| Klebsiella pneumoniae is an important opportunistic pathogen that causes a substantial burden of nosocomial infections including respiratory,urinary, bloodstream and wound infections.An increasing proportion of nosocomial infections are caused by multidrug-resistant K.pneumoniae strains.K.pneumoniae strains that are particularly well adapted for human infection may harbor additional genomic islands that confer infection-related positive attributes.The HPI pathogenicity island that codes for the virulence-associated yersiniabactin siderophore has been identified in some strains of K.pneumoniae.Similarly,strains of K.pneumoniae associated with primary hepatic abscesses have been found to harbor the integrative conjugative element ICEKp1.The majority of bacterial species exhibit highly variable genomes comprising core conserved regions that are frequently interrupted by transposons,integrons, prophages or genomic islands(GIs).Chromosomal rearrangements,deletions and nucleotide polymorphisms add to the picture of genomic plasticity.Horizontally acquired islands potentially allow host strains to exploit entirely new niches through the acquisition of new virulence factors,metabolic pathways,antimicrobial resistance mechanisms or cell signaling systems.In many cases,GIs are flanked by specific DNA sequences such as direct repeats(DR) or insertion sequences and carry genes encoding integrases or transposases.In addition,islands typically exhibit distinct sequence features as compared to the core genome backbone,such as anomalous codon usage,discordant G+C content and dinucleotide bias.GIs can be obtained or lost by lateral gene transfer and those that exhibit site-specific integration frequently map to att sites within or directly adjacent to tRNA genes.The tRNAcc tool of MobilomeFINDER was used to identify genomic islands by comparative analysis of the contents and contexts of tRNA sites in two sequenced K. pneumoniae genomes,strain MGH78578 and strain Kp342.Eight out of 87 tRNA and tmRNA genes surveyed were found to harbor a putative island in one of the two sequenced genomes.(1) Genomic islands insertion in K.pneumoniae clinical isolates at 4 tRNA loci In this study a PCR-based screen was employed to investigate four tRNA genes (arg6,asn34,met56 and phe55 locus) in fifty one multidrug-resistant K.pneumoniae clinical isolates from sputum,urine,wound and bile.The vast majority of these predicted hotspots were confirmed to be occupied by putative alien islands,16 insertions in arg6 tRNA locus,9 insertions in asn34 tRNA locus,7 insertions in met56 tRNA locus and 51 insertions in phe55 tRNA locus.In our study,we found that the phe55 tRNA locus was the most hot spot for genomic island insertion in K.pneumoniae clinical isolates.There are 44 of 51 clinical isolates carrying a 3.7 kb insertion named KpGI-1,1 of 51 carrying a 6.4 kb insertion named KpGI-2 at phe55 tRNA locus.A 12.6 kb genomic island named KpGI-3 was inserted at the phe tRNA locus in K.pneumoniae MGH78578.In our study,the clinical isolates HS04053 carried an altered KpGI-3 island,which carried all the genes in KpGI-3 except the integrase gene and carried a large fragment not found in KpGI-3.It showed that this island might be from the KpGI-3 and got some changes during gene lateral transfer.(2) Novel genomic island KpGI-1 and KpGI-2 identification3.7 kb KpGI-1 and 6.4 kb KpGI-2 was sequenced and analysis by using blast at NCBI.The data showed that the KpGI-1 islet possessed a reduced GC content(46.7%) and KpGI-2 possessed a markedly abnormal GC content(38.03%) as compared to the genome average of K.pneumoniae strains MGH78578(57.5%).Four intact or partial 163 bp direct repeat sequences(DRs) were identified at the extremities of KpGI-1. Varying numbers of near identical repeat sequences were also found at one or both ends of the other two phe55-associated islands identified,KpGI-2 in HS04160 and KpGI-3 in MGH78578.The KpGI-1 orf2 coded for a predicted protein with a transposase8 family conserved domain(pfam01527) and KpGI-2 bored a 223-bp truncated phage integrase gene.All these showed that KpGI-1 and KpGI-2 was the novel genomic island in K.pneumoniae clinical isolates.KpGI-1 harbored four putative protein coding sequences.However,the best Blastp match found in GenBank exhibited only 35%identity.Except the orf2 harbored a transposase conserved domain,the putative product of orf3 contained an acetyltransferase domain(pfam00583).KpGI-2 bore five predicted protein coding sequences,orf4 possessed the DEXDc(DEAD-like helicases superfamily)(CDD accession no.cd00046) and HELICc(Helicase superfamily c-terminal domain) conserved domains(pfam00271)(Fig 1D).These domains contain the ATP-binding regions involved in ATP-dependent RNA or DNA unwinding,suggesting that orf4 may play a role in DNA replication or transcription,orf5 harobored a central conserved motif HPFXXGNG that is present in members of Fic protein family (pfam02661) that was shown to mediate(?)ilamentation(?)nduced by(?)AMP and that was proposed to play a role in regulation of cell division via folate metabolism.(3) KpGI-2 might originate from S.enteriaThe fic gene in E.coli K-12 was identified more than twenty years ago by Kawamukai and colleagues on the basis of a temperature-sensitive mutation that gave rise to bacterial filamentation following incubation at an elevated temperature in medium containing cAMP.In our study,we found that fic gene was highly conserved during E.coli strains,K.pneumoniae strains and S.enteria strains.But K. pneumoniae strains harbor two distinct fic homologues;kpn03747 and kpn03553 in hospital infection-associated strain MGH78578.In our study,we found that all the 51 K.pneumoniae clinical isolates harbored the gene kpn03747 whereas 47 of 51 K. pneumoniae clinical isolates harbored the gene kpn03553.HS04160 carrying island KpGI-2 was lack of the fic gene kpn03553.The ORF5KpGI-2 exhibited 97%identity to amino acid sequences of a Fic protein in the partially sequenced S.enterica Weltevreden HIN05-537.Analysis of the relevant S.enterica Weltevreden HIN05-537 contig revealed that it contained a 14.7 kb,fic-bearing,phenylalanine tRNA-associated genomic island,strongly substantiating the idea of a cross-genus acquisition of KpGI-2 and its fic gene.(4) Effect of orfs of KpGI-2 on cell growthKawamukai M et al had reported that a mutant form of the fic gene in E.coli K-12 was responsible for cAMP-induced inhibition of cell division and cell filamentation which led to cell growth inhibition.Given that ORF5 contained the Fic protein motif and exhibited high level similarity with a Fic protein of Salmonella Weltevreden,we analysed clones carrying the entire KpGI-2,orf2-3,orf4 and orf5 using an equivalent cell filamentation assay to that described by Kawamukai et al.We found that cAMP-specific cell filamentation was evident with all four clones and not just the two that harbored orf5 Furthermore,DH5αalso harbored the fic gene b3361 which resulted in cell filamentation suggesting that the effect may be non-specific to the cloned KpGI-2 genes.To further investigate the nature of these clones we performed cell viability experiments in media with or without cAMP supplementation.We found that cell growth was inhibited in all the strains after incubation in media containing 5mM cAMP compared with growth in media lacking this nucleoside.Interestingly,strains that harbored pUC4(orf4) or pUC5(orf5) exhibited more than a 10×relative increase in viable cell numbers at 6 hours after cAMP induction as compared to the strains that contained the intact KpGI-2 or pUC19 only.Cell viability kinetics for the KpGI-2 clone and the pUC19 vector control strain were very similar under both conditions of growth with a minor decline in viable cell numbers from t= 0 hours in cAMP-containing media.The strain that harbored pUC2-3(orf2-3) demonstrated the most dramatic cell viability phenotype,exhibiting a rapid,marked and time-dependent reduction in viable cell numbers on exposure to cAMP incubation.Intriguingly,the strain that contained the intact pUCKp appeared to exhibit a neutral cell viability phenotype comparable to that of the vector only control suggesting functional complementation between orf2-3(negative impact on cell viability) and orf4 and/or orf5(positive impact on cell viability). |
