters is lower than the mean GC content (57.7 %) of the K. pneumoniae NTUH-K2044 genome (Wu et al., 2009), especially in the region between wzc and gnd (Fig. 2a⇓).

All of the cps gene clusters in K. pneumoniae contain six conserved genes at the 5′ end of the cps region in the following order: galF, orf2, wzi, wza, wzb and wzc. Genes encoding glucose-6-phosphate dehydrogenase (gnd) and UDP-glucose dehydrogenase (ugd) were found downstream of all cps gene clusters except cps_kpK57 and cps_kpA1517, in which the ugd is still unknown (Fig. 2a⇑). Our data also reveal that most gnd and ugd are separated by manCB, rmlBADC or manCBrmlBADC genes (Fig. 2a⇑).

Sugar nucleotide synthesis genes in the cps clusters

Mannose, rhamnose and glucuronic acid are common components of capsular and O antigens. The genes encoding enzymes for the biosynthesis of sugar nucleotide precursors in the K. pneumoniae capsule are found in the cps gene cluster (Figs 2a⇑ and 3⇓). Products of manC and manB are involved in the synthesis of GDP-d-mannose. RmlA, RmlB, RmlC and RmlD proteins are responsible for the conversion of glucose 1-phosphate to dTDP-l-rhamnose (Fig. 3⇓). The genes involved in GDP-d-mannose and dTDP-l-rhamnose synthesis are arranged in the order manCB and rmlBADC, respectively. However, manCB are present in seven cps and rmlBADC are present in only three cps (Fig. 2a⇑, Table 2⇑). manCB and rmlBADC are absent in K. pneumoniae NK29 (cps_kpB), while K. pneumoniae VGH916 (cps_kpK14) contains manCB and rmlBADC genes (Fig. 2a⇑ and Table 2⇑). GDP-d-mannose is synthesized from fructose 6-phosphate by three enzymes, ManA, ManB and ManC (Fig. 3⇓). However, the manA gene is not located in the cps gene cluster of K. pneumoniae (Fig. 2a⇑).

The rhamnose pathway is widely distributed in bacteria (reviewed by Giraud & Naismith, 2000). dTDP-l-rhamnose, the precursor of rhamnose, is synthesized by glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-d-glucose-4,6-dehydratase (RmlB), dTDP-6-deoxy-d-glucose-3,5-epimerase (RmlC) and dTDP-6-deoxy-l-mannose-dehydrogenase (RmlD) from glucose 1-phosphate (Köplin et al., 1993) (Fig. 3⇑). These genes are clustered in the order rmlBADC in K. pneumoniae (Fig. 2a⇑). However, these genes are in the order rmlACBD in the Gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155 (Graninger et al., 2002). Since the enzymes for rhamnose synthesis are absent in humans, inhibitors for RmlA, RmlB, RmlC and RmlD activities have been screened for chemotherapy (Graninger et al., 2002; Ma et al., 2001; Giraud & Naismith, 2000). Other than these, galF and ugd genes were also found in the 5′ end and the 3′ end of K. pneumoniae cps gene clusters, respectively (Fig. 2a⇑). GalF protein modulates the activity of GalU to elevate the cellular concentration of UDP-glucose in E. coli and to increase the capsule production of E. coli K30 (Marolda & Valvano, 1996; Rahn & Whitfield, 2003). The ugd gene encodes UDP-glucose dehydrogenase, which converts UDP-glucose into UDP-glucuronic acid, the substrate for capsule synthesis.

Fucose is widely distributed in bacteria, plants and animals (Albermann et al., 2000). GDP-d-mannose 4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxy-d-mannose 3,5-epimerase/reductase (WcaG) are needed for the conversion of GDP-d-mannose to GDP-l-fucose, the precursor of fucose. The colanic acid of the capsule of both E. coli and K. pneumoniae K1 contains fucose. The gmd and wcaG genes are located in the gene clusters responsible for E. coli colanic acid (Stevenson et al., 1996; Andrianopoulos et al., 1998) and K. pneumoniae K1 capsule biosynthesis (Fig. 2a⇑) (Chuang et al., 2006). The enzyme homologous to WcaG is also found in humans (formerly called FX protein) (Tonetti et al., 1996). The WcaG involved in the K. pneumoniae K1 capsule synthesis shows 29 % identity to FX protein (accession number AAC50786) and 77 % identity to the corresponding gene product (accession number AAC77843) of the E. coli colanic acid biosynthesis gene cluster.

The protein encoded by glf found in K. pneumoniae VGH916 (K14) shows 42 % identity to Glf (accession number AAC98417) of K. pneumoniae serotype O1 and 63 % identity to the Glf (accession number AAB88403) of E. coli K-12. Glf catalyses the conversion of UDP-galactopyranose into UDP-galactofuranose (Nassau et al., 1996; Lee et al., 1996). The enzymic activity (Nassau et al., 1996; Lee et al., 1996) and crystal structure (Sanders et al., 2001) of Glf of E. coli have been characterized. The presence of a glf homology suggested that K. pneumoniae K14 capsule contains galactofuranose sugar. Owing to the absence of Glf in humans, Glf is useful in drug development (Lee et al., 1996).

Membrane and periplasmic proteins in the cps gene clusters of K. pneumoniae

Membrane proteins are important in the biosynthesis of E. coli group 1 and K. pneumoniae CPSs. Wzi, Wza, Wzc, Wzx, Wzy, WbaP and WcaJ are well-characterized membrane proteins (Stevenson et al., 1996; Whitfield & Roberts, 1999; Rahn & Whitfield, 2003; Beis et al., 2004; Collins et al., 2006; Dong et al., 2006; Whitfield, 2006). The gene organization and sequence of wzi, wza and wzc are conserved among E. coli group 1 and K. pneumoniae cps gene clusters (Fig. 2a⇑). On the other hand, the sequences of wzx and wzy in the cps gene clusters are more diverse. Therefore, the transposon containing the leaderless and promoterless β-lactamase gene was used to characterize the genes encoding Wzx, Wzy and novel membrane proteins of K. pneumoniae cps. The EZ-Tn5 <blaM/R6Kγori> transposon was used to identify the genes encoding membrane and periplasmic proteins in cps_kpA, cps_kpB, cps_kpK9 and cps_kpK62. Fosmids pCCKPA, pCCKPB, pCCK9CPS and pCCK62CPS containing the full-length cps gene cluster of cps_kpA, cps_kpB, cps_kpK9 and cps_kpK62, respectively, were tagged with the EZ-Tn5 <blaM/R6Kγori> transposon in vitro, transformed into E. coli Epi300, and then plated on LB plates with ampicillin. Genes encoding membrane or periplasmic proteins fused in-frame with β-lactamase allow E. coli to survive on plates with ampicillin. Ninety-six transformants of each fosmid were purified and sequenced. Sequencing analysis revealed that 79, 80, 78 and 80 transposon insertion sites were successfully identified for pCCKPA, pKPPB, pCCK9CPS and pCCK62CPS, respectively (Supplementary Table S2). By using blaM, we identified eight genes (wzi, wza, wzc, wbaP, wzy, orfZ, wzx and manC) of cps_kpA, six genes (wzi, wza, wzc, wbaP, kpb2 and gnd) of cps_kpB, six genes (wzi, wza, wzc, kp9D, wzx and rmlA) of cps_kpK9, and nine genes (orf2, wzi, wza, wzc, wbaP, kp62C, wzy, kp62F and kp62I) of cps_kpK62 that encoded membrane or periplasmic proteins (Supplementary Table S2). Wzi and Wza were identified more frequently than the other genes. WbaP is a membrane protein with four hydrophobic segments at the N terminal (Stevenson et al., 1996; Jiang et al., 2001; Whitfield, 2006). In cps_kpA, wzx and wzy were identified in four out of 79 transformants. wbaP was also identified in three out of four cps including cps_kpA, cps_kpB and cps_kpK62 at low frequency (Supplementary Table S2).

wzx and wzy genes of K. pneumoniae cps gene clusters

Synthesis of E. coli groups 1 and 4 and K. pneumoniae CPSs are Wzy-dependent (Whitfield, 2006). Wzx functions as a flippase and exports repeat-unit oligosaccharides to the periplasmic region. Wzy in turn polymerizes these repeating units (Whitfield & Roberts, 1999). It is known that the sequence similarity of wzx and wzy is very low (Jiang et al., 2001), and because of this low similarity, wzx and wzy genes should be serotype-specific. Therefore, serotyping by PCR targeting wzx and wzy for E. coli O antigen and Streptococcus pneumoniae K antigen has been developed (DebRoy et al., 2005; Kong et al., 2005). Due to the low-level of similarity of wzx and wzy genes among different gene clusters, sequence similarity and transmembrane helix profiles have been used in wzx and wzy identification (Jiang et al., 2001; Mazur et al., 2005; Nakhamchik et al., 2007; Cunneen & Reeves, 2008).

In this study, protein families of putative Wzx and Wzy in K. pneumoniae cps gene clusters were analysed by searching the Pfam database (Finn et al., 2006; Bateman et al., 2004). Additionally, the sequence similarity of Wzx and Wzy from K. pneumoniae cps was analysed by blastp and psi-blast (Altschul et al., 1997), while transmembrane helices were predicted using HMMTOP (Tusnády & Simon, 1998) and TMpred (Hofmann & Stoffel, 1993) (Table 3⇓). Proteins listed in Table 3⇓ were found to contain a number of transmembrane helices ranging from nine to 14 (predicted by HMMTOP). The Pfam protein family and homologue of each protein were determined by searching the Pfam and GenBank databases. Proteins that belong to the Pfam families Polysacc_synt (PF01943) and Wzy_C (PF04932), were named as Wzx and Wzy, respectively. Other Wzx and Wzy proteins with no significant match to a Pfam family were identified according to their homologues. We found that nine cps gene clusters that contained ORFs encoding Wzx belonged to the Polysacc_synt family (Table 3⇓). Although Wzx of cps_kpK1 did not belong to the Polysacc_synt family, it showed 20 % identity to O-antigen flippase of Francisella tularensis (YP_170390) (Chuang et al., 2006). Moreover, Wzx of cps_kpK9 and Wzy of cps_kpK62 were indeed membrane proteins characterized using <blaM/R6Kγori> transposon insertion (Supplementary Table S2). In addition, Kpb2 and Kp62I were identified using transposon insertion (Supplementary Table S2).

Wzx protein, a flippase, is essential for capsule biosynthesis and should be present in K. pneumoniae cps, but no Wzx-like protein was identified in cps_kpC and cps_kpK62 gene clusters. Kpc4 of cps_kpC and Kp62I of cps_kpK62 belonged to the MVIN (PF03023) protein family, and both MVIN and Polysacc_synt belonged to the MviN_MATE integral membrane protein superfamily. Since no other Wzx-like protein was identified in cps_kpC and cps_kpK62, Kpc4 and Kp62I are likely candidates for Wzx.

Synthesis of K. pneumoniae capsule is a Wzy-dependent process (Whitfield, 2006). Five out of 12 cps gene clusters contain ORFs encoding Wzy which belongs to structural motif Pfam04932 (Wzy_C), including magA of cps_kpK1.Wzy of cps_kpK2, cps_kpK52 and cps_kpA1517 have low similarity to Wzy of E. coli and Salmonella enterica (Table 3⇑). Due to the sequence diversity of Wzy, we did not identify Wzy in cps_kpB, cps_kpK5, cps_kpK9 and cps_kpK14. The number of transmembrane helices predicted by HMMTOP in Kpb2, Kp5A, Kp9D and Kp14E was similar to that of Wzy(s) identified in K. pneumoniae cps gene clusters (Table 3⇑). Our data suggest that the similarity of wzx and wzy is extremely low in K. pneumoniae cps; therefore, these two genes and GTs can be used for serotyping by PCR.

Transferase genes in K. pneumoniae capsule biosynthesis gene clusters

Both WbaP and WcaJ are first GTs that transfer sugar to the lipid acceptor and initiate the synthesis of the capsular repeat (Whitfield, 2006). Either the wbaP gene or the wcaJ gene existed in a given K. pneumoniae cps (Fig. 2a⇑, Table 1⇑). Unlike other GTs, WbaP and WcaJ are membrane proteins with four hydrophobic segments at the N terminal (Stevenson et al., 1996; Jiang et al., 2001; Whitfield, 2006). EZ-Tn5 <blaM/R6Kγori> transposon insertion analysis also suggests that the WbaPs are indeed membrane proteins (Supplementary Table S2). Moreover, according to transposon insertion analysis, none of the other putative GTs identified by searching the Pfam database is a membrane protein.

It has been shown that WbaP is the UDP-Gal : undecaprenolphosphate galactose-1-phosphate transferase of S. enterica B and E. coli K30 (Liu et al., 1993; Drummelsmith & Whitfield, 1999). The WbaPs of K. pneumoniae NK8, NK29, NK245, VGH484, VGH698, MGH78578, A1142 and A1517 share 98, 71, 74, 74, 72, 76, 77 and 74 % identity with WbaP of E. coli K30 (AAD21565), respectively. A previous study has suggested that WcaJ (AAC77848) functions as a UDP-glucose lipid carrier transferase in the biosynthesis of colanic acid in E. coli K-12 (Stevenson et al., 1996). The WcaJs of K. pneumoniae NTUH-K2044, VGH404, VGH916 and Chedid share 63, 61, 63 and 62 % identity with WcaJ of E. coli K-12 (AAC77848), respectively.

GTs of the K. pneumoniae cps gene clusters were identified by searching the Pfam protein families database (Finn et al., 2006; Bateman et al., 2004). Putative GTs determined by searching Pfam are listed in Table 4⇓. The results show that cps_kpK1 contains three, cps_kpA four, cps_kpB four, cps_kpC six, cps_kpK5 two, cps_kpK9 four, cps_kpK14 three, cps_kpK62 five, cps_kpK2 three, cps_kpK52 four, cps_kpK57 four and cps_kpA1517 four genes that encode GTs (Table 4⇓). Earlier studies indicate that the DXD motif is conserved in some GT families (Liu & Mushegian, 2003; Breton et al., 1998; Wiggins & Munro, 1998) and is essential for their enzymic activities (Li et al., 2001; Götting et al., 2004; Klutts et al., 2007; Persson et al., 2007). Amino acid sequence analysis of GTs in K. pneumoniae cps gene clusters revealed that most GTs identified by Pfam HMM contained the DXD motifs (Table 4⇓). All WbaPs in the cps gene clusters of K. pneumoniae were found to contain a conserved DXD motif in the C-terminal region. Moreover, the sequence of the DXD motif was DVD, except in WbaP of K. pneumoniae A1142, where it was DID (Table 4⇓). In contrast to WbaP, two out of four WcaJs identified in K. pneumoniae VGH404 and Chedid were found to contain a DVD motif (Table 4⇓).

Since the structure of the K. pneumoniae K1 capsule is →4)-[2,3-(S)-pyruvate]-β-d-GlcpA-(1→4)-α-l-Fucp-(1→3)-β-d-Glcp(1→ (Chuang et al., 2006), we expect that a pyruvyl transferase (Kp1A) and three GTs (Kp1B, WcaI and WcaJ) are involved in repeating-unit synthesis. We also found that ORF15 of cps_kpA1517 was a putative pyruvyl transferase which belonged to the Pfam PS_pyruv_trans family. In addition to GTs and pyruvyl transferase, acyltransferases were also identified in K. pneumonia cps. Kp1C of cps_kpK1, ORF13 of cps_kpK2, Kp5E of cps_kpK5 and OrfY of cps_kpA belonged to Pfam family Hexapep (PF00132). Some bacterial acetyl and acyltransferases contain Hexapep repeats, including DapD, GlmU and LacA (Williams & Raetz, 2007). In addition, sequence analysis indicated that Kp52I of cps_kpK52 and Kp62F of cps_kpK62 belonged to Pfam family Acyl_transf_3. The Kp52I of cps_kpK52 showed 34 % identity to acyltransferase 3 of Ralstonia metallidurans CH34 (ZP_00593471). ORF14 of cps_kpK57 showed 30 % identity to acyltransferase family protein of Pseudomonas fluorescens (YP_261319). However, acylation of Klebsiella K52 CPS was not noted in an earlier study (Stenutz et al., 1997). In contrast to K52, Klebsiella K5 CPS is acetylated at glucopyranose (Cescutti et al., 1995). In this study we were unable to clarify whether cps_kpK5 contains gene(s) responsible for Klebsiella K5 CPS acetylation. The relationship between genes and acylation in K. pneumoniae CPS biosynthesis still requires further investigation.

Lateral gene transfer and recombination

Both E. coli group 1 cps and the cps gene clusters in K. pneumoniae contain six (galF, orf2, wzi, wza, wzb and wzc) and two conserved genes (gnd and ugd) at the 5′ end and 3′ end, respectively. The average pairwise sequence identities of galF, orf2, wzi, wza, wzb, wzc, gnd and ugd genes were 97.7±2.0, 95.0±3.2, 87.0±3.7, 80.1±2.6, 65.5±4.3, 66.2±4.3, 92.9±3.2 and 97.3±0.6 %, respectively (Fig. 4⇓). Average pairwise sequence identities of galF, orf2, wzi, wza, wzb, wzc and gnd were results from 12 cps gene clusters of K. pneumoniae, whereas the value for ugd was the result from 10 cps gene clusters, not including cps_kpK57 and cps_kpKpA1517 of K. pneumoniae A1142 and A1517, respectively. The average pairwise sequence identities of the 5′-end conserved genes (galf, orf2, wzi, wza, wzb and wzc) gradually decreased toward the centre of the cps gene clusters (Fig. 4⇓). A similar phenomenon has also been observed in the Streptococcus pneumoniae CPS gene clusters (Jiang et al., 2001).

Sequence analysis showed that the pairwise sequence divergence of 12 wzi, wza, wzb and wzc genes in K. pneumoniae cps was significantly higher than that of the gnd gene. The gnd gene, located at the 3′ end of the cps, encoding an NADP-dependent 6-phosphogluconate dehydrogenase, is involved in the pentose phosphate pathway. 6-Phosphogluconate dehydrogenase converts 6-phosphogluconate into ribulose 5-phosphate and produces NADPH (Nelson & Selander, 1994). The pentose phosphate pathway provides NADPH and ribose 5-phosphate for reductive biosynthesis and nucleotide biosynthesis. The fact that the diversity of wzi, wza, wzb, wzc and gnd genes was higher than that of other housekeeping genes indicated that the diversity could be due to recombination (Nelson & Selander, 1994). Previously, the capsule of K. pneumoniae K20 has been shown to be identical to that of E. coli K30 (Laakso et al., 1988; Homonylo et al., 1988), and the capsule of E. coli K42 is serologically identical to that of Klebsiella K63 (Niemann et al., 1978). In the present study, sequence analysis indicated that the cps gene cluster of K. pneumoniae of NK8 is identical to that of E. coli K30 (accession number AF104912). This suggests that gene substitution has occurred in an existing cps gene cluster. These clues and the low GC content (Fig. 2a⇑) strongly suggest that the K. pneumoniae cps gene cluster was acquired from a low-GC organism via lateral gene transfer and recombination (Rahn et al., 1999).

K. pneumoniae cps gene expression

Transcriptional regulation of the cps gene cluster is important for capsule synthesis and pathogenicity of K. pneumoniae (Lai et al., 2003). RmpA2, RcsAB and RfaH modulate the differential expression of the cps gene clusters (Lai et al., 2003; Rahn & Whitfield, 2003). Loss of the conjugative plasmid pLVPK, which carries the rmpA2 gene, dramatically reduces the mucoidy and pathogenicity of K. pneumoniae CG43 (Lai et al., 2003). Previous studies suggest that cps_kpK2 includes three promoters (Arakawa et al., 1995; Lai et al., 2003), named as P1, P2 and P3 in this study (Fig. 2b⇑). Since the 12 K. pneumoniae cps have conserved genetic organization, the transcriptional organization and regulation mechanism would be similar to that of cps_kpK2. Promoters P1 and P2 are located upstream of galF and wzi, respectively (Fig. 2b⇑). The transcript driven by promoter P1 consisted of galF and orf2. The polycistronic mRNA driven by promoter P2 from wzi to gnd consisted of genes of capsule repeat unit synthesis and polymerization, as well as surface assembly (Fig. 2b⇑).

An earlier study indicates that a transcriptional terminator structure possibly exists just downstream of gnd (Arakawa et al., 1995), suggesting the existence of P3 downstream of gnd (Fig. 2b⇑). uge, encoding uridine diphosphate galacturonate 4-epimerase, is located downstream of ugd in the K. pneumoniae NTUH-K2044 and MGH78578 genomes. The transcriptional direction of ugd is opposite to that of uge, suggesting that transcription by P3 could terminate at a site downstream of ugd (Fig. 2b⇑). Since manCB and rmlBADC could be dispersed between gnd and ugd in K. pneumoniae cps, the possibility of another promoter located downstream of P3 cannot be ruled out.

RmpA2, an activator of capsule biosynthesis, can bind to P1 and P2 and activate gene expression (Lai et al., 2003). An RcsAB box (TAAGATTATTCTCA) was found immediately upstream of the galF gene in the K. pneumoniae NTUKH2044 (K1) and MGH78578 (K52) genomes. An RcsAB box was also identified in the K. pneumoniae Chedid (K2) (Arakawa et al., 1995) and E. coli K30 group 1 capsule gene clusters (Rahn & Whitfield, 2003). Meanwhile, rcsA (KP3552), rcsB (KP3872) and rcsC (KP3873) genes are present in the genome of K. pneumoniae NTUKH2044 (Wu et al., 2009). We speculate that both RmpA2 and RcsAB are required for high-level capsule synthesis of K. pneumoniae.

In E. coli K30 cps, a transcriptional terminator with a hairpin loop structure has been identified between wzc and wbaP. Transcription will be influenced by this terminator. RfaH, an antiterminator, is involved in distal genes expression (Rahn & Whitfield, 2003). The JUMPStart sequence was identified in all 12 K. pneumoniae cps immediately upstream of wzi. Moreover, rfaH (KP0198) is present in the genome of K. pneumoniae NTUKH2044 (Wu et al., 2009). Therefore, the JUMPStart-RfaH antitermination mechanism could modulate K. pneumoniae cps expression.

CPSs of K. pneumoniae

Much of the information about CPS synthesis in K. pneumoniae is modelled after the E. coli pathways. We have predicted gene functions of the available sequences of the K. pneumoniae cps clusters and correlated them with the CPS structures (Supplementary Table S3). The structural repeating unit of CPSs of E. coli K30 consists of →2)-α-d-Manp-(1→3)-β-d-Galp-(1→ chains carrying β-d-GlcpA-(1→3)-α-d-Galp-(1→ branches at position 3 of the mannoses (Chakraborty et al., 1980). The tetrasaccharide repeating unit from the K30 capsule of E. coli is synthesized on UndP by four GTs (WbaP, WbaZ, WcaO and WcaN) (Whitfield & Paiment, 2003). The repeating units containing mannose are synthesized by the products of manA, manB and manC, of which only manB and manC are present in cps_kpA. Although cps_kpK1 also contains manB and manC, mannose is not included in the structural unit of the K1 capsule (Chuang et al., 2006). However, it consists of fucose, which is synthesized from GDP-d-mannose by Gmd and WcaG. These findings may reinforce the correlation of genes and capsule components. Furthermore, colanic acid of E. coli contains two fucose units, and the gene cluster responsible for colanic acid synthesis indeed contains gmd and wcaG genes (Stevenson et al., 1996; Andrianopoulos et al., 1998).

Since the K1 capsule also contains pyruvate, synthesis of K. pneumoniae NTUH-K2044 capsule requires pyruvyl transferase. An earlier study shows that kp1A of cps_kpK1 encodes a putative pyruvyl transferase (Chuang et al., 2006). In addition to pyruvyl transferase, three more GTs found in cps_kpK1 (Fig. 2a⇑, Table 3⇑) may be engaged in the synthesis of K1 capsule repeating units by sequential addition of pyruvate and three sugars to UndP.

The structural unit of K2 polysaccharide of K. pneumoniae has been characterized as →3)-β-d-Glcp-(1→4)-β-d-Manp-(1→4)-α-d-Glcp-(1→ chains carrying α-d-GlcpA-(1→ branches at position 3 of the mannoses (Corsaro et al., 2005). The capsular hexasaccharide repeating unit from Klebsiella type 52 contains two rhamnoses (Stenutz et al., 1997). GDP-d-mannose and dTDP-l-rhamnose biosynthesis genes are present in cps_kpK2 and cps_kpK52, respectively (Fig. 2a⇑). The presence of manCB and rmlBADC could reflect the existence of mannose and rhamnose in the capsule of K2 and K52. Moreover, the structural unit of K5 polysaccharide of K. pneumoniae has been characterized as →4)-β-d-GlcpA-(1→4)-β-d-Glcp-(1→3)-β-d-Manp-(1→ with a pyruvic acetal group acetylated at the C-2 position of the glucopyranose (Cescutti et al., 1995). The GDP-d-mannose biosynthesis gene was found in cps_kpK5 (Fig. 2a⇑). manCB and GDP-l-fucose biosynthesis genes (gmd and wcaG) are both found in cps_kpK1; however, only fucose is incorporated into the repeat unit of the K1 capsule. In contrast to other cps gene clusters of K. pneumoniae, except cps_kpK57 and cps_kpA1517, cps_kpB does not contain mannose and rhamnose sugar nucleotide biosynthesis genes. Therefore, the capsule of K. pneumoniae NK29 might not contain mannose and rhamnose.

The repeating units of capsules usually include uronic acids (Corsaro et al., 2005), and glucuronic acid is found in the repeating unit of K1, K2, K5 and K52 isolated from K. pneumoniae (Cescutti et al., 1995; Stenutz et al., 1997; Corsaro et al., 2005; Chuang et al., 2006). The presence of ugd could explain the prevalence of glucuronic acids in the capsules. Moreover, Wzc could increase Ugd activity by phosphorylation (Grangeasse et al., 2003), and phosphorylated Ugd could produce UDP-glucuronic acids from UDP-glucose (Fig. 4⇑). However, galacturonic acid is present in Klebsiella K57 CPS instead of glucuronic acid (Pan et al., 2008).

Conclusions

Serotyping has been widely used for the epidemiological study of K. pneumoniae. However, the procedure of serotyping is laborious and time-consuming. Recently, methods for the molecular typing of Klebsiella species have been developed (Brisse et al., 2004; Fang et al., 2007; Pan et al., 2008) to supplant the existing serotyping method. In principle, molecular typing of K. pneumoniae is easier to perform than conventional serotyping. Nevertheless, successful typing requires a panel of probes that can easily detect all common strain types. Our study provides seven new cps gene clusters of K. pneumoniae strains that will facilitate the development of PCR-based identification methods.