Replicative resolution of integron cassette insertion

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC × attI reaction.     

Phylogenetic and evolutionary analysis of Vibrio parahaemolyticus and Vibrio alginolyticus isolates based on toxR gene sequence

The Vibrio genus contains a large number of closely related bacterial species differing, in some cases, less than 1% in 16S rRNA gene sequence. The present study evaluated the usefulness of toxR gene for phylogenetic and evolution analysis on Vibrio isolates of environmental or clinical origin belonging to the two closely related species V. parahaemolyticus and V. alginolyticus. The phylogenetic analysis based on toxR gene, contrary to 16S rRNA gene, allowed a clear differentiation of the isolates belonging to the two species and showed the presence of two separate, statistically supported clusters in V. alginolyticus (subgroup A and B). Such division, partially reflected in the biochemical features of the isolates, could not be explained by spatial and/or temporal distance in the isolation, leading to the hypothesis of two distinct, co-existing clusters in the V. alginolyticus isolates analysed. The evolutionary analysis on the toxR sequence showed that while the substitutions inferred from the alignment of V. parahaemolyticus are best explained by the negative/neutral selection model, in V. alginolyticus--and particularly in subgroup B--is acting a positive evolutionary pressure. The site detected as under diversifying selection (P164L) could be related to conformational changes of ToxR protein.     

ACID: annotation of cassette and integron data

Background  

Although integrons and their associated gene cassettes are present in ~10% of bacteria and can represent up to 3% of the genome in which they are found, very few have been properly identified and annotated in public databases. These genetic elements have been overlooked in comparison to other vectors that facilitate lateral gene transfer between microorganisms.     

金属离子抑制细菌整合子捕获耐药基因盒的机制研究

【目的】探索金属离子对整合子捕获耐药基因盒的影响及其相关机制。【方法】在大肠埃希菌中构建一个1类整合子捕获耐药基因盒的体内模型。通过实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)测定不同浓度银(0.3、0.9、1.5μg/mL的Ag+)和铜离子(5、50、100、150、210μg/mL的Cu²⁺)干预后实验组和无金属离子干预对照组整合子整合频率,并用表型筛选法验证。利用质谱法测量细菌吸收的金属离子浓度,并进一步通过转录组测序方法分析银离子抑制细菌整合子捕获耐药基因盒的分子机制。【结果】qPCR和表型筛选法的结果表明,0.9μg/mL银离子组整合频率为9.42×10^-5(6.49×10^-5,1.44×10^-4),1.5μg/mL银离子组整合频率为7.29×10^-5(4.45×10^-5,9.03×10^-5),与对照组2.59×10^-4(2.24×10^-4,3.33×10^-4)相比整合频率明显降低,差异有统计学意义(P<0.001)。而不同浓度铜离子组与对照组没有明显差异。转录组测序方法对银离子作用前后大肠杆菌基因表达水平进行对比分析,通过GO功能和KEGG通路富集发现,差异表达基因与甲基半乳糖苷转运(methylgalactoside transport)、麦芽糖转运(maltose transport)、磷酸烯醇式丙酮酸-甘油磷酸转移酶活性(phosphoenolpyruvate-glycerone phosphotransferase activity)和甘油激酶活性(glycerone kinase activity)等功能有关以及涉及氨基酸糖和核苷酸糖代谢(amino sugar and nucleotide sugar metabolism)、鞭毛组装(flagellar assembly)、阳离子抗菌肽(CAMP)耐药性[cationicantimicrobial peptide(CAMP)resistance]、果糖和甘露糖代谢和磷酸糖转移酶系统[phosphotransferase system(PTS)]等代谢通路。通过蛋白互作网络筛选出前12个枢纽基因(ptsG、malE、lamB、lacZ、malK、basR、ais、ugd、nagE、metN、malQ和malF)。【结论】一定浓度银离子可以抑制整合子整合耐药基因盒。铜离子对整合频率影响不明显,因此不是所有具有杀菌性的金属离子都能对整合频率产生影响。银离子抑制细菌整合子捕获耐药基因盒可能是通过影响麦芽糖转运和碳分解代谢来调控。然而,碳分解代谢和麦芽糖转运过程很复杂,需要进一步研究。目前尚无细菌整合子转录组学相关研究,这为解决细菌耐药性问题提供了一个新的途径。     

Identification and analysis of integrons and cassette arrays in bacterial genomes

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

Identification and analysis of the sap genes from Vibrio fischeri belonging to the ATP-binding cassette gene family required for peptide transport and resistance to antimicrobial peptides

Partial nucleotide sequences of the sapD and sapF genes of the sap operon (GenBank Accession No. AF178651) from Vibrio fischeri ATCC 7744 have been determined, and the peptide transport system of ATP-binding proteins SapD and SapF encoded by the genes have been deduced. Alignment and comparison of the Sap proteins of V. fischeri, Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae Rd show that these proteins are homologous. The sap operon residing in the genome enables V. fischeri to transport peptides and resist antimicrobial peptides. Nucleotide sequence and functional analyses confirm that the specific regulatory-region-like sequence R&R* that resides inside the sapD gene and before the sapF gene functions in gene expression and regulation; also, it is regulated by the LuxR-AI complex of the V. fischeri lux regulon. The putative upstream activator binding sequences SigmaUASI, SigmaUASII, SigmaUASIII TGTCGACTTGGGCCTCGCTGTCCGTATGCACA (72nd to 103rd bp), TGTCCGTATGCACA (90th to 103rd bp), and TGTTCAAGTACCAGAAAGACA (111st to 133rd bp) in the R&R* sequence, which are similar to the two-component regulator binding sequence TGT-N(8-12)-ACA and the LuxR-AI binding sequence ACCTGTAGGATCGTACAGGT in the regulatory region of the V. fischeri lux regulon, might be the specific sequences recognized by the LuxR-AI complex for enhancement.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.     

Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Identification and characteristic analysis of the ampC gene encoding beta-lactamase from Vibrio fischeri

Vibrio fischeri ATCC 7744 is an ampicillin resistant (Amp(r)) marine luminous bacterium. The MIC test indicates that V. fischeri is highly resistant to penicillins, and susceptible to cephalosporins. V. fischeri ampC gene was cloned and identified. Nucleotide sequence of an unidentified ufo gene and the ampC, ppiB genes (GenBank Accession No. AY438037) has been determined; whereas the ampC gene encodes the beta-lactamase (AmpC) and the ppiB gene encodes the peptidyl-prolyl cis-trans isomerase B. Alignment and comparison show that V. fischeri beta-lactamase is homologous to the related species'. The specific amino acid residues STFK (62nd to 65th), SDN (122nd to 124th), and D (155th) located 34 residues downstream from the SDN loop of the class A beta-lactamases are highly conserved, but the KTG is not found. V. fischeri ampC gene encoding beta-lactamase has a calculated M(r) 31,181 and comprises 283 amino acid residues (pI 5.35). There is a signal peptide of 18 amino acid residues MKIKPFLFGLIVLANNAI in the pro-beta-lactamase, which functioned for secretion; thus, the matured protein only has M(r) 29,197 and comprises 265 amino acid residues (pI 4.95). SDS-PAGE and the beta-lactamase functional assays elicit that the M(r) of the beta-lactamases are close to 29kDa. IEF and the beta-lactamase functional assays show that the beta-lactamases' pI are close to 4.8 as predicted. The results elucidate that V. fischeri ampC gene and the cloned ampC gene in Escherichia coli are the same one. The gene order of the ampC and the related genes is -ufo-(P*-intern)-ampC-ppiB--> (P*-intern: intern promoter for sub-regulation), whereas the P*-intern promoter displays the function to lead the ampC gene's expression for stress response.     

Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04

The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.     

Mutational and evolutionary analysis of an archaebacterial gene

The bacteriorhodopsin precursor gene of H. halobium is shown to contain 71 potential sites of mutation to premature stop codons, encompassing all detectable transitions and transversions. This same gene contains 24 potential frameshift hot-spots, all of the G and/or C type, nine possible dam methylation sites, and a unique mec methylation site which may act as a deamination mutagenesis hot-spot. While the gene contains pre-dominantly GC-rich codons, there is appreciable usage of other types of codons for particular amino acids. The bacteriorhodopsin precursor gene does not contain an abnormally high proportion of RNY-type, primitive codons, suggesting that even if distinct from the eubacteria, and an ancient group, modern-day archaebacteria have, nevertheless, undergone extensive evolution.     

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.