Sequencing and analysis of the large virulence plasmid pLVPK of
Klebsiella pneumoniae CG43
Ying-Tsong Chen
a, Hwan-You Chang
b, Yi-Chyi Lai
b, Chao-Chi Pan
b, Shih-Feng Tsai
c,d,
Hwei-Ling Peng
a,*
aDepartment of Biological Science and Technology, National Chiao Tung University, 75, Po-Ai Street, Hsinchu, Taiwan, ROC bInstitute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, ROC
cInstitute of Genetics, National Yang-Ming University, Taipei, Taiwan, ROC
dDivision of Molecular and Genomic Medicine, National Health Research Institutes, Taipei, Taiwan, ROC
Received 18 February 2004; received in revised form 26 April 2004; accepted 6 May 2004
Available online
Received by J.A. Engler
Abstract
We have determined the entire DNA sequence of pLVPK, which is a 219-kb virulence plasmid harbored in a bacteremic isolate of Klebsiella pneumoniae. A total of 251 open reading frames (ORFs) were annotated, of which 37% have homologous genes of known function, 31% match the hypothetical genes in the GenBank database, and the remaining 32% are novel sequences. The obvious virulence-associated genes carried by the plasmid are the capsular polysaccharide synthesis regulator rmpA and its homolog rmpA2, and multiple iron-acquisition systems, including iucABCDiutA and iroBCDN siderophore gene clusters, Mesorhizobium loti fepBC ABC-type transporter, and Escherichia coli fecIRA, which encodes a Fur-dependent regulatory system for iron uptake. In addition, several gene clusters homologous with copper, silver, lead, and tellurite resistance genes of other bacteria were also identified. Identification of a replication origin consisting of a repA gene lying in between two sets of iterons suggests that the replication of pLVPK is iteron-controlled and the iterons are the binding sites for the repA to initiate replication and maintain copy number of the plasmid. Genes homologous with E. coli sopA/sopB and parA/parB with nearby direct DNA repeats were also identified indicating the presence of an F plasmid-like partitioning system. Finally, the presence of 13 insertion sequences located mostly at the boundaries of the aforementioned gene clusters suggests that pLVPK was derived from a sequential assembly of various horizontally acquired DNA fragments.
D 2004 Elsevier B.V. All rights reserved.
Keywords: rmpA; Siderophore; Heavy-metal resistance; Tellurite resistance; Iteron
1. Introduction
Klebsiella pneumoniae is an important cause of com-munity-acquired bacterial pneumonia, occurring particular-ly in chronic alcoholics and commonparticular-ly results in a high fatality rate if untreated. Nevertheless, the vast majority of K. pneumoniae infections are associated with hospitaliza-tion. It has been estimated that K. pneumoniae causes up to 8% of all nosocomial bacterial infections in developed countries, and its colonization in hospitalized patients
appears to be associated with the use of antibiotics (Scha-berg et al., 1991). Recently, the prevalence of multiple-drug-resistant K. pneumoniae strains has significantly re-stricted the availability of antibiotics for effective treatment of the bacterial infections.
Despite its significance, our knowledge of the pathoge-nicity of the bacterium is rather limited. Clinically isolated K. pneumoniae usually produces large amounts of capsular polysaccharides (CPS) as reflected by the formation of glistening mucoid colonies. The CPS provides the bacterium an anti-phagocytic ability and prevents the bacteria from being killed by serum bactericidal factors(Simmons-Smit et al., 1986). Additional virulence-associated factors identified so far in K. pneumoniae include lipopolysaccharides, several adhesins, and iron-acquisition systems(Simmons-Smit et al., 1986; Nassif and Sansonetti, 1986). The small numbers of
0378-1119/$ - see front matterD 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.05.008
Abbreviations: bp, base pair(s); IS, insertion sequence(s); kb, kilo-base(s); LB, Luria – Bertani (medium); ORF, open reading frame; ori, origin of DNA replication.
* Corresponding author. Tel.: +886-3-5742910; fax: +886-3-5742909. E-mail address: [email protected] (H.-L. Peng).
known virulence-associated factors rather limit the possible targets for drug development, thus making the intervention of bacterial infection rather difficult.
Several strategies including in vivo expression techno-logy, subtractive DNA hybridization, and signature-tagged mutagenesis have been adopted to identify virulence-asso-ciated genes in K. pneumoniae. These efforts have allowed the identification of many novel genes that might be important for the bacterium to infect humans. For instance, by using the in vivo expression technology, we have identified the presence of a plasmid-borne iron-acquisition gene cluster in K. pneumoniae that is primarily expressed in the hosts(Lai et al., 2001). Nevertheless, further investiga-tion of the funcinvestiga-tional roles of these novel sequences has been significantly hampered by the lack of the complete genome sequence of K. pneumoniae.
Most of the blood isolates of K. pneumoniae harbor a large plasmid of 200 kb in size (Peng et al., 1991). The plasmid has been demonstrated to contain the aerobactin siderophore biosynthesis genes and curing of the plasmid would result in an avirulent phenotype(Nassif and Sanso-netti, 1986). In our laboratory, we also found that the loss of pLVPK, a plasmid of the similar size harbored in K. pneumoniae CG43, a highly virulent clinical isolate of K2 serotype (Lai et al., 2003), resulted in a loss of colony mucoidy, the ability to synthesize aerobactin, and a 1000-fold decrease of virulence. It is conceivable that the plasmid is likely to carry many additional virulence-asso-ciated genes and complete sequencing of the plasmid would hence be the most straightforward way for their identification. We herein report the 219-kb sequence and annotation of this large virulence plasmid from K. pneumo-niae CG43.
2. Materials and methods 2.1. Sequencing of pLVPK
The DNA of pLVPK was isolated from K. pneumoniae CG43 by using a Qiagen Plasmid Purification kit and fragmented by sonication. The DNA fragments were then resolved on a 0.7% low melting point agarose gel and DNA of size ranging from 2.0 to 3.0 kb were recovered, blunt-repaired by Bal31 nuclease, and subsequently cloned into the pUC18 vector. A total of 2304 clones were sequenced from both ends to achieve approximately 11-fold coverage of the plasmid. Sequences were assembled initially using the Phred/Phrap program (Ewing et al., 1998) with optimized parameters and the quality score was set to >20. When all the sequences assembled into 11 major contigs (>20 reads; >2 kb), the Consed program(Gordon et al., 1998)was then used for the final sequence closure (autofinishing). Finally, several gaps among contigs were closed either by primer walking on selected clones, which were identified by analysis on the forward and the reverse links of each of
the contigs, or by sequencing the DNA amplicons generated by PCR.
2.2. Gene prediction and annotation
GLIMMER 2.02 (Delcher et al., 1999), a program that searches for protein coding regions, was used to identify those ORFs possessing more than 30 codons. Overlapping and closely clustered ORFs were manually inspected. The predicted polypeptide sequences were used to search the protein database with the BLAST (NCBI database), and the clusters of orthologous groups (COGs) of proteins database was used to identify families to which the predicted proteins were related. Mobile elements and repetitive sequences were identified using pairwise com-parison with the known insertion sequences. The presence of tRNA sequences was identified by the program tRNAs-can-SE (Lowe and Todd, 1997). The G + C nucleotide composition analysis was made by GCWin of the G-Language package (Arakawa et al., 2003).
2.3. Drug susceptibility assay
Tellurite, copper, silver, and lead susceptibility for the strains were determined essentially as described (Meno-haran et al., 2003). E. coli, K. pneumoniae CG43, and its derivatives were propagated at 37 jC in Luria – Bertani (LB) broth. The overnight-grown cells were spread onto LB plates and the 3MM paper discs (5-mm diameter) impreg-nated with aliquots of a serial dilution of K2TeO3, CuSO4,
AgNO3, and Pb(NO3)2solutions were placed on top of each
of the plates. The plates were then incubated at 37 jC for another 12 h and the inhibition zone was measured. Iron-acquisition activity was assayed using iron-deprived M9 plates (with 200 AM 2,2V-dipyridyl) and the paper discs were impregnated with a serial dilution of FeCl3 solution.
After spreading the overnight-grown bacteria onto the plates, the iron-loaded discs were then placed on top of each of the plates. The plates were incubated at 37 jC for 12 h, and the growth zones around the paper discs were measured.
2.4. Nucleotide sequence accession number
The nucleotide sequences reported in this paper have been submitted to GenBank under the accession no. AY378100.
3. Results and discussion 3.1. General overview
The entire DNA sequence consists of 219,385 bp forming a circular plasmid (Fig. 1). The size and the predicted restriction enzyme cutting sites are consistent with the
ex-Y.-T. Chen et al. / Gene 337 (2004) 189–198 190
perimental findings using pulse-field gel electrophoresis. The plasmid contains 251 ORFs, as determined by the Glimmer program. The possible functions of these ORFs were subse-quently analyzed by comparing the sequence to the current nonredundant 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 = f 55%). The G + C content plotted along the pLVPK sequence with a window size of 1000 bp is shown inFig. 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 Fig. 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.
3.2. 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 com-plements 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.,
Fig. 1. Map of the pLVPK plasmid. The circle map shows the major features of the plasmid including the replication origins (ori), partition regions (par, sop), major gene clusters, and IS elements (IS). The positions of some of the ORFs and gene clusters are depicted in box and their contents were labeled. Gene clusters, which are homologous unknown gene clusters reported from other species, are labeled with the species name respectively. The G + C% contents along the plasmid are shown in colors from low% (red) to high% (purple).
1989). The gene rmpA2, which was named because of its high similarity with rmpA, was identified later (Wacharo-tayankun et al., 1993). 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 et al. was a truncated form of rmpA2. Our sequencing result shows that rmpA and rmpA2, which share 81% nucleotide sequence homology (78% identity in the 194 comparable amino acids), are actually two independent loci 29 kb apart(Fig. 3a). Southern hybridization analysis of the plasmid using an rmpA2 probe also confirmed the presence of two copies of the gene(Fig. 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 mainte-nance of the plasmid stability. Interestingly, the G + C content of the vagCD genes ( f 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.
3.3. Iron-acquisition systems
The capability of iron acquisition is generally a pre-requisite 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 3V sequences of E. coli K12 tRNALysand 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 S. enter-ica, is known to participate in the uptake of catecholate-type
Fig. 2. Base composition of pLVPK. The G + C content along the sequence of pLVPK is calculated with a window of 1000 bp. The horizontal line indicates 50% G + C content, and the selected ORFs are shown as open boxes drawn to the exact scale. The region that contained the genes similar to that of the B. fungorum gene cluster is labeled. The iut and iro siderophore gene clusters, the lead-resistance gene clusters (pbr), and the region of rmpA and rmpA2 genes, respectively, are also indicated.
Y.-T. Chen et al. / Gene 337 (2004) 189–198 192
siderophores. Recently, similar gene cluster contained in a PAI was also found either on the chromosome or a trans-missible 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 side-rophore utilization activity(Sorsa et al., 2003).
A two-gene operon that encodes an ABC-type transporter related to M. loti FepBC was noted on pLVPK at nucleotide positions 77,450 – 80,256. The identity between the pLVPK genes and FepBC is 38% and 44%, respectively. These genes also share significant homology with many ABC transport-ers mediating translocation of iron, siderophores, and heme (Koster, 2001). Although the contribution of this putative ABC transporter in the uptake of iron remains unclear, it is undoubtedly advantageous for the bacteria to have multiple 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 are no observable fecABCDE homologs in pLVPK. This phenomenon is not that unusual. As shown in Fig. 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.
Fig. 3. Comparison maps of the rmpA and rmpA2 and their neighboring genes. (a) The restriction map of rmpA2 and rmpA. (b) Southern analysis of pLVPK (lane 2). Southern hybridization with a probe prepared from the PCR product of the 636-bp rmpA2 coding sequence is shown at the right. Two EcoRI fragments of 6.9 and 1.6 kb, respectively, representing the fragment containing rmpA and rmpA2 are detected. The 3.8- and 14.6-kb fragments in lane 2 represent the rmpA- and rmpA2-containing HindIII fragments.
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. 3.4. 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 Fig. 5 (152,306 – 177,234 bp), were identified in the pLVPK that
are related to metal resistance phenotype in K. pneumo-niae. These gene clusters include homologs of the lead-resistance genes pbrRSABC of Ralstonia metallidurans CH34 (Borremans et al., 2001), the copper-resistance genes pcoEABCDRS of E. coli plasmid pRJ1004 (Brown et al., 1995), and the silver-resistance gene cluster silC-BAPsilRSE 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, remains 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
Fig. 4. Comparison of the organization of fecIRA genes of pLVPK with similar iron transport and signaling systems. The arrows indicate the transcription orientation of the genes. The genes in the fecIRA gene cluster of the pLVPK are very similar to that of the E. coli fec operon, however, lacking the fecBCDE genes.
Fig. 5. The heavy-metal-resistant gene clusters. The silver-resistance sil gene cluster, in comparison with that of the S. enterica serovar Typhimurium pMG101
(Gupta et al., 1999), the copper-resistance pco gene cluster, in comparison with that of the E. coli pRJ1004(Brown et al., 1995), and the lead-resistance pbr gene cluster, in comparison with that of the R. metallidurans pMOL30, are shown(Borremans et al., 2001). The homologous gene clusters are depicted in solid black and the transcriptional orientation of the individual ORFs are shown by arrows.
Y.-T. Chen et al. / Gene 337 (2004) 189–198 194
protein, pbrC for a putative prolipoprotein signal pepti-dase, pbrD that confers lead sequestration, and pbrR that regulates the transcription of pbrABCD (Borremans et al., 2001). Unlike that of the R. metallidurans, the pbr gene clusters of pLVPK contain only the efflux system (pbrABC) and regulator encoding genes (pbrR) (Fig. 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 (19,890 – 34,588 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(Fig. 6a). A recent study suggests that the Ter -contain-ing pathogenicity island in enterohemorrhagic E. coli iso-lates was acquired from plasmid. With considerable degree of sequence homology (75 – 98% amino acid sequence similarity respectively with that of the E. coli O157 ter-ZABCDE), the ter genes of the pLVPK are likely horizon-tally 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 (Fig. 6b), suggesting that the tellurite resistance of the bacteria is determined by the ter gene cluster of pLVPK rather than the tehB homolog.
3.5. Replication and plasmid maintenance
DNA sequence analysis also revealed a single plasmid replication region of 1756 bp (217,448 – 219,203 bp), which consists of repA and sequence elements with char-acteristics of plasmid replicons that employ an iteron-based
Fig. 6. The tellurite resistance genes in K. pneumoniae CG43. (a) The ter gene cluster of pLVPK similar to terZABCDE of E. coli O157:H7 EDL933 O-island #43, a region of the EDL933 chromosome not homologous to E. coli K-12 MG1655. The homologous regions are shown in hatched and solid arrow. (b) Effects of the tehB deletion and curing of pLVPK on the tellurite resistance of the bacteria. Tellurite resistance assays of K. pneumoniae CG43S3, the pLVPK-cured bacteria (CG-101), and the tehB mutant CG43S3T1 using the discs immersed, respectively, with 20 and 40 Ag K2TeO3are shown. The arrows indicate the
replication initiation and control mechanism (Chattoraj, 2000). The repA product showed a high sequence similar-ity to a number of plasmid replication initiation proteins, including RepFIB of S. enterica serovar Typhi R27 plas-mid (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 Fig. 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, 4 21-bp and 13 42-bp direct repeats, located, respectively, at the upstream and downstream of the repA locus (Fig. 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).
3.6. Segregation control machineries
A region (203,493 – 203,994 bp) consisting of 11 copies of a 43-bp repeat (5V-gggaccacggtcccacctgcatcgtcgtttaggtttt-cagcct-3V) is believed to be required for segregation control of the plasmid. Next to the 43-bp direct repeat pattern, positioned are the genes encoding sopA and sopB homolo-gous. The organization is comparable to that of the sop operon, which governs the partition of the F plasmid(Yates et al., 1999). In addition to sopAB, genes showing sequence similarity with parAB of E. coli P1 phage were identified. It has been shown previously that the corresponding partition-ing site in the P1 parAB system is composed of direct or inverted repeats (Davis and Austin, 1988). We also noted
Fig. 7. The repA and the iteron sequences at the replication origin. (a) Sequence alignment of the repA from pLVPK (LVPK), R27 of S. enterica serovar Typhi (R27), pHMT-1 of Y. pestis (PMT1), and pHCM2 of S. enterica serovar Typhi (PHCM2). Leucine residues near the N-terminus are marked with asterisks, and the two-helix regions corresponding to the helix-turn-helix DNA-binding domain are shown. (b) The replication origin including the repA structural gene and two iteron sequences nearby of the pLVPK. The sequences of the two sets of iterons, iteron-1 (217,448 – 218,020 bp) and iteron-2 (219,102 – 219,203 bp), are shown with each row, respectively, representative for each of the adjacent repeat units.
Y.-T. Chen et al. / Gene 337 (2004) 189–198 196
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. Nev-ertheless, how these two partitioning systems contribute to the maintenance of pLVPK remains to be confirmed. 3.7. Heterogeneity
Pathogenic bacteria have obtained a significant propor-tion of their genetic diversity by acquisipropor-tion of DNA from other organisms. Many of the gene clusters identified in pLVPK are homologous to the unknown gene clusters in the other organisms. Although with unknown functions, the homologs of the gene clusters contained in the 9-kb region from nucleotide 2522 to 11,618 and the 5.9-kb region from nucleotide 13,997 to 19,886 were found, respectively, in the genome of B. fungorum and Y. pestis KIM. A gene cluster which encodes a putative ABC transporter system (117,432 – 113,670 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 (46,979 – 51,336 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 3V-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.
4. Conclusions
The 219-kb plasmid contains 251 ORFs, of which 37%
have homologous genes of known function, 31% match the hypothetical genes in GenBank, and the remaining 32% are novel sequences.
Virulence-associated genes identified include the CPS synthesis regulator gene rmpA2 and its homolog rmpA and multiple iron-acquisition system genes iucABC-DiutA, iroBCDN, fepBC, and fecIRA.
Gene clusters homologous with copper silver, lead, and
tellurite-resistant genes were identified. After curing of the plasmid, resistance of the bacteria to either lead or tellurite was found to decrease. However, no apparent effect was observed on the bacterial susceptibility to silver and copper.
Identification of a repA gene lying in between two sets of
iterons suggests that the replication of the plasmid is iteron-controlled. Furthermore, the presence of two regions similar to the sop and par genes indicates the presence of an F plasmid-like partitioning system.
Acknowledgements
This work was supported in part by National Science Council of the Republic of China (NSC92-2311-B009-001 to HLP and 92-2311-B009-001 to HYC). We are grateful to N. Venkatesan and J. Vatsyayan for critical reading of the manuscript.
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