Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess - PubMed (original) (raw)

Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess

Tzu-Lung Lin et al. J Bacteriol. 2008 Jan.

Abstract

Genomic heterogeneity has been shown to be associated with Klebsiella pneumoniae strains causing pyogenic liver abscesses (PLA) and metastatic infections. In order to explore the mechanism responsible for genomic heterogeneity in K. pneumoniae, we compared the complete genomic sequences of strains NTUH-K2044 and MGH78578. An approximately 76-kbp DNA fragment located adjacent to an asparagine (asn) tRNA gene was present in NTUH-K2044 but not in MGH78578. This fragment could be divided into three regions with different functions, and structurally it resembled a functional integrative and conjugative element (ICE), ICEEc1, in Escherichia coli. The 5' region of this fragment contained genes similar to a high-pathogenicity island (HPI) of Yersinia pestis and Yersinia pseudotuberculosis. The middle region was similar to part of a large plasmid in K. pneumoniae, and the 3' region contained genes responsible for DNA conjugative transfer. Therefore, this DNA fragment was designated ICEKp1. Precise excision and extrachromosomal circularization of ICEKp1 were detected in K. pneumoniae wild-type strain NTUH-K2044. ICEKp1 could integrate into the asn tRNA loci of the chromosome of another K. pneumoniae isolate. The prevalence of ICEKp1 was higher in PLA strains (38 of 42 strains) than in non-tissue-invasive strains (5 of 32 strains). Therefore, ICEKp1 may contribute to the transmission of the HPI and result in K. pneumoniae PLA infection-associated genomic heterogeneity.

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Figures

FIG. 1.

Seventy-six-kilobase pair insertion in K. pneumoniae strain NTUH-K2044. The dashed line indicates that that there was an insertion in K. pneumoniae strain NTUH-K2044 compared with strain MGH78578. The insertion was near the asparagine tRNA gene (the asn tRNA genes are indicated by gray arrows [asnV tRNA gene in E. coli ECOR31 and asn3 tRNA gene in K. pneumoniae NTUH-K2044 and MGH78578]). The cross-hatched box indicates the attachment site (attO) composed of a 17-bp direct repeat (DR). The black arrows indicate the int and fyuA genes, which were located in the 5′ and 3′ ends of the HPI. In contrast to the 34,480-bp fragment located adjacent to the HPI core in E. coli strain ECOR31, a 42,533-bp fragment was located in the right border of HPI in K. pneumoniae strain NTUH-K2044.

FIG. 2.

Genetic alignment of the middle and 3′ regions of ICE_Kp1_. The black triangle indicates the 17-bp direct repeat (DR) and the end of ICE_Kp1_. The large arrows indicate the locations and orientations of ORFs. The ORF numbers and the designations of ORFs are indicated below and above the arrows, respectively. The middle region exhibited similarity to part of large plasmid pLVPK in K. pneumoniae strain CG43. The 3′ region contained three functionally distinct segments (segments 3′-1, 3′-2, and 3′-3). The small arrows indicate the locations and orientations of primers used to study the prevalence of ICE_Kp1_.

FIG. 3.

Functional analysis of ICE_Kp1_. (A) Comparison of oriT sequences of ICE_Kp1_ and ICE_Ec1_. Asterisks indicate identical nucleotides in the oriT sequences of ICE_Kp1_ and ICE_Ec1_. The arrows above the sequences indicate the orientations and locations of two inverted repeats (IR1 and IR2) and one direct repeat (DR1). (B) DNA mobilization of ICE_Kp1_. The arrows in the diagram at the top indicate the simplified structure of ICE_Kp1_, which contains three regions. K. pneumoniae wild-type strain NTUH-K2044 and mutant strains harboring plasmid pACYC184-oriT were mated with E. coli strain HB101. A plus sign indicates that the pACYC184-oriT plasmid was mobilized to the recipient. A minus sign indicates that mobilization of plasmid pACYC184-oriT did not occur. DR, direct repeat.

FIG. 4.

Precise excision and extrachromosomal circularization of ICE_Kp1_. (A) Integration and excision model of ICE_Kp1_. The precise excision and extrachromosomal circularization of ICE_Kp1_ were mediated by recombination of attO sequences (indicated by a black box). Arrows 1, 1′, 2, 2′, 3, 3′, 4, and 4′ represent the primers outside the attL and attR sequences used to detect the precise excision and extrachromosomal circularization of ICE_Kp1_. (B) Extrachromosomal circular form of ICE_Kp1_ detected by nested PCR. PCR using primers 1 and 4 followed by nested PCR using primers 1′ and 4′ detected the attB site after excision (left gel). PCR using primers 2 and 3 followed by nested PCR using primers 2′ and 3′ detected the attP site in the extrachromosomal circular form (right gel). (C) Sequences of the chromosome and extrachromosomal junctions. Sequences of the chromosomal (1/4→1′/4′) and extrachromosomal (2/3→2′/3′) junctions were compared and aligned with the sequences of the left and right borders of ICE_Kp1_ and the sequences without an insertion in MGH78578. The 17-bp direct repeat is indicated by uppercase letters.

FIG. 5.

Self-transmission of ICE_Kp1_. The transfer of ICE_Kp1_ from the NTUH-K2044 rmpA mutant (donor) to K. pneumoniae strain N4252 or E. coli strain HB101 (recipient) in four randomly selected transconjugants was analyzed by PCR. Chromosomal genes (magA, kfu, and all) outside ICE_Kp1_ were used to differentiate the donor and the recipient. Genes (int, ybtU, iroN, virB1, and mobB) located in ICE_Kp1_ were used to examine the presence of ICE_Kp1_.

FIG. 6.

Integration of ICE_Kp1_. (A) (Top) Alignment of four asn tRNA genes of E. coli and K. pneumoniae strains NTUH-K2044 and MGH78578. The arrows indicate the locations and orientations of asn tRNA genes. An inversion between the asn_2 and asn_4 tRNA genes was found in strain MGH78578 (indicated by dashed lines). (Bottom) Alignment of sequences of the 17-bp direct repeat (DR) and asn tRNA genes of E. coli and K. pneumoniae. Asterisks indicate identical nucleotides in the E. coli and K. pneumoniae asn tRNA genes. (B) (Top) Integration of ICE_Kp1 adjacent to asn tRNA genes (asn1 tRNA, for example). The asn tRNA is indicated by gray arrows, and the cross-hatched box indicates the 17-bp direct repeat (DR). The small arrows indicate the orientations of primers. The asn tRNA gene was detected by primers flanking the asn1 tRNA gene (asn1-F and asn1-R). The left junction of the ICE_Kp1 insertion was detected by a primer flanking the asn1 tRNA gene combined with a primer in the left end of ICE_Kp1_ (asn1-F and ybtS-R inverse). The right junction of the ICE_Kp1_ insertion was detected by a primer flanking the asn1 tRNA gene combined with a primer in the right end of ICE_Kp1_ (asn1-R and 3′). (Bottom) PCR analysis of the integration site of ICE_Kp1_ in the four N4252 transconjugants. (C) Southern hybridization of EcoRV-digested DNA from various strains with asn tRNA gene (left gel) and int (right gel) probes. The arrows indicate the positions of asn tRNA gene fragments with or without an ICE_Kp1_ insertion.

FIG. 7.

Mobility of ICE_Kp1_ in strain NTUH-K2044. (A) PCR analysis of the location of ICE_Kp1_ in strains NTUH-K2044 and NTUH-K2044 ICE_Kp1_−. (B) Southern hybridization of EcoRV-digested DNA from strains NTUH-K2044 and NTUH-K2044 ICE_Kp1_− with asn tRNA gene (left gel) and int (right gel) probes. The arrows indicate the positions of asn tRNA gene fragments with or without ICE_Kp1_ insertion.

FIG. 8.

Comparison of sequences from the left and right parts of the middle region and the large plasmid. Asterisks indicate identical nucleotides in nucleotides 35579 to 35862 and 54053 to 54336 in the accession number AB298504 sequence and nucleotides 193069 to 193352 in the pLVPK plasmid sequence.

References

1. Abbott, S. 2003. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae, p. 684-700. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. American Society for Microbiology Press, Washington, DC.
1. Antonenka, U., C. Nolting, J. Heesemann, and A. Rakin. 2005. Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid. Mol. Microbiol. 57727-734. - PubMed
1. Antonenka, U., C. Nolting, J. Heesemann, and A. Rakin. 2006. Independent acquisition of site-specific recombination factors by asn tRNA gene-targeting genomic islands. Int. J. Med. Microbiol. 296341-352. - PubMed
1. Bach, S., A. de Almeida, and E. Carniel. 2000. The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae. FEMS Microbiol. Lett. 183289-294. - PubMed
1. Baumler, A. J., T. L. Norris, T. Lasco, W. Voight, R. Reissbrodt, W. Rabsch, and F. Heffron. 1998. IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica. J. Bacteriol. 1801446-1453. - PMC - PubMed

Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess - PubMed (original) (raw)