Transposases are responsible for the target specificity of IS1397 and ISKpn1 for two different types of palindromic units (PUs)

Insertion sequences (IS)1397 and ISKpn1, found in Escherichia coli and Klebsiella pneumoniae, respectively, are IS3 family members that insert specifically into short palindromic repeated sequences (palindromic units or PUs). In this paper, we first show that although PUs are naturally absent from extrachromosomal elements, both ISs are able to transpose from the chromosome or from a plasmid into PUs artificially introduced into target plasmids. We also show that ISKpn1 target specificity is restricted to K.pneumoniae Z¹ PU type, whereas IS1397 target specificity is less stringent since the IS targets the three E.coli Y, Z¹ and Z² PU types indifferently. Experiments of transposition of both ISs driven by both transposases demonstrate that the inverted repeats flanking the ISs are not responsible for this target specificity, which is entirely due to the transposase itself. Implications on ISs evolution are presented.     

Palindromic unit-independent transposition of IS1397 in Yersinia pestis

Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes of Escherichia coli and several other enterobacteria. In the latter, IS1397, an E. coli insertion sequence specific to PUs, transposes into PUs with sequences close to the E. coli consensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that for Yersinia pestis, a species deprived of PUs, IS1397 can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397 transposase.     

Bacterial Interspersed Mosaic Elements (Bimes) Are a Major Source of Sequence Polymorphism in Escherichia Coli Intergenic Regions Including Specific Associations with a New Insertion Sequence

A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS1397. Remarkably, 14 out of 14 IS1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.     

Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes

Members of the recently identified IS 1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS 1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS 1111 family members. This region may determine an RNA used to guide the IS to its specific target.     

多重耐药肺炎克雷伯菌获得性耐药相关基因和可移动遗传元件检测与指标聚类分析

目的 探讨一组多重耐药肺炎克雷伯菌(MDR-KPN)中获得性耐药相关基因和可移动遗传元件遗传标记的存在状况以及二者的相关性.方法 收集2008年8月至2010年5月浙江省杭州市和湖州市6所医院共47株MDR-KPN,采用聚合酶链反应(PCR)的方法分析74种获得性耐药基因和24种可移动遗传元件遗传标记,并用指标聚类分析(SPSS法)分析获得性耐药相关基因和可移动遗传元件遗传标记的相关性.结果 47株MDR-KPN共检出5种β-内酰胺类获得性耐药基因、6种氨基糖苷类获得性耐药基因、3种喹诺酮类获得性耐药基因、6种其他获得性耐药基因、1种整合子遗传标记、2种转座子遗传标记、4种插入序列遗传标记、2种接合性质粒遗传标记和1种噬菌体原标记;指标聚类分析(SPSS法)将上述阳性检出基因分成A、B两大簇.结论 指标聚类分析提示获得性耐药相关基因和可移动遗传元件密切相关;由Ⅰ类整合子( intI1)、插入序列(IS26、ISEcp1、ISKpn6)、耐药质粒(trbC)介导的TEM-1和KPC是本组菌株的特征.在肺炎克雷伯菌中做指标聚类分析为国内首次报道.     

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.     

Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.     

高毒力肺炎克雷伯菌致病及耐药分子机制研究进展

近年来,肺炎克雷伯菌已成为医院内感染及社区获得性感染的常见致病菌,临床标本分离率仅次于大肠埃希菌.根据毒力特征差异,肺炎克雷伯菌可分为经典肺炎克雷伯菌和高毒力肺炎克雷伯菌2种类型.高毒力肺炎克雷伯菌是引起化脓性肝脓肿的主要病原菌,其感染可出现内源性转移,包括眼、肺和中枢神经系统;此外还与原发性肝外感染有关,包括菌血症、...     

Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression

The structural gene encoding cyclodextrin-glycosyltransferase of Klebsiella pneumoniae strain M5a1 was cloned; it is expressed both in Escherichia coli and in K. pneumoniae and the gene product is secreted into the extracellular space. Determination of the nucleotide sequence revealed an open reading frame coding for a single polypeptide of 655 amino acid (aa) residues. The enzyme is synthesized as a precursor with an N-terminal signal peptide of 30 aa residues, which is proteolytically processed between two alanine residues during export. The primary structure of CGT bears homology with the sequences of amylases from both prokaryotic and eukaryotic origins.     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

Genome Sequence of Klebsiella pneumoniae LZ, a Potential Platform Strain for 1,3-Propanediol Production

Klebsiella pneumoniae LZ is a bacterium isolated from soil which can produce 1,3-propanediol from glycerol. Here we present a 5,431,750-bp assembly of its genome sequence. We annotated 9 coding sequences (CDSs) responsible for glycerol fermentation to 1,3-propanediol, 19 CDSs encoding glycerol utilization, and 134 CDSs related to its virulence and defense.     

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.     

Identification of Klebsiella pneumoniae by DNA hybridization and fatty acid analysis.

On the basis of the idea that DNA sequences encoding cell surface-exposed regions of outer membrane proteins are genus or species specific, two oligonucleotide probes which were based on the PhoE protein of Klebsiella pneumoniae were evaluated. In slot blot hybridizations and in polymerase chain reactions, no cross-hybridizations were observed with non-Klebsiella strains. When the probes were tested on 75 different K-antigen reference Klebsiella strains, 16 strains were not recognized although they did produce PhoE protein under phosphate starvation. To determine whether these 16 strains belong to (a) different species, the reference strains were also tested for the ability to produce indole and to grow at 10 degrees C and their whole-cell fatty acid patterns were analyzed by gas chromatography. A strong correlation was observed among (i) reaction with the probes, (ii) the inability to produce indole, (iii) the inability to grow at 10 degrees C, and (iv) the presence of the hydroxylated fatty acid C14:0-2OH. From these results we conclude that the two oligonucleotides are specific for the species K. pneumoniae. Furthermore, analysis of fatty acid patterns appears to be a useful tool to distinguish K. pneumoniae from other Klebsiella species.     

肺炎克雷伯菌KP1＿4563基因突变株的构建及其生物膜表型分析

KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。     

长沙某医院产质粒AmpC酶型肺炎克雷伯菌的多重PCR检测与分析

为了了解湖南长沙某医院临床分离的肺炎克雷伯菌中质粒介导AmpC β-内酰胺酶的产生情况及其基因型,收集了该医院2008年3月至2010年10月临床分离的多重耐药肺炎克雷伯菌104株,用头孢西丁纸片扩散法对这些菌株进行表型初筛,用多重PCR确定ampC耐药基因型;结果发现其中有19株对头孢西丁纸片不敏感,疑为产AmpC酶菌株;再经多重PCR扩增,有12株菌分别在约400 bp（11株）和约350 bp（1株）出现了阳性条带,特异性PCR证明此12株菌分别携带了DHA型（11株）和ACC型（1株）ampC耐药基因;产质粒介导AmpC酶肺炎克雷伯菌的分离率为11.5%（12/104）。该医院产质粒介导AmpC酶肺炎克雷伯菌的分离率较高,应对其检测与监测给予足够重视,以指导临床合理选用抗菌药物。     

克氏肺炎杆菌NiFe-氢酶基因的克隆与序列分析

采用CLUSTAL-W软件对Swiss-Prot蛋白数据库中已报道的NiFe-氢酶大亚基氨基酸序列进行比对分析,找到保守区并根据此设计兼并引物。利用其中一对引物进行PCR得到一条大小约为1000bp的DNA序列,并根据此序列设计引物进行反向PCR得到整个NiFe-氢酶的序列。再利用生物信息学软件对此氢酶的序列进行二、三级结构预测及大小亚基的对接(docking)。结果表明克氏肺炎杆菌的NiFe-氢酶属于一类膜结合放氢酶(Ech氢酶)。     

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.     

Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae.

Strains of Klebsiella pneumoniae are known to express two morphologically and functionally distinct filaments, the type 3 and the type 1 fimbriae. The gene (mrkD) encoding the adhesion of K. pneumoniae type 3 fimbriae was identified by transcomplementation analysis with the pap fimbrial gene cluster of Escherichia coli. The nucleotide sequence of the mrkD gene was determined. In addition, the determinant coding for the K. pneumoniae type 1 fimbrial adhesion was identified, and its nucleotide sequence was deduced. The predicted amino acid sequences of the K. pneumoniae adhesion proteins are compared, and similarities with the major fimbrial structural proteins (MrkA and FimA) are discussed.