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The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands

The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands Volume 1 † Number 1 † March 2008 10.1093/biohorizons/hzn006 ......................................................................................................................................................................................................................................... Research article The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands Jon Jurriaan van Aartsen* Department of Infection Immunity and Inflammation, University of Leicester, Leicester, UK. * Corresponding author: Lab 212, Department of Infection Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, University Rd, Leicester LE1 9HN. Tel: þ44 (0)116 2523056. Email: jjv1@le.ac.uk Supervisor: Kumar Rajakumar, Department of Infection Immunity and Inflammation, University of Leicester, Leicester, UK. ........................................................................................................................................................................................................................................ Klebsiella sp. cause a wide range of human infections, particularly nosocomial septicaemia, pneumonia and urinary tract infections. Like other Enterobacteriaceae, Klebsiella are likely to possess plastic genomes comprised of core regions interspersed with horizontally acquired genomic islands. As phenylalanine tRNA genes are known to be occupied by islands in other Enterobacteriaceae, we utilized PCR-based screening and chromosome walking techniques to examine the pheV locus in Klebsiella isolates from blood stream and urinary tract infections. We hypothesized that this gene was an integration hotspot that served as a repository for novel genetic material in Klebsiella. The pheV site in Klebsiella KR116 and KR164 harboured an islet encoding four genes, two with similarity to genes within an island downstream of pheR in Salmonella enterica serovar Typhi CT18. In KR173 the locus contained a larger, potentially intact version of this island and harboured an integrase gene similar to that in the S. Typhi CT18 island. However, the Klebsiella and Salmonella islands were clearly distinguishable by strain-specific segments and organizational variation. On the basis of available sequence and restriction fragment length polymorphism data, three other Klebsiella isolates were found to possess an entirely distinct entity that resembled a 12.6 kb pheV associated island in K. pneumoniae MGH78578. This island was predicted to encode a P pilus-like structure, a probable virulence factor on the basis of parallels with E. coli. A unique and intriguing feature of Klebsiella pheV loci was the presence of multiple tandem repeats of up to 163 bp immediately downstream of pheV and a truncated copy at the opposite end of the islands. The tRNA proximal repeats were variable in number and size between isolates, while the solitary downstream repeats varied in length. These elements may represent genetic debris of previous recombination events. In conclusion, the pheV locus of Klebsiella exhibited consider- able variability between strains and harboured at least two distinct island types that could play important roles in adaptation and/or virulence. Functional characterization of this genetic armory will help unravel basic microbial and pathogenesis processes and may in time lead to improvements in the diagnosis, prevention and treatment of Klebsiella infections. Key words: Klebsiella, phenylalanine, tRNA, genomic island, pathogenicity island, genome plasticity. ........................................................................................................................................................................................................................................ highly variable complement of strain-specific genes. Introduction Genome variability plays an important role in the evolution The Klebsiella genus contains a diverse group of commensal and adaptive ability of bacteria, allowing for loss and/or and pathogenic species. K. pneumoniae and K. oxytoca are acquisition of functions via mutational changes or horizontal 4, 5 the most frequently implicated species in nosocomial and gene transfer. The flexible genome often harbours seg- community acquired Klebsiella infections, which include ments of recognizable mobile genetic elements, such as trans- pneumonia, septicaemia, urinary tract infections and posons, phages, plasmids and archetypal integrative genomic 1, 2 wound infections. islands, that may confer enhanced antibiotic resistance, Bacterial genomes consist of two parts: the core and flex- pathogenicity or ecological fitness. ible genome. The core genome is shared by nearly all strains tRNA loci commonly serve as insertion sites for of the same species and encodes proteins involved in basic mobile elements as they are highly conserved between bac- cellular function. The remainder is the flexible genome, a teria and thus allow for a greater degree of promiscuous ......................................................................................................................................................................................................................................... The Author 2008. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 51 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... 7, 8 21 21 movement. The phe tRNA genes in several members of with ampicillin (100 mgml ) or kanamycin (50 mgml ) the Enterobacteriaceae family have been found to harbour when required. pathogenicity islands, large 10 to 200 kb clusters of strain- Preparation and manipulation of DNA specific genes some of which confer defined virulence 9, 10 traits. The pheV locus of uropathogenic E. coli strain Genomic DNA was isolated by a modified phenol/chloro- J96 contains a 170 kb island (PAI I ) that encodes a P form extraction protocol. Plasmid DNA was prepared by J96 11, 12 18, 19 pilus, an essential virulence factor in pyelonephritis. standard alkaline lysis. Restriction enzymes (Roche In enteropathogenic E. coli this same locus is occupied by Diagnostics) and T4 DNA ligase (Promega) were used the LEE pathogenicity island that is crucial for the attach- according to manufacturer’s instructions. Genomic libraries ment and effacing phenotype responsible for much of the were constructed by overnight ligation of digested genomic resulting pathology. Similarly, the pheV locus of DNA to appropriately digested pBluescriptII KSþ . S. flexneri serotype 2a is occupied by the she island which Chemically competent E. coli DH5a were prepared and encodes multiple genes involved in Shigella pathogen- transformed according to standard methods. Standard sub- 13 – 15 18 esis. Additionally, a large-scale analysis of sequenced cloning methods were utilized. genomes using the Islander algorithm, which searches the tRNAcc analysis and PCR primer design genome for potential islands next to tRNA sites bordered 17, 20 by direct repeats and containing an integrase gene, has also tRNAcc was run using default parameters. 87 tRNA identified phenylalanine tRNA genes as insertion ‘hotspots’ loci from K. pneumoniae MGH78578 were mapped to hom- in several other bacteria. These findings were confirmed ologous tRNA loci in K. pneumoniae Kp342, a partially by tRNAcc, an algorithm that evaluates the content and sequenced genome. Subsequently, the tRNAcc subprogram context of tRNA and tmRNA genes across two or more ExtractFlank was used to obtain and align 2 kb upstream genomes by identifying the conserved segments that flank and downstream conserved flanking regions corresponding potential island integration sites. to these tRNA loci from both genomes. Genomic islands Although much effort has been focused on identifying were identified as non-homologous regions lying between E. coli, Salmonella and Shigella genomic islands, little is conserved upstream and downstream flanks. Primers to known about the Klebsiella flexible genome and its constitu- amplify across the pheV locus, 55pheU (CGTGCTTT ent islands, as only one Klebsiella genome, that of TAGCGCAATGT) and 55pheD (GACATAACCATTTAC K. pneumoniae MGH78578, has been sequenced completely CCACTCGT), were designed using the upstream and down- to date. On the basis that pheV is a known integration target stream pheV flanking consensus sequences, respectively in other Enterobacteriaceae and assuming that genomic (M. Patel and H.Y. Ou, personal communication). islands move at low frequency between bacteria sharing an tRIP PCR, SGSP PCR reactions and sequencing ecological niche and that once acquired integrate at the same locus through site-specific recombination, we targeted tRIP PCR (tRNA site interrogation for pathogenicity islands, Klebsiella phenylalanine tRNA loci, equivalent to that at prophages and other genomic islands PCR) reactions were 3.72 Mb in K. pneumoniae MGH78578 which we have performed in a volume of 20 ml using 1.25 U GoTaq termed pheV, for investigation. We aimed to identify novel DNA polymerase (Promega), 0.4 ml of 10.0 mM dNTP, islands, uncover likely horizontal gene transfer events and 20 pmol of primers 55 pheU and 55 pheD, and 10 ng of explore the genome plasticity of Klebsiella. Specifically, by genomic DNA as template. Cycling conditions comprised o o targeting clinical Klebsiella isolates we hoped to discover 30 cycles of 30 s at 95.0 C, 30 s at 59.0 C and 3 min at pathogenicity islands bearing novel virulence genes, which 72.0 C. SGSP PCR (single genome specific primer PCR) could potentially be harnessed as targets for a new gener- was performed similarly, but used genomic libraries based ation of pathotype-specific diagnostic tools, prophylactic on five distinct restriction enzymes (EcoRI, BamHI, PstI, measures and/or therapeutics strategies. HindIII, HincII) as template instead. Additionally, either the primer 55 pheU or 55 pheD was used in conjunction with a vector specific primer (T3 or T7) to amplify the island extremities adjacent to the upstream and downstream Material and methods conserved flanks, respectively. SGSP PCR cycling conditions when using T7 comprised an initial 10 cycles of 30 s at o o o Bacterial strains, plasmids and media 95.0 C, 30 s at 67.4 C (decreased by 1 C each cycle) and Bacterial strains and plasmids used in this study are listed in 4 min at 72.0 C. This was followed by 20 cycles of 30 s at o o o Table 1. Clinical Klebsiella isolates were obtained from 95.0 C, 30 s at 57.4 C and 4 min at 72.0 C. When using o o blood and urine cultures at Leicester Royal Infirmary and T3 the annealing temperatures of 67.4 C and 57.4 C were 8 8 o o stored in 220 C/ 2 80 C glycerol stocks. Strains were decreased to 63.0 C and 53.0 C, respectively. PCR ampli- grown at 37 C in LB medium or LB agar, supplemented cons were gel purified using the DNA Spin Gel Extraction ......................................................................................................................................................................................................................................... 52 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Table 1. Bacterial strains and plasmids used Bacterial strain/ Additional information Reference plasmid ........................................................................................................................................................................................................................................ Bacterial strains Klebsiella KR116 K. pneumoniae, BSI This study KR162 K. pneumoniae, BSI This study KR163 K. pneumoniae, BSI This study KR164 Klebsiella species, BSI This study KR168 K. pneumoniae, BSI This study KR169 K. pneumoniae, BSI This study KR173 K. pneumoniae, BSI This study KR310 Klebsiella species, UTI, ESBL This study KR311 Klebsiella species, UTI, ESBL This study KR312 Klebsiella species, UTI, possible ESBL This study KR313 Klebsiella species, UTI, ESBL This study KR314 Klebsiella species, UTI, ESBL This study KR315 Klebsiella species, UTI, ESBL This study KR357 K. pneumoniae, BSI This study KR358 Klebsiella species, UTI, ESBL This study KR360 Klebsiella species, UTI, ESBL This study E. coli 2 2 þ 2 DH5a F f80dlacZ DM15 D(lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rK -mK ) phoA supE44 l thi-1 gyrA96 relA118 2 R TOP10 F mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacX74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (Str ) endA1 Invitrogen nupG Plasmids pBluescriptII KSþ High copy number vector, Amp 50 pWSK129 Low copy number vector, Km 26 w r r pJVABSc1 3.7kb tRIP PCR fragment KR116_pheV cloned into pCR4-TOPO , Amp ,Km This study pJVABSc2 3.7kb EcoRI fragment of pJVABSc1 cloned into EcoRI site of pWSK129, Km This study pJVABSc3 Plasmid remaining after deletion of 1.1kb XbaI fragment from pJVABSc2, Km This study pJVABSc4 Plasmid remaining after deletion of 2.3kb BamHI fragment from pJVABSc2, Km This study pJVABSc5 1.1kb XbaI fragment of pJVABSc2 cloned into XbaI site in pBluescriptII KS þ , Amp This study pJVABSc6 2.3kb BamHI fragment of pJVABSc2 cloned into BamHI site in pBluescriptII KS þ , Amp This study r r BSI, blood stream isolate; UTI, urinary tract isolate; ESBL, extended spectrum beta lactamase; Amp , ampicillin resistance; Km , kanamycin resistance. /PCR DNA purification kit (Yorkshire Bioscience) and DNA nucleotide sequences were aligned using ClustalX with sequencing was performed by MWG Biotech. default parameters. Sequence analysis Both local and online databases were searched for nucleotide Results and amino acid similarities using Blastn, Blastp, Blastx, 21 20 Interrogation of pheV loci in KR116 and KR164 reveals a tBlastn and tBlastx. The MobilomeFINDER and novel genomic islet Islander databases were explored to identify whether simi- larity hits occurred within known genomic islands. Protein Ten of 16 Klebsiella strains produced pheV tRIP PCR ampli- coding sequence (CDS) prediction was performed using cons of 0.5 kb, confirming these loci were unoccupied. Four 22 23 Glimmer 3.02 and CDD identified protein domains. strains (KR162, KR163, KR169 and KR173) produced no Tandem Repeat Finder and Blastn were used to localize tRIP PCR amplicon and two (KR116 and KR164) yielded upstream and downstream repeats, respectively. Repeat an 3.7 kb product. ......................................................................................................................................................................................................................................... 53 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... KR116_pheV, the amplicon corresponding to the KR116 had 93 – 99% aa identity to the matching predicted KR173 pheV site, was ligated into pCR4-TOPO (Invitrogen) and protein. However, the minor size discrepancy between subcloned into pWSK129 for sequencing. Sequence analy- these regions suggested a possible short insertion within sis revealed that this segment harboured four predicted CDS the KR173 integrase gene (Fig. 2). Additionally, in KR173 and was novel to Klebsiella (Fig. 1). KR116_pheV_1 coded there was a small 270 bp UA segment which did not match for a 51 amino acid (aa) protein with high homology to the Salmonella island and was predicted to encode a part of a non-functional putative transposase in Yersinia hypothetical protein that lacked Blastp homologs. The last pestis. The second CDS, KR116_pheV_2, was predicted to 172 bp of the KR173 UA matched the S. Typhi CT18 encode a novel 181aa protein with no Blastn, tBlastn or island 2 kb further downstream of the integrase bearing Blastp matches and no conserved domains. The protein Blastn hit. encoded by KR116_pheV_3 (174aa) strongly matched a The first 1.4 kb of the KR173 pheV island DA matched putative acetyltransferase in Salmonella enterica Typhi an equivalent region in KR116; approximately 900 bp of this CT18, an association supported by the detection of a Gcn5 common region, which encoded two hypothetical proteins, related N-acetyltransferase (GNAT) domain at the protein’s exhibited strong similarity to a portion of island DNA amino terminus. KR116_pheV_4 was predicted to encode 12.1 kb downstream of pheR in S. Typhi CT18. However, a 79aa protein that harboured a truncated version of a unlike KR116_pheV_4, the slightly larger KR173 homolog, domain of unknown function present in a S. Typhi CT18 like that of Salmonella, was predicted to encode the full hypothetical protein. Interestingly, the two corresponding domain of unknown function. Nucleotide sequence from S. Typhi CT18 genes were themselves located within a further within the KR173 island did not exhibit DNA 133.7 kb pheR associated genomic island (H.Y. Ou, personal matches to KR116_pheV, S. Typhi CT18 or other communication). The matching region of the Klebsiella islet Genbank entries. The sequence was predicted to code for a had 85% nucleotide identity to the S. Typhi CT18 island. 303aa hypothetical protein with a very low homology Sample sequencing of the KR164 pheV islet revealed Blastp hit to a Lactococcus lactis cremoris MG1363 96% nucleotide sequence identity to that of KR116, protein; there were no conserved domains identified. strongly suggesting that the two strains harboured near iden- tical islets at this genomic location. Restriction pattern similarities at three loci: a prevalent Klebsiella island coding for type 1 pili? KR173 harbours a large integrase bearing element Three tRIP PCR negative strains (KR162, KR163 and KR169) within the pheV locus that potentially harboured elements at the pheV site produced SGSP PCR amplicons produced using as template EcoRI comparable restriction patterns with SGSP PCR. (2.7 kb), HindIII (1.5 kb) and BamHI (1.3 kb) Chromosome walking into the UA produced 3.5 kb and genomic libraries of KR173 were selectively sequenced to 2.1 kb fragments with BamHI and PstI genomic libraries, chromosome walk into the putative island integrated into respectively. Similarly, DA analysis using PstI libraries pro- the KR173 pheV gene (Fig. 2). Blastn revealed that the duced 1.8 kb fragments, while the use of EcoRI libraries defined portions of the upstream arm (UA) of the KR173 generated 4.0 kb products. In silico SGSP PCR analysis on island had high homology to corresponding regions of the the K. pneumoniae MGH78578 genome revealed the ampli- pheR island in S. Typhi CT18, the same island mentioned con sizes from the three test strains matched those expected previously in relation to the KR116 and KR164 islets. This from K. pneumoniae MGH78578, which harboured a region of the Salmonella island encoded a P4 integrase that 12.6 kb genomic island immediately downstream of the Figure 1. An outline of the novel 3.7 kb Klebsiella genomic islet present at the KR116 and KR164 pheV loci with predicted CDS and Blastp similarity hits. The grey rectangle highlights the full conserved domain of unknown function in the S. Typhi CT18 hypothetical protein homolog of KR116_pheV_4. UF, conserved upstream flank; DF, conserved downstream flank; DR, direct repeat; I, identity; E, expect value. ......................................................................................................................................................................................................................................... 54 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. The genetic organization of the KR173 pheV locus genomic island and its associated Blast similarity hits in KR116 and S. Typhi CT18. UF, con- served upstream flank; DF, conserved downstream flank; DR, direct repeat; I, identity; E, expect value. pheV gene. In total, ten SGSP PCR products representative of The sequence data and restriction patterns confirmed that the three test strains were sequenced and confirmed to have at least 7.5 kb of the 12.6 kb MGH78578 island was present very high nucleotide homology (95%) to that of the in all three test strains (Fig. 3 and Table 2). KPN_03400 MGH78578 island. The only major discrepancy was an putatively coded for a full length (397aa) P4 integrase, extra 163 bp repeat in MGH78578 (see in what follows). with 67% amino acid identity to an Enterobacter sp. 638 Figure 3. An illustration of genes present on the K. pneumoniae MGH78578 pheV island. The areas of the island that have been confirmed to occur in KR162, KR163 and/or KR169 by sequence data and/or SGSP PCR restriction patterns are depicted at the top of the diagram. Details of the products encoded for by each of the labelled island genes are found in Table 2. The Yersinia frederiksenii ATC33641 Blastp homologs are pictured in the order that they appear on the genome; they are positioned in tandem and in the same order as in MGH78578. However, in MGH78578 this structure is inter- rupted by KPN_03404 and KPN_03405. PapD , PapD and PapD correspond to three distinct PapD proteins that share a high level of sequence similarity. 1 2 3 UF, conserved upstream flank; DF, conserved downstream flank. ......................................................................................................................................................................................................................................... 55 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... Table 2. Genes present on an island downstream of pheV in K. pneumoniae MGH78578 GenBank Function Coordinates O L Blastp Left Right Best homolog GenPept S E I ........................................................................................................................................................................................................................................ b 235 KPN_03399 tRNA-phe 178 ! 78 Klebsiella pneumoniae W70- tRNA-phe DQ643993.1 78 2e 100 KPN_03400 Putative phage 873 1736 ! 288 Enterobacter sp. 638- P4 phage YP_001175202.1 399 1e 67 P4-integrase integrase KPN_03401 Putative fimbrial usher 4576 7185 ! 870 Y. frederiksenii ATCC33641- PapC ZP_00827700.1 875 0 53 protien KPN_03402 Putative P pilus assembly 7217 7927 237 Y. frederiksenii ATCC33641 - PapD ZP_00827699.1 170 5e 40 protein KPN_03403 Putative P pilus assembly 7990 8784 265 Y. frederiksenii ATCC33641 - PapD ZP_00827698.1 167 8e 42 protein KPN_03404 Putative fimbrial protein 8777 9355 193 Y. intermedia ATCC29909 - FimA ZP_00831721.1 62 2e 31 KPN_03405 Putative fimbrial protein 9374 9877 168 Y. intermedia ATCC29909 - FimA ZP_00831721.1 52 1e 28 KPN_03406 Putative P pilus assembly 9906 10637 244 Y. frederiksenii ATCC33641 - PapD ZP_00827696.1 148 2e 39 protein KPN_03407 Putative fimbrial 10688 11203 172 Y. frederiksenii ATCC33641 - ZP_00827695.1 134 7e 29 associated protien Hypothetical protein O, orientation; L, length in amino acids; S, score; E, expect value; I, percentage identity. Coordinates are given in base pair values relative to the start of pheV. Data for tRNA-phe comprises length in nucleotides, and best homolog by Blastn analysis and its associated GenBank entry. integrase. KPN_03401 to KPN_03403 and KPN_03406 had The typical full length repeat unit was identified as a low identity (39 – 53%) to protein homologs in Yersinia fre- 163 bp sequence that started with 18 bp of the 3’ terminus deriksenii ATCC33641 that were deduced to code for sub- of pheV. ClustalX alignment of the full set of identified units of a P pilus system, a type 1 pilus involved in repeats revealed that, with the exception of Kp342_UR3, 27, 28 adhesion. Analysis of the Y. frederiksenii chromosome all UA repeat segments in this six strain panel exhibited showed that the putative P pilus genes were positioned in limited nucleotide variation and were highly homologous tandem with each other and in the same order as in (Fig. 5). KR310 possessed an empty pheV site and only K. pneumoniae MGH78578. Additionally, these genes had a single 81 bp truncated repeat sequence. In contrast, were located 5 kb downstream of a lambda like integrase, Kp342 also had an empty site but possessed a three repeat suggesting that the Yersinia counterparts were also part of sequence of 386 bp in total. Furthermore, even though a larger acquired island. KP_03407 putatively encoded a KR173 harboured the large novel island identified in this hypothetical protein. The presence of a similar gene in a study it lacked any recognizable upstream repeats, perhaps second related but quite distinct cluster suggested that these with the exception of the 18 bp 3’ terminus of its pheV homologues may play a role in fimbrial biogenesis as well. In gene. These observations suggested that the UA repeat MGH78578, the P pilus cluster was interrupted by the inser- number did not correlate with the size or type of island inte- tion of two near identical tandem genes, KPN_03404 and grated at the pheV locus. KPN_03405, whose products had 28 – 31% homology to The solitary DA repeats that were found also possessed the Yersinia intermedia ATC29909 FimA protein, further strong nucleotide similarity to the 163 bp consensus supporting the hypothesis that these genes code for a novel sequence, although they were much shorter than their UA Klebsiella fimbrial appendage. counterparts. In KR116 and KR173, the downstream repeats contained 17 bp of the 3’ end of pheV and continued Variable tandem repeats downstream of pheV tRNA with a further 63 – 69 bp of repeat sequence; those in KR162 genes and MGH78578 did not contain any pheV sequence at all and were even shorter in length. KR310 and Kp342 did Analyses of DNA sequences from various K. pneumoniae not possess any downstream repeats. Additionally, none of pheV loci revealed multiple repeats existed immediately the DA repeats lay at the very extremity of the island arm, downstream of the tRNA gene in the majority of strains ana- as identified based on the boundary with highly conserved lysed and that a solitary repeat sequence often lay within the downstream flanking core genome. Between 67 and 74 bp opposite end of the island as well (Fig. 4). ......................................................................................................................................................................................................................................... 56 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Figure 4. A diagram comparing the repeats present in the upstream and downstream extremities of elements present at the pheV locus in six Klebsiella strains. The full length repeat unit was identified to be a 163 bp sequence that started with 18 bp of the 3’ end of pheV. Downstream repeats bore much nucleotide similarity to the repeat unit, although they were much shorter and occurred as isolated units. UR indicates upstream direct repeat number x. DR represents downstream direct repeat number x. The triangles represent the area of the direct repeat that corresponds to the 3’ terminus of pheV. UF, conserved upstream flank; DF, conserved downstream flank. separated the single DA repeat and the predicted core that novel bacterial genes were commonly localized to genome. However, this could have been an artefact resulting genomic islands. from incorrect island/flank boundary determination by com- KR116_pheV_3 was predicted to code for a hypothetical parative genome analysis. This is known to occur particu- protein harbouring a GNAT domain, a signature motif of larly when only two input genomes are used for tRNAcc an enzyme superfamily widely distributed in all kingdoms. analysis, as was the case in this study. One group of GNAT proteins, the aminoglycoside trans- ferases, chemically modify aminoglycosides, thus resulting in resistance to members of this class of antimicrobials. Discussion However, preliminary antibiotic susceptibility assays per- formed on KR116 have failed to show an increase in resist- Genome plasticity is of major importance in bacterial evol- ance to gentamicin or kanamycin (J.J.v.A., unpublished ution and has frequently been associated with intraspecies data) alluding to a possible alternative function for phenotype variation. This paper has provided strong evi- KR116_pheV_3. A second group of GNAT enzymes, rep- dence that the Klebsiella pheV tRNA gene is an island inte- resented by the E. coli GlmU protein, function as gration hotspot. The presence of these elements gives rise to a glucosamine-6-phosphate N-acetyltransferases. GlmU pro- variable genomic region that serves as a repository for novel duces UDP-N-acetylglucosamine, an essential precursor of genes. Of the 16 strains investigated, six were found to two biofilm components: peptidoglycans and lipopolysac- harbour one of three different islands within their pheV charides. In Klebsiella urinary tract and respiratory tract locus. Two entities, one potentially being a markedly trun- infections, the ability to form biofilms on abiotic surfaces cated version of the other, were entirely novel to Klebsiella. is of recognized importance. Inhibition of GlmU reduces The discovery of several islands at this locus was in perfect biofilm formation and bacterial colonization, and hence agreement with our hypothesis and with the findings of pathogenicity, raising the intriguing possibility that Germon et al. who reported frequent occupation of the KR116_pheV_3 may play a direct role in virulence. E. coli pheV locus. Similarly, the presence within these Nevertheless, bioinformatics analysis alone could not attri- elements of previously undiscovered DNA sequences was bute a specific function to KR116_pheV_3. However, given consistent with the report by Hsiao et al. who observed ......................................................................................................................................................................................................................................... 57 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... Figure 5. A ClustalX alignment of all the direct repeats found at the pheV loci of six Klebsiella strains and illustrated in Fig. 4. Using KR116_UR1 as the reference unit, identical bases in other repeats were shaded grey. The alignment revealed that all upstream arm repeats in this panel of strains, except Kp342_UR3, exhibited limited nucleotide variation and were highly homologous. The repeats have been labelled STRAIN_URX or STRAIN_DRX, where URX denotes upstream direct repeat number X and DRX represents downstream direct repeat number X. that this gene is located within the potentially unstable common island ancestor present within the wider gene flexible genome of multiple strains, it is likely that it is pool shared by Salmonella and Klebsiella. Over time, each being retained as a consequence of selection pressure that island has differentiated and evolved independently as a arises from its yet to be defined biological role. function of the different environmental and host-derived The KR173 pheV island is likely to be an example of a selection pressures that are exerted on these two types of bac- cross genus lateral transfer event. Both the Salmonella and teria. The available evidence also suggests that the KR116/ Klebsiella islands encoded a putative P4-related integrase, a KR164 islet represents a remnant of the larger KR173 type of enzyme which has been experimentally determined pheV island that has resulted from an imprecise excision or to control island excision and integration. Island encoded deletion event; similar phenomena affecting other genomic integrases are widely regarded as playing an essential role islands have been reported previously. in horizontal island transfer events and genomic island evol- A 12.6 kb pheV island was identified in K. pneumoniae ution. The high level of nucleotide similarity of the term- MGH78578 and although it harboured a large cluster of inal regions of both islands and their presence within genes encoding P pilus subunits, including PapD, PapC and phenylalanine tRNA genes in two distinct hosts, further sub- FimA, it did not appear to encode all subunits. P pili are stantiates the idea of inter genus movement. Additionally, the essential virulence factors in E. coli that cause pyelonephri- Klebsiella pheV and Salmonella pheR genes possess an tis, raising the prospect that the island may contribute to identical nucleotide sequence, thus providing an identical disease causation. The consequences of an incomplete set substrate for the two integrases during site-specific recombi- of P pilus subunits on the bacterial phenotype remains to nation. We hypothesize that these elements arose from a be tested, but it must be noted that the ‘missing’ subunits ......................................................................................................................................................................................................................................... 58 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... may be coded for elsewhere on the genome. Additionally, be central to defining the detailed mechanisms of pathogen- multiple genes putatively encoded the same or closely esis utilized by this important human pathogen. In light of related subunits. The incentive for maintaining this level of increasing antimicrobial resistance, a comprehensive under- apparent redundancy is unknown, but may involve increased standing of the genes, proteins and molecular events involved ability to evade the immune system by varying antigens pre- in pathogen survival, transmission and disease causation is sented on the cell surface. Apart from an extra 163 bp essential if we are to develop a new generation of thera- repeat, the extremities of the MGH78578 pheV island peutic, prognostic and diagnostic tools. Indeed this is likely were almost identical to pheV associated islands in KR162, to be crucial if we are to continue to successfully manage KR163 and KR169. However, it is quite possible that both community-acquired and nosocomial infections well repeat structure variation may be indicative of significant into the future. divergence further within a genomic island, an area hidden from our current interrogation tools. Future studies using a Acknowledgements yeast recombinational island capture system will be invalu- 20, 37 able in addressing this matter. We thank Hong-Yu Ou for his assistance and support with A unique and intriguing feature of Klebsiella pheV loci tRNAcc. We thank Dr Peter Munthali for providing the was the presence of multiple tandem repeats. With the excep- clinical Klebsiella strains from the Leicester Royal tion of tandem insertion sequence elements, the E. coli LEE Infirmary, and James Lonnen, Ewan Harrison and Mansi pathogenicity island is the only other island described as har- Patel for laying the foundations on which this work was bouring terminally located, large, directly oriented repeats started. We also thank the Institute for Genomic Research 7, 38 (136 bp). The presence of other repeat motifs elsewhere (TIGR) and Washington University in St Louis for their in the Klebsiella genomes remains to be determined, though policy of making preliminary sequence data publicly avail- the pheV 163 bp repeat sequence is specific to this particular able and acknowledge the use in this study of unpublished locus. The differing numbers of UA repeats is reminiscent of genome data corresponding to Kp342 and MGH78578, many other variable number tandem repeat (VNTR) loci respectively. 39 – 42 that have been described in bacteria; these are thought to originate from slipped strand mispairing or recombination Funding events. It is widely accepted that some VNTR loci can affect phase variation, either by modifying promoters that The author was supported by a Wolfson Foundation Award modulate transcription or by altering protein amino acid Intercalated BSc Studentship. sequence. 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Methods Enzymol 216: 483–495. ........................................................................................................................................................................................................................................ Submitted on 27 September 2007; accepted on 17 December 2007 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands

van Aartsen, Jon Jurriaan
Bioscience Horizons , Volume 1 (1) – Mar 1, 2008
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Volume 1 † Number 1 † March 2008 10.1093/biohorizons/hzn006 ......................................................................................................................................................................................................................................... Research article The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands Jon Jurriaan van Aartsen* Department of Infection Immunity and Inflammation, University of Leicester, Leicester, UK. * Corresponding author: Lab 212, Department of Infection Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, University Rd, Leicester LE1 9HN. Tel: þ44 (0)116 2523056. Email: jjv1@le.ac.uk Supervisor: Kumar Rajakumar, Department of Infection Immunity and Inflammation, University of Leicester, Leicester, UK. ........................................................................................................................................................................................................................................ Klebsiella sp. cause a wide range of human infections, particularly nosocomial septicaemia, pneumonia and urinary tract infections. Like other Enterobacteriaceae, Klebsiella are likely to possess plastic genomes comprised of core regions interspersed with horizontally acquired genomic islands. As phenylalanine tRNA genes are known to be occupied by islands in other Enterobacteriaceae, we utilized PCR-based screening and chromosome walking techniques to examine the pheV locus in Klebsiella isolates from blood stream and urinary tract infections. We hypothesized that this gene was an integration hotspot that served as a repository for novel genetic material in Klebsiella. The pheV site in Klebsiella KR116 and KR164 harboured an islet encoding four genes, two with similarity to genes within an island downstream of pheR in Salmonella enterica serovar Typhi CT18. In KR173 the locus contained a larger, potentially intact version of this island and harboured an integrase gene similar to that in the S. Typhi CT18 island. However, the Klebsiella and Salmonella islands were clearly distinguishable by strain-specific segments and organizational variation. On the basis of available sequence and restriction fragment length polymorphism data, three other Klebsiella isolates were found to possess an entirely distinct entity that resembled a 12.6 kb pheV associated island in K. pneumoniae MGH78578. This island was predicted to encode a P pilus-like structure, a probable virulence factor on the basis of parallels with E. coli. A unique and intriguing feature of Klebsiella pheV loci was the presence of multiple tandem repeats of up to 163 bp immediately downstream of pheV and a truncated copy at the opposite end of the islands. The tRNA proximal repeats were variable in number and size between isolates, while the solitary downstream repeats varied in length. These elements may represent genetic debris of previous recombination events. In conclusion, the pheV locus of Klebsiella exhibited consider- able variability between strains and harboured at least two distinct island types that could play important roles in adaptation and/or virulence. Functional characterization of this genetic armory will help unravel basic microbial and pathogenesis processes and may in time lead to improvements in the diagnosis, prevention and treatment of Klebsiella infections. Key words: Klebsiella, phenylalanine, tRNA, genomic island, pathogenicity island, genome plasticity. ........................................................................................................................................................................................................................................ highly variable complement of strain-specific genes. Introduction Genome variability plays an important role in the evolution The Klebsiella genus contains a diverse group of commensal and adaptive ability of bacteria, allowing for loss and/or and pathogenic species. K. pneumoniae and K. oxytoca are acquisition of functions via mutational changes or horizontal 4, 5 the most frequently implicated species in nosocomial and gene transfer. The flexible genome often harbours seg- community acquired Klebsiella infections, which include ments of recognizable mobile genetic elements, such as trans- pneumonia, septicaemia, urinary tract infections and posons, phages, plasmids and archetypal integrative genomic 1, 2 wound infections. islands, that may confer enhanced antibiotic resistance, Bacterial genomes consist of two parts: the core and flex- pathogenicity or ecological fitness. ible genome. The core genome is shared by nearly all strains tRNA loci commonly serve as insertion sites for of the same species and encodes proteins involved in basic mobile elements as they are highly conserved between bac- cellular function. The remainder is the flexible genome, a teria and thus allow for a greater degree of promiscuous ......................................................................................................................................................................................................................................... The Author 2008. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 51 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... 7, 8 21 21 movement. The phe tRNA genes in several members of with ampicillin (100 mgml ) or kanamycin (50 mgml ) the Enterobacteriaceae family have been found to harbour when required. pathogenicity islands, large 10 to 200 kb clusters of strain- Preparation and manipulation of DNA specific genes some of which confer defined virulence 9, 10 traits. The pheV locus of uropathogenic E. coli strain Genomic DNA was isolated by a modified phenol/chloro- J96 contains a 170 kb island (PAI I ) that encodes a P form extraction protocol. Plasmid DNA was prepared by J96 11, 12 18, 19 pilus, an essential virulence factor in pyelonephritis. standard alkaline lysis. Restriction enzymes (Roche In enteropathogenic E. coli this same locus is occupied by Diagnostics) and T4 DNA ligase (Promega) were used the LEE pathogenicity island that is crucial for the attach- according to manufacturer’s instructions. Genomic libraries ment and effacing phenotype responsible for much of the were constructed by overnight ligation of digested genomic resulting pathology. Similarly, the pheV locus of DNA to appropriately digested pBluescriptII KSþ . S. flexneri serotype 2a is occupied by the she island which Chemically competent E. coli DH5a were prepared and encodes multiple genes involved in Shigella pathogen- transformed according to standard methods. Standard sub- 13 – 15 18 esis. Additionally, a large-scale analysis of sequenced cloning methods were utilized. genomes using the Islander algorithm, which searches the tRNAcc analysis and PCR primer design genome for potential islands next to tRNA sites bordered 17, 20 by direct repeats and containing an integrase gene, has also tRNAcc was run using default parameters. 87 tRNA identified phenylalanine tRNA genes as insertion ‘hotspots’ loci from K. pneumoniae MGH78578 were mapped to hom- in several other bacteria. These findings were confirmed ologous tRNA loci in K. pneumoniae Kp342, a partially by tRNAcc, an algorithm that evaluates the content and sequenced genome. Subsequently, the tRNAcc subprogram context of tRNA and tmRNA genes across two or more ExtractFlank was used to obtain and align 2 kb upstream genomes by identifying the conserved segments that flank and downstream conserved flanking regions corresponding potential island integration sites. to these tRNA loci from both genomes. Genomic islands Although much effort has been focused on identifying were identified as non-homologous regions lying between E. coli, Salmonella and Shigella genomic islands, little is conserved upstream and downstream flanks. Primers to known about the Klebsiella flexible genome and its constitu- amplify across the pheV locus, 55pheU (CGTGCTTT ent islands, as only one Klebsiella genome, that of TAGCGCAATGT) and 55pheD (GACATAACCATTTAC K. pneumoniae MGH78578, has been sequenced completely CCACTCGT), were designed using the upstream and down- to date. On the basis that pheV is a known integration target stream pheV flanking consensus sequences, respectively in other Enterobacteriaceae and assuming that genomic (M. Patel and H.Y. Ou, personal communication). islands move at low frequency between bacteria sharing an tRIP PCR, SGSP PCR reactions and sequencing ecological niche and that once acquired integrate at the same locus through site-specific recombination, we targeted tRIP PCR (tRNA site interrogation for pathogenicity islands, Klebsiella phenylalanine tRNA loci, equivalent to that at prophages and other genomic islands PCR) reactions were 3.72 Mb in K. pneumoniae MGH78578 which we have performed in a volume of 20 ml using 1.25 U GoTaq termed pheV, for investigation. We aimed to identify novel DNA polymerase (Promega), 0.4 ml of 10.0 mM dNTP, islands, uncover likely horizontal gene transfer events and 20 pmol of primers 55 pheU and 55 pheD, and 10 ng of explore the genome plasticity of Klebsiella. Specifically, by genomic DNA as template. Cycling conditions comprised o o targeting clinical Klebsiella isolates we hoped to discover 30 cycles of 30 s at 95.0 C, 30 s at 59.0 C and 3 min at pathogenicity islands bearing novel virulence genes, which 72.0 C. SGSP PCR (single genome specific primer PCR) could potentially be harnessed as targets for a new gener- was performed similarly, but used genomic libraries based ation of pathotype-specific diagnostic tools, prophylactic on five distinct restriction enzymes (EcoRI, BamHI, PstI, measures and/or therapeutics strategies. HindIII, HincII) as template instead. Additionally, either the primer 55 pheU or 55 pheD was used in conjunction with a vector specific primer (T3 or T7) to amplify the island extremities adjacent to the upstream and downstream Material and methods conserved flanks, respectively. SGSP PCR cycling conditions when using T7 comprised an initial 10 cycles of 30 s at o o o Bacterial strains, plasmids and media 95.0 C, 30 s at 67.4 C (decreased by 1 C each cycle) and Bacterial strains and plasmids used in this study are listed in 4 min at 72.0 C. This was followed by 20 cycles of 30 s at o o o Table 1. Clinical Klebsiella isolates were obtained from 95.0 C, 30 s at 57.4 C and 4 min at 72.0 C. When using o o blood and urine cultures at Leicester Royal Infirmary and T3 the annealing temperatures of 67.4 C and 57.4 C were 8 8 o o stored in 220 C/ 2 80 C glycerol stocks. Strains were decreased to 63.0 C and 53.0 C, respectively. PCR ampli- grown at 37 C in LB medium or LB agar, supplemented cons were gel purified using the DNA Spin Gel Extraction ......................................................................................................................................................................................................................................... 52 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Table 1. Bacterial strains and plasmids used Bacterial strain/ Additional information Reference plasmid ........................................................................................................................................................................................................................................ Bacterial strains Klebsiella KR116 K. pneumoniae, BSI This study KR162 K. pneumoniae, BSI This study KR163 K. pneumoniae, BSI This study KR164 Klebsiella species, BSI This study KR168 K. pneumoniae, BSI This study KR169 K. pneumoniae, BSI This study KR173 K. pneumoniae, BSI This study KR310 Klebsiella species, UTI, ESBL This study KR311 Klebsiella species, UTI, ESBL This study KR312 Klebsiella species, UTI, possible ESBL This study KR313 Klebsiella species, UTI, ESBL This study KR314 Klebsiella species, UTI, ESBL This study KR315 Klebsiella species, UTI, ESBL This study KR357 K. pneumoniae, BSI This study KR358 Klebsiella species, UTI, ESBL This study KR360 Klebsiella species, UTI, ESBL This study E. coli 2 2 þ 2 DH5a F f80dlacZ DM15 D(lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rK -mK ) phoA supE44 l thi-1 gyrA96 relA118 2 R TOP10 F mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacX74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (Str ) endA1 Invitrogen nupG Plasmids pBluescriptII KSþ High copy number vector, Amp 50 pWSK129 Low copy number vector, Km 26 w r r pJVABSc1 3.7kb tRIP PCR fragment KR116_pheV cloned into pCR4-TOPO , Amp ,Km This study pJVABSc2 3.7kb EcoRI fragment of pJVABSc1 cloned into EcoRI site of pWSK129, Km This study pJVABSc3 Plasmid remaining after deletion of 1.1kb XbaI fragment from pJVABSc2, Km This study pJVABSc4 Plasmid remaining after deletion of 2.3kb BamHI fragment from pJVABSc2, Km This study pJVABSc5 1.1kb XbaI fragment of pJVABSc2 cloned into XbaI site in pBluescriptII KS þ , Amp This study pJVABSc6 2.3kb BamHI fragment of pJVABSc2 cloned into BamHI site in pBluescriptII KS þ , Amp This study r r BSI, blood stream isolate; UTI, urinary tract isolate; ESBL, extended spectrum beta lactamase; Amp , ampicillin resistance; Km , kanamycin resistance. /PCR DNA purification kit (Yorkshire Bioscience) and DNA nucleotide sequences were aligned using ClustalX with sequencing was performed by MWG Biotech. default parameters. Sequence analysis Both local and online databases were searched for nucleotide Results and amino acid similarities using Blastn, Blastp, Blastx, 21 20 Interrogation of pheV loci in KR116 and KR164 reveals a tBlastn and tBlastx. The MobilomeFINDER and novel genomic islet Islander databases were explored to identify whether simi- larity hits occurred within known genomic islands. Protein Ten of 16 Klebsiella strains produced pheV tRIP PCR ampli- coding sequence (CDS) prediction was performed using cons of 0.5 kb, confirming these loci were unoccupied. Four 22 23 Glimmer 3.02 and CDD identified protein domains. strains (KR162, KR163, KR169 and KR173) produced no Tandem Repeat Finder and Blastn were used to localize tRIP PCR amplicon and two (KR116 and KR164) yielded upstream and downstream repeats, respectively. Repeat an 3.7 kb product. ......................................................................................................................................................................................................................................... 53 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... KR116_pheV, the amplicon corresponding to the KR116 had 93 – 99% aa identity to the matching predicted KR173 pheV site, was ligated into pCR4-TOPO (Invitrogen) and protein. However, the minor size discrepancy between subcloned into pWSK129 for sequencing. Sequence analy- these regions suggested a possible short insertion within sis revealed that this segment harboured four predicted CDS the KR173 integrase gene (Fig. 2). Additionally, in KR173 and was novel to Klebsiella (Fig. 1). KR116_pheV_1 coded there was a small 270 bp UA segment which did not match for a 51 amino acid (aa) protein with high homology to the Salmonella island and was predicted to encode a part of a non-functional putative transposase in Yersinia hypothetical protein that lacked Blastp homologs. The last pestis. The second CDS, KR116_pheV_2, was predicted to 172 bp of the KR173 UA matched the S. Typhi CT18 encode a novel 181aa protein with no Blastn, tBlastn or island 2 kb further downstream of the integrase bearing Blastp matches and no conserved domains. The protein Blastn hit. encoded by KR116_pheV_3 (174aa) strongly matched a The first 1.4 kb of the KR173 pheV island DA matched putative acetyltransferase in Salmonella enterica Typhi an equivalent region in KR116; approximately 900 bp of this CT18, an association supported by the detection of a Gcn5 common region, which encoded two hypothetical proteins, related N-acetyltransferase (GNAT) domain at the protein’s exhibited strong similarity to a portion of island DNA amino terminus. KR116_pheV_4 was predicted to encode 12.1 kb downstream of pheR in S. Typhi CT18. However, a 79aa protein that harboured a truncated version of a unlike KR116_pheV_4, the slightly larger KR173 homolog, domain of unknown function present in a S. Typhi CT18 like that of Salmonella, was predicted to encode the full hypothetical protein. Interestingly, the two corresponding domain of unknown function. Nucleotide sequence from S. Typhi CT18 genes were themselves located within a further within the KR173 island did not exhibit DNA 133.7 kb pheR associated genomic island (H.Y. Ou, personal matches to KR116_pheV, S. Typhi CT18 or other communication). The matching region of the Klebsiella islet Genbank entries. The sequence was predicted to code for a had 85% nucleotide identity to the S. Typhi CT18 island. 303aa hypothetical protein with a very low homology Sample sequencing of the KR164 pheV islet revealed Blastp hit to a Lactococcus lactis cremoris MG1363 96% nucleotide sequence identity to that of KR116, protein; there were no conserved domains identified. strongly suggesting that the two strains harboured near iden- tical islets at this genomic location. Restriction pattern similarities at three loci: a prevalent Klebsiella island coding for type 1 pili? KR173 harbours a large integrase bearing element Three tRIP PCR negative strains (KR162, KR163 and KR169) within the pheV locus that potentially harboured elements at the pheV site produced SGSP PCR amplicons produced using as template EcoRI comparable restriction patterns with SGSP PCR. (2.7 kb), HindIII (1.5 kb) and BamHI (1.3 kb) Chromosome walking into the UA produced 3.5 kb and genomic libraries of KR173 were selectively sequenced to 2.1 kb fragments with BamHI and PstI genomic libraries, chromosome walk into the putative island integrated into respectively. Similarly, DA analysis using PstI libraries pro- the KR173 pheV gene (Fig. 2). Blastn revealed that the duced 1.8 kb fragments, while the use of EcoRI libraries defined portions of the upstream arm (UA) of the KR173 generated 4.0 kb products. In silico SGSP PCR analysis on island had high homology to corresponding regions of the the K. pneumoniae MGH78578 genome revealed the ampli- pheR island in S. Typhi CT18, the same island mentioned con sizes from the three test strains matched those expected previously in relation to the KR116 and KR164 islets. This from K. pneumoniae MGH78578, which harboured a region of the Salmonella island encoded a P4 integrase that 12.6 kb genomic island immediately downstream of the Figure 1. An outline of the novel 3.7 kb Klebsiella genomic islet present at the KR116 and KR164 pheV loci with predicted CDS and Blastp similarity hits. The grey rectangle highlights the full conserved domain of unknown function in the S. Typhi CT18 hypothetical protein homolog of KR116_pheV_4. UF, conserved upstream flank; DF, conserved downstream flank; DR, direct repeat; I, identity; E, expect value. ......................................................................................................................................................................................................................................... 54 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. The genetic organization of the KR173 pheV locus genomic island and its associated Blast similarity hits in KR116 and S. Typhi CT18. UF, con- served upstream flank; DF, conserved downstream flank; DR, direct repeat; I, identity; E, expect value. pheV gene. In total, ten SGSP PCR products representative of The sequence data and restriction patterns confirmed that the three test strains were sequenced and confirmed to have at least 7.5 kb of the 12.6 kb MGH78578 island was present very high nucleotide homology (95%) to that of the in all three test strains (Fig. 3 and Table 2). KPN_03400 MGH78578 island. The only major discrepancy was an putatively coded for a full length (397aa) P4 integrase, extra 163 bp repeat in MGH78578 (see in what follows). with 67% amino acid identity to an Enterobacter sp. 638 Figure 3. An illustration of genes present on the K. pneumoniae MGH78578 pheV island. The areas of the island that have been confirmed to occur in KR162, KR163 and/or KR169 by sequence data and/or SGSP PCR restriction patterns are depicted at the top of the diagram. Details of the products encoded for by each of the labelled island genes are found in Table 2. The Yersinia frederiksenii ATC33641 Blastp homologs are pictured in the order that they appear on the genome; they are positioned in tandem and in the same order as in MGH78578. However, in MGH78578 this structure is inter- rupted by KPN_03404 and KPN_03405. PapD , PapD and PapD correspond to three distinct PapD proteins that share a high level of sequence similarity. 1 2 3 UF, conserved upstream flank; DF, conserved downstream flank. ......................................................................................................................................................................................................................................... 55 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... Table 2. Genes present on an island downstream of pheV in K. pneumoniae MGH78578 GenBank Function Coordinates O L Blastp Left Right Best homolog GenPept S E I ........................................................................................................................................................................................................................................ b 235 KPN_03399 tRNA-phe 178 ! 78 Klebsiella pneumoniae W70- tRNA-phe DQ643993.1 78 2e 100 KPN_03400 Putative phage 873 1736 ! 288 Enterobacter sp. 638- P4 phage YP_001175202.1 399 1e 67 P4-integrase integrase KPN_03401 Putative fimbrial usher 4576 7185 ! 870 Y. frederiksenii ATCC33641- PapC ZP_00827700.1 875 0 53 protien KPN_03402 Putative P pilus assembly 7217 7927 237 Y. frederiksenii ATCC33641 - PapD ZP_00827699.1 170 5e 40 protein KPN_03403 Putative P pilus assembly 7990 8784 265 Y. frederiksenii ATCC33641 - PapD ZP_00827698.1 167 8e 42 protein KPN_03404 Putative fimbrial protein 8777 9355 193 Y. intermedia ATCC29909 - FimA ZP_00831721.1 62 2e 31 KPN_03405 Putative fimbrial protein 9374 9877 168 Y. intermedia ATCC29909 - FimA ZP_00831721.1 52 1e 28 KPN_03406 Putative P pilus assembly 9906 10637 244 Y. frederiksenii ATCC33641 - PapD ZP_00827696.1 148 2e 39 protein KPN_03407 Putative fimbrial 10688 11203 172 Y. frederiksenii ATCC33641 - ZP_00827695.1 134 7e 29 associated protien Hypothetical protein O, orientation; L, length in amino acids; S, score; E, expect value; I, percentage identity. Coordinates are given in base pair values relative to the start of pheV. Data for tRNA-phe comprises length in nucleotides, and best homolog by Blastn analysis and its associated GenBank entry. integrase. KPN_03401 to KPN_03403 and KPN_03406 had The typical full length repeat unit was identified as a low identity (39 – 53%) to protein homologs in Yersinia fre- 163 bp sequence that started with 18 bp of the 3’ terminus deriksenii ATCC33641 that were deduced to code for sub- of pheV. ClustalX alignment of the full set of identified units of a P pilus system, a type 1 pilus involved in repeats revealed that, with the exception of Kp342_UR3, 27, 28 adhesion. Analysis of the Y. frederiksenii chromosome all UA repeat segments in this six strain panel exhibited showed that the putative P pilus genes were positioned in limited nucleotide variation and were highly homologous tandem with each other and in the same order as in (Fig. 5). KR310 possessed an empty pheV site and only K. pneumoniae MGH78578. Additionally, these genes had a single 81 bp truncated repeat sequence. In contrast, were located 5 kb downstream of a lambda like integrase, Kp342 also had an empty site but possessed a three repeat suggesting that the Yersinia counterparts were also part of sequence of 386 bp in total. Furthermore, even though a larger acquired island. KP_03407 putatively encoded a KR173 harboured the large novel island identified in this hypothetical protein. The presence of a similar gene in a study it lacked any recognizable upstream repeats, perhaps second related but quite distinct cluster suggested that these with the exception of the 18 bp 3’ terminus of its pheV homologues may play a role in fimbrial biogenesis as well. In gene. These observations suggested that the UA repeat MGH78578, the P pilus cluster was interrupted by the inser- number did not correlate with the size or type of island inte- tion of two near identical tandem genes, KPN_03404 and grated at the pheV locus. KPN_03405, whose products had 28 – 31% homology to The solitary DA repeats that were found also possessed the Yersinia intermedia ATC29909 FimA protein, further strong nucleotide similarity to the 163 bp consensus supporting the hypothesis that these genes code for a novel sequence, although they were much shorter than their UA Klebsiella fimbrial appendage. counterparts. In KR116 and KR173, the downstream repeats contained 17 bp of the 3’ end of pheV and continued Variable tandem repeats downstream of pheV tRNA with a further 63 – 69 bp of repeat sequence; those in KR162 genes and MGH78578 did not contain any pheV sequence at all and were even shorter in length. KR310 and Kp342 did Analyses of DNA sequences from various K. pneumoniae not possess any downstream repeats. Additionally, none of pheV loci revealed multiple repeats existed immediately the DA repeats lay at the very extremity of the island arm, downstream of the tRNA gene in the majority of strains ana- as identified based on the boundary with highly conserved lysed and that a solitary repeat sequence often lay within the downstream flanking core genome. Between 67 and 74 bp opposite end of the island as well (Fig. 4). ......................................................................................................................................................................................................................................... 56 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... Figure 4. A diagram comparing the repeats present in the upstream and downstream extremities of elements present at the pheV locus in six Klebsiella strains. The full length repeat unit was identified to be a 163 bp sequence that started with 18 bp of the 3’ end of pheV. Downstream repeats bore much nucleotide similarity to the repeat unit, although they were much shorter and occurred as isolated units. UR indicates upstream direct repeat number x. DR represents downstream direct repeat number x. The triangles represent the area of the direct repeat that corresponds to the 3’ terminus of pheV. UF, conserved upstream flank; DF, conserved downstream flank. separated the single DA repeat and the predicted core that novel bacterial genes were commonly localized to genome. However, this could have been an artefact resulting genomic islands. from incorrect island/flank boundary determination by com- KR116_pheV_3 was predicted to code for a hypothetical parative genome analysis. This is known to occur particu- protein harbouring a GNAT domain, a signature motif of larly when only two input genomes are used for tRNAcc an enzyme superfamily widely distributed in all kingdoms. analysis, as was the case in this study. One group of GNAT proteins, the aminoglycoside trans- ferases, chemically modify aminoglycosides, thus resulting in resistance to members of this class of antimicrobials. Discussion However, preliminary antibiotic susceptibility assays per- formed on KR116 have failed to show an increase in resist- Genome plasticity is of major importance in bacterial evol- ance to gentamicin or kanamycin (J.J.v.A., unpublished ution and has frequently been associated with intraspecies data) alluding to a possible alternative function for phenotype variation. This paper has provided strong evi- KR116_pheV_3. A second group of GNAT enzymes, rep- dence that the Klebsiella pheV tRNA gene is an island inte- resented by the E. coli GlmU protein, function as gration hotspot. The presence of these elements gives rise to a glucosamine-6-phosphate N-acetyltransferases. GlmU pro- variable genomic region that serves as a repository for novel duces UDP-N-acetylglucosamine, an essential precursor of genes. Of the 16 strains investigated, six were found to two biofilm components: peptidoglycans and lipopolysac- harbour one of three different islands within their pheV charides. In Klebsiella urinary tract and respiratory tract locus. Two entities, one potentially being a markedly trun- infections, the ability to form biofilms on abiotic surfaces cated version of the other, were entirely novel to Klebsiella. is of recognized importance. Inhibition of GlmU reduces The discovery of several islands at this locus was in perfect biofilm formation and bacterial colonization, and hence agreement with our hypothesis and with the findings of pathogenicity, raising the intriguing possibility that Germon et al. who reported frequent occupation of the KR116_pheV_3 may play a direct role in virulence. E. coli pheV locus. Similarly, the presence within these Nevertheless, bioinformatics analysis alone could not attri- elements of previously undiscovered DNA sequences was bute a specific function to KR116_pheV_3. However, given consistent with the report by Hsiao et al. who observed ......................................................................................................................................................................................................................................... 57 Research article Bioscience Horizons † Volume 1 † Number 1 † March 2008 ......................................................................................................................................................................................................................................... Figure 5. A ClustalX alignment of all the direct repeats found at the pheV loci of six Klebsiella strains and illustrated in Fig. 4. Using KR116_UR1 as the reference unit, identical bases in other repeats were shaded grey. The alignment revealed that all upstream arm repeats in this panel of strains, except Kp342_UR3, exhibited limited nucleotide variation and were highly homologous. The repeats have been labelled STRAIN_URX or STRAIN_DRX, where URX denotes upstream direct repeat number X and DRX represents downstream direct repeat number X. that this gene is located within the potentially unstable common island ancestor present within the wider gene flexible genome of multiple strains, it is likely that it is pool shared by Salmonella and Klebsiella. Over time, each being retained as a consequence of selection pressure that island has differentiated and evolved independently as a arises from its yet to be defined biological role. function of the different environmental and host-derived The KR173 pheV island is likely to be an example of a selection pressures that are exerted on these two types of bac- cross genus lateral transfer event. Both the Salmonella and teria. The available evidence also suggests that the KR116/ Klebsiella islands encoded a putative P4-related integrase, a KR164 islet represents a remnant of the larger KR173 type of enzyme which has been experimentally determined pheV island that has resulted from an imprecise excision or to control island excision and integration. Island encoded deletion event; similar phenomena affecting other genomic integrases are widely regarded as playing an essential role islands have been reported previously. in horizontal island transfer events and genomic island evol- A 12.6 kb pheV island was identified in K. pneumoniae ution. The high level of nucleotide similarity of the term- MGH78578 and although it harboured a large cluster of inal regions of both islands and their presence within genes encoding P pilus subunits, including PapD, PapC and phenylalanine tRNA genes in two distinct hosts, further sub- FimA, it did not appear to encode all subunits. P pili are stantiates the idea of inter genus movement. Additionally, the essential virulence factors in E. coli that cause pyelonephri- Klebsiella pheV and Salmonella pheR genes possess an tis, raising the prospect that the island may contribute to identical nucleotide sequence, thus providing an identical disease causation. The consequences of an incomplete set substrate for the two integrases during site-specific recombi- of P pilus subunits on the bacterial phenotype remains to nation. We hypothesize that these elements arose from a be tested, but it must be noted that the ‘missing’ subunits ......................................................................................................................................................................................................................................... 58 Bioscience Horizons † Volume 1 † Number 1 † March 2008 Research article ......................................................................................................................................................................................................................................... may be coded for elsewhere on the genome. Additionally, be central to defining the detailed mechanisms of pathogen- multiple genes putatively encoded the same or closely esis utilized by this important human pathogen. In light of related subunits. The incentive for maintaining this level of increasing antimicrobial resistance, a comprehensive under- apparent redundancy is unknown, but may involve increased standing of the genes, proteins and molecular events involved ability to evade the immune system by varying antigens pre- in pathogen survival, transmission and disease causation is sented on the cell surface. Apart from an extra 163 bp essential if we are to develop a new generation of thera- repeat, the extremities of the MGH78578 pheV island peutic, prognostic and diagnostic tools. Indeed this is likely were almost identical to pheV associated islands in KR162, to be crucial if we are to continue to successfully manage KR163 and KR169. However, it is quite possible that both community-acquired and nosocomial infections well repeat structure variation may be indicative of significant into the future. divergence further within a genomic island, an area hidden from our current interrogation tools. Future studies using a Acknowledgements yeast recombinational island capture system will be invalu- 20, 37 able in addressing this matter. We thank Hong-Yu Ou for his assistance and support with A unique and intriguing feature of Klebsiella pheV loci tRNAcc. We thank Dr Peter Munthali for providing the was the presence of multiple tandem repeats. With the excep- clinical Klebsiella strains from the Leicester Royal tion of tandem insertion sequence elements, the E. coli LEE Infirmary, and James Lonnen, Ewan Harrison and Mansi pathogenicity island is the only other island described as har- Patel for laying the foundations on which this work was bouring terminally located, large, directly oriented repeats started. We also thank the Institute for Genomic Research 7, 38 (136 bp). The presence of other repeat motifs elsewhere (TIGR) and Washington University in St Louis for their in the Klebsiella genomes remains to be determined, though policy of making preliminary sequence data publicly avail- the pheV 163 bp repeat sequence is specific to this particular able and acknowledge the use in this study of unpublished locus. The differing numbers of UA repeats is reminiscent of genome data corresponding to Kp342 and MGH78578, many other variable number tandem repeat (VNTR) loci respectively. 39 – 42 that have been described in bacteria; these are thought to originate from slipped strand mispairing or recombination Funding events. It is widely accepted that some VNTR loci can affect phase variation, either by modifying promoters that The author was supported by a Wolfson Foundation Award modulate transcription or by altering protein amino acid Intercalated BSc Studentship. sequence. 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Methods Enzymol 216: 483–495. ........................................................................................................................................................................................................................................ Submitted on 27 September 2007; accepted on 17 December 2007 .........................................................................................................................................................................................................................................

Journal

Bioscience Horizons – Oxford University Press

Published: Mar 1, 2008

Keywords: Key words Klebsiella phenylalanine tRNA genomic island pathogenicity island genome plasticity

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