Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43
1.3 Results and Discussions
General overview
The entire DNA sequence consists of 219,385 bp forming a circular plasmid (Figure 1). The size and the predicted restriction enzyme cutting sites are consistent with the experimental findings using pulse-field gel electrophoresis. The plasmid contains 251 ORFs, as determined by the Glimmer program (Appendix I). The possible functions of these ORFs were subsequently analyzed by comparing the sequence to the current non-redundant protein database of the National Center for Biotechnology Information using BLAST software through the Internet.
Approximately 37% of the 251 ORFs have significant amino acid sequence similarity (>60%) with the genes of known function in GenBank or with protein domains or motifs in protein databases. Despite their lack of homology to the known genes, the deduced amino acid sequences of 31% of the ORFs matched the hypothetical genes in the database. The remaining 32% had lower or no significant sequence similarities (<20%) with those in the database and their functions could not be assigned.
The average G+C content of the plasmid is 50.35%, which is somewhat lower than that of the K. pneumoniae MGH78578 genome (G+C = ~55%). The G+C content plotted along the pLVPK sequence with a window size of 1000 bp is shown in Figure 2. Four regions (Box 1~4) with a significant high G+C content in comparison with the average of the whole plasmid sequence were identified. The Box 1 consists of 9 ORFs showing 56~90% sequence similarity to an unknown gene cluster in Burkholderia fungorum genome. The second and third high G+C regions contain two iron
acquisition systems: iut and iro genes, respectively. The fourth box covered the lead-resistant pbr gene cluster and its nearby transposase gene. Two low G+C content regions are also marked in Figure 2, which include the two mucoidy regulator encoding genes, rmpA (34.6%) and rmpA2 (31.9%). The values of G+C at the third codon are even lower with 29.2% for rmpA and 28% for rmpA2.
Virulence-associated genes
The BLAST search revealed an 18-kb region, which is highly similar to the SHI-2 pathogenicity island (PAI) of Shigella flexneri (Moss et al., 1999). The SHI-2 like region includes the iron acquisition genes iucABCDiutA, vagCD, the unknown function ORF shiF, and rmpA2, a known virulence-associated gene in K. pneumoniae (Lai et al., 2003). Elsewhere the PAI-like region, a rmpA2 homolog, rmpA, and two additional gene clusters associated with iron metabolism were also found.
One interesting finding in pLVPK is the presence of rmpA and rmpA2, two genes encoding regulatory proteins for CPS synthesis in K. pneumoniae. CPS has been known to be a major virulence factor in K pneumoniae that protects the bacterium from the bactericidal activity of serum complements and macrophages (Simmons-Smit et al., 1986). The gene rmpA was first identified in K. pneumoniae as a determinant controlling the CPS biosynthesis (Nassif et al., 1989). The gene rmpA2, which was named because of its high similarity with rmpA, was identified later (Wacharotayankun et al., 1996). Since the major difference between these two gene products is that the RmpA2 has an extended N-terminal region, it has been generally thought that rmpA and rmpA2 are the same gene, and the rmpA reported earlier by Nassif and colleagues was a truncated form of rmpA2. Our sequencing result shows
in the 194 comparable amino acids), are actually two independent loci 29 kb apart (Figure 3a). Southern hybridization analysis of the plasmid using an rmpA2 probe also confirmed the presence of two copies of the gene (Figure 3b). The finding not only clarified that rmpA is not a part of rmpA2, but also demonstrated that both the genes are plasmid-borne. Our laboratory has recently found that RmpA2 protein directly interacts with the promoters of the K2 CPS biosynthesis genes through its carboxyl terminal helix-turn-helix motif-containing portion (Lai et al., 2003). Thus, we believe that RmpA could also interact with the cps gene promoter, although how it activates the cps gene expression and the interplay between these two Rmp proteins remain to be investigated.
The K. pneumoniae vagCD products exhibit 94% and 84% amino acid sequence identities with that of the VagC and VagD on pR64 of Salmonella enterica serovar Dublin. Like the vagCD of pR64, the two genes are also overlapped by one nucleotide. It has been proposed that VagC and VagD might be involved in the coordination of plasmid replication and cell division and disruption of the vagC locus would reduce the bacterial virulence (Pullinger and Lax, 1992). The high sequence similarity suggests that vagCD genes on the pLVPK also participate in the maintenance of the plasmid stability. Interestingly, the G+C content of the vagCD genes (~70%) is significantly higher than that of the rmpA2 (31.9%), which is located only 1.1 kb away, implying that rmpA2 and vagCD were recruited onto pLVPK independently.
Iron acquisition systems
The capability of iron acquisition is generally a prerequisite for a pathogen to establish infection when entering the hosts. In pLVPK, two siderophore-mediated iron
acquisition systems, iucABCDiutA and iroBCDN, were identified. The iucABCDiutA operon, which was first reported on pCoIV-K30 in E. coli (Ambrozic et al., 1998), consists of five genes responsible for synthesis and transport of the hydroxymate siderophore aerobactin. The presence of the aerobactin synthesis and utilization genes has also been reported for Salmonella, and Shigella spp., indicating that the genes are freely transferable within the Enterobacteriaceae. This notion is also consistent with the finding that the iucABCDiutA gene cluster is flanked by two transposable elements, IS630 and IS3, and 3’ sequences of E. coli K12 tRNALys and tRNATrp, which have been proposed to play a role in the horizontal transfer of PAIs between bacterial pathogens (Hou, 1999).
The iroBCDEN gene cluster, first described in Salmonella enterica, is known to participate in the uptake of catecholate-type siderophores. Recently, similar gene cluster contained in a PAI was also found either on the chromosome or a transmissible plasmid in the uropathogenic E. coli (Sorsa et al., 2003). It should be mentioned that the iro gene cluster in pLVPK lacks iroE gene. Nevertheless, the absence of iroE gene probably would not affect the utilization of catecholate siderophore by the bacterium since it has been demonstrated in E. coli that an iroE mutation does not hinder the siderophore utilization activity (Sorsa et al., 2003).
A two-gene operon that encodes a ABC-type transporter related to Mesorhizobium loti FepBC was noted on pLVPK at nucleotide positions 77450..80256. The identity between the pLVPK genes and FepBC is 38% and 44%, respectively. These genes also share significant homology with many ABC transporters mediating translocation of iron, siderophores, and heme (Koster, 2001).
Although the contribution of this putative ABC transporter in the uptake of iron
acquisition systems in order to obtain iron from the frequently changing environment.
Finally, a gene cluster similar to E. coli fecIRA, which is responsible for regulating the uptake of ferric citrate in a Fe2+-Fur dependent manner was identified approximately 3 kb upstream of the iroBCDN. In E. coli, fecIR genes are within a large gene cluster with fecABCDE that are the structural genes for iron citrate uptake and are thought to be the target of FecIR regulatory system (Braun et al., 2003).
However, there is no observable fecABCDE homologs in pLVPK. This phenomenon is not that unusual. As shown in Figure 4, the homologs of fecIRA, but not fecBCDE, have been identified experimentally in Bordetella spp. as well as in several other bacterial species. It is not clear what the target genes are for these FecIRA-like regulatory systems in these bacteria (Braun et al., 2003). One possibility is that a fecABCDE gene cluster could be located on K. pneumoniae chromosome.
Alternatively, the FepBC-like ABC-type iron transporter encoding genes on pLVPK could be the target gene of the FecIRA regulators.
It should be pointed out here that the pLVPK fecR open reading frame is disrupted by an in-frame termination codon. FecR is an inner membrane protein that senses whether FecA, the outer membrane ferric citrate receptor, is bound to the substrate, and in response activates FecI, which is known as a transcription factor.
Deletion analysis of the fecR in E. coli has shown that a minimum of 59 amino acids in length of the FecR N-terminal derivative is still able to activate the FecI and subsequently a constitutive expression of the downstream target genes (Ochs et al., 1995). Thus, despite the presence of an internal stop codon, the fecR of pLVPK may still be capable of encoding a truncated but functional product and may result in a constitutive iron acquisition phenotype in K. pneumoniae CG43.
The hydroxamate-bioassay with the aerobactin indicator strain E. coli LG1522
showed that the plasmid-cured strain, CG43-101, loses the aerobactin activity in comparison with its parental strain CG43. In addition, the iron acquisition activity assay revealed that CG43-101 apparently has a smaller growth zone around the iron-loaded disc. These results indicated that the iron-acquisition capability of the bacteria could mostly be attributed to the plasmid pLVPK.
Genes related to metal resistance
Heavy metals at certain concentrations in the cell may form unspecific complex compounds leading to a toxic effect. Many genes for the maintenance of the heavy metal ion homeostasis have been identified in bacteria. Three physically linked gene clusters, as shown in Figure 5 (152306..177234 bp), were identified in the pLVPK that are related to metal resistance phenotype in K. pneumoniae. These gene clusters include homologs of the lead-resistance genes pbrRSABC of Ralstonia metallidurans CH34 (Borrenmans et al., 2001), the copper-resistance genes pcoEABCDRS of E. coli plasmid pRJ1004 (Brown et al., 1995), and the silver-resistance gene cluster silCBAPsilRSE of S. enterica serovar Typhimurium (Gupta et al., 1999). By using disk diffusion assay, we have found that the resistance against silver and copper ions between K. pneumoniae CG43 and a plasmid-cured strain, CG43-101 remain the same.
A putative lead resistance gene cluster, pbrRABC, showed a 63~71% deduced amino acid sequence identity with that of the R. metallidurans pbrTRABCD genes.
The R. metallidurans lead resistance operon, carried on a large plasmid, pMOL30, contains pbrT for Pb2+ uptake; pbrA, for Pb2+ efflux; pbrB for a putative integral membrane protein; pbrC for a putative prolipoprotein signal peptidase; pbrD that
(Borremans et al., 2001). Unlike that of the R. metallidurans, the pbr gene clusters of pLVPK contains only the efflux system (pbrABC) and regulator encoding genes (pbrR) (Figure 5), which suggest a simple lead-efflux mechanism similar to that of the CadA ATPase of Staphylococcus aureus and the ZntA ATPase of E. coli (Rensing et al., 1998). In contrast to the indifference of copper and silver ion resistance, the lead susceptibility increased in the disk diffusion assay after curing of the plasmid. The pbr genes in the pLVPK may contribute to the adaptation of K. pneumoniae in lead polluted human inhabitants.
A gene cluster encoding E. coli terZABCDE homolog was also identified. The terZABCDE has been shown previously to be a part of a PAI, which also contains integrase, prophage, and urease genes in E. coli EDL933 (Taylor et al., 2002). This gene cluster also provides the resistance to bacteriophage infection as well as resistance to pore-forming colicins. Although terBCDE are sufficient for the tellurite resistance property, the functions of each of these genes are unknown. The 14.7 kb region (19890..34588 bp) containing terZABCDE genes and 12 putative ORFs of pLVPK are comparable to the ter genes-containing region in the E. coli O157 genome.
The homology is interrupted downstream of the terZABCDE region by an E. coli pTE53 tellurite resistance terF homolog and IS903 gene (Figure 6a). A recent study suggests that the Ter-containing pathogenicity island in enterohemorrhagic E. coli isolates was acquired from plasmid. With considerable degree of sequence homology (75~98% amino acid sequence similarity respectively with that of the E. coli O157 terZABCDE), the ter genes of the pLVPK are likely horizontally acquired. It has been speculated that the ter system most likely plays other functional roles such as protection against host defenses so as to be stably maintained in the bacterium (Taylor et al., 2002).
A chromosomally located ORF which showed 77% amino acid sequence identity with the E. coli tellurite resistant gene tehB (Taylor et al., 2002) has also been recently isolated in our laboratory from K. pneumoniae CG43. Deletion of the tehB-like gene had no apparent effect on tellurite resistance of the bacteria (Figure 6b) suggesting that the tellurite resistance of the bacteria is determined by the ter gene cluster of pLVPK rather than the tehB homolog.
Replication and plasmid maintenance
DNA sequence analysis also revealed a single plasmid replication region of 1,756 bp (217448..219203 bp), which consists of repA and sequence elements with characteristics of plasmid replicons that employ an iteron-based replication initiation and control mechanism (Chattoraj, 2000). The repA product showed a high sequence similarity to a number of plasmid replication initiation proteins, including RepFIB of Salmonella enterica serovar Typhi R27 plasmid (60% identity), RepFIB of E. coli O103:H2 (43% identity), RepA of Yersinia pestis KIM plasmid pMT-1 (42% identity), and RepA of S. enterica serovar Typhi plasmid pHCM2 (42% identity). As shown in the multiple sequence alignment in Figure 7a, RepA appears to be an initiator for plasmid replication, which is able to bind the flanking repeated sequences through its DNA binding structures, a winged-helix domain and a leucine-zipper motif (Chattoraj, 2000). We have also found two sets of iterons, four 21 bp and thirteen 42 bp direct repeats, located respectively at the upstream and downstream of the repA locus (Figure 7b). The sequences are most likely the specific binding sites for the RepA protein to initiate replication of the plasmid and also control the plasmid copy number (Chattoraj, 2000).
A region (203493..203994 bp) consisting of 11 copies of a 43-bp repeat (5’-gggaccacggtcccacctgcatcgtcgtttaggttttcagcct-3’), is believed to be required for segregation control of the plasmid. Next to the 43-bp direct repeat pattern are inverted repeats (Davis and Austin, 1988). We also noted that a 66-bp direct repeat upstream of the parAB homologs is found, which indicates that they also contribute to the partitioning control of the pLVPK. It is reasonable that such a large plasmid has meticulous maintenance systems. Nevertheless, how these two partitioning systems contribute to the maintenance of pLVPK remaine to be confirmed.
Heterogeneity
Pathogenic bacteria have obtained a significant proportion of their genetic diversity by acquisition of DNA from other organisms. Many of the gene clusters putative ABC transporter system (117432..113670 bp) is also identified for which the deduced amino acid sequences are similar to those of the putative ABC transporter system of Streptomyces coelicolor A3. A region (46979..51336 bp) comparable to the
phage infection inhibition pif region of E. coli F plasmid was also identified. The boundary sequences of these gene clusters, as well as that of the PAI-like region, are mobile elements including insertion sequences and short pieces of 3’-sequences of tRNA genes. With the involvement of the transposons and the tRNA sequences, horizontal gene transfers have made possible these gene clusters to be introduced into the plasmid and hence affect the ecological and pathological characteristics of bacteria.