Fengjun Sun

Production of plasmid-encoding NDM-1 in clinical Raoultella ornithinolytica and Leclercia adecarboxylata from China

Raoultella ornithinolytica YNKP001 and Leclercia adecarboxylata P10164, which harbor conjugative plasmids pYNKP001-NDM and pP10164-NDM, respectively, were isolated from two different Chinese patients, and their complete nucleotide sequences were determined. Production of NDM-1 enzyme by these plasmids accounts for the carbapenem resistance of these two strains. This is the first report of blaNDM in L. adecarboxylata and third report of this gene in R. ornithinolytica. pYNKP001-NDM is very similar to the IncN2 NDM-1-encoding plasmids pTR3, pNDM-ECS01, and p271A, whereas pP10164-NDM is similar to the IncFIIY blaNDM-1-carrying plasmid pKOX_NDM1. The blaNDM-1 genes of pYNKP001-NDM and pP10164-NDM are embedded in Tn125-like elements, which represent two distinct truncated versions of the NDM-1-encoding Tn125 prototype observed in pNDM-BJ01. Flanking of these two Tn125-like elements by miniature inverted repeat element (MITE) or its remnant indicates that MITE facilitates transposition and mobilization of blaNDM-1 gene contexts.

H-NS is a repressor of major virulence gene loci in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a leading cause of seafood-associated diarrhea and gastroenteritis, harbors three major virulence gene loci T3SS1, Vp-PAI (T3SS1+tdh2) and T6SS2. As showing in this study, the nucleoid-associated DNA-binding regulator H-NS binds to multiple promoter-proximal regions in each of the above three loci to repress their transcription, and moreover H-NS inhibits the cytotoxicitiy, enterotoxicity, hemolytic activity, and mouse lethality of V. parahaemolyticus. H-NS appears to act as a major repressor of the virulence of this pathogen. Date presented here would promote us to gain a deeper understanding of H-NS-mediated silencing of horizontally acquired virulence loci in V. parahaemolyticus.

Molecular Characterization of Direct Target Genes and cis-Acting Consensus Recognized by Quorum-Sensing Regulator AphA in Vibrio parahaemolyticus

Background AphA is the master quorum-sensing (QS) regulator operating at low cell density in vibrios. Molecular regulation of target genes by AphA has been characterized in Vibrio harveyi and V. cholerae, but it is still poorly understood in V. parahaemolyticus. Methodology/Principal Findings The AphA proteins are extremely conserved in V. parahaemolyticus, Vibrio sp. Ex25, Vibrio sp. EJY3, V. harveyi, V. vulnificus, V. splendidus, V. anguillarum, V. cholerae, and V. furnissii. The above nine AphA orthologs appear to recognize conserved cis-acting DNA signals which can be represented by two consensus constructs, a 20 bp box sequence and a position frequency matrix. V. parahaemolyticus AphA represses the transcription of ahpA, qrr4, and opaR through direct AphA-target promoter DNA association, while it inhibits the qrr2-3 transcription in an indirect manner. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and AphA-binding sites (containing corresponding AphA box-like sequences) were determined for the three direct AphA targets ahpA, qrr4, and opaR in V. parahaemolyticus. Conclusions/Significance AphA-mediated repression of ahpA, qrr2-4, and opaR was characterized in V. parahaemolyticus by using multiple biochemical and molecular experiments. The computational promoter analysis indicated the conserved mechanism of transcriptional regulation of QS regulator-encoding genes ahpA, qrr4, and opaR in vibrios.

Fur Is a Repressor of Biofilm Formation in Yersinia pestis

Background Yersinia pestis synthesizes the attached biofilms in the flea proventriculus, which is important for the transmission of this pathogen by fleas. The hmsHFRS operons is responsible for the synthesis of exopolysaccharide (the major component of biofilm matrix), which is activated by the signaling molecule 3′, 5′-cyclic diguanylic acid (c-di-GMP) synthesized by the only two diguanylate cyclases HmsT, and YPO0449 (located in a putative operonYPO0450-0448). Methodology/Principal Findings The phenotypic assays indicated that the transcriptional regulator Fur inhibited the Y. pestis biofilm production in vitro and on nematode. Two distinct Fur box-like sequences were predicted within the promoter-proximal region of hmsT, suggesting that hmsT might be a direct Fur target. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that Fur specifically bound to the hmsT promoter-proximal region for repressing the hmsT transcription. In contrast, Fur had no regulatory effect on hmsHFRS and YPO0450-0448 at the transcriptional level. The detection of intracellular c-di-GMP levels revealed that Fur inhibited the c-di-GMP production. Conclusions/Significance Y. pestis Fur inhibits the c-di-GMP production through directly repressing the transcription of hmsT, and thus it acts as a repressor of biofilm formation. Since the relevant genetic contents for fur, hmsT, hmsHFRS, and YPO0450-0448 are extremely conserved between Y. pestis and typical Y. pseudotuberculosis, the above regulatory mechanisms can be applied to Y. pseudotuberculosis.

Dissemination of IMP-4-encoding pIMP-HZ1-related plasmids among Klebsiella pneumoniae and Pseudomonas aeruginosa in a Chinese teaching hospital

A total of 26 blaIMP-4-carrying strains of Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from 2009 to 2013 in a Chinese teaching hospital, and these strains can be assigned into multiple sequence types or allelic profiles as determined by multilocus sequence typing. Of these strains, P. aeruginosa P378 and K. pneumoniae 1220 harbor the IMP-4-encoding plasmids pP378-IMP and p1220-IMP, respectively, whose complete nucleotide sequences are determined to be genetically closely related to the IncN1-type plasmid pIMP-HZ1. pP378-IMP/p1220-IMP-like plasmids are hinted to be present in all the other blaIMP-4-carrying strains, indicating the dissemination of pIMP-HZ1-related plasmids among K. pneumoniae or P. aeruginosa of different genotypes in this hospital. pP378-IMP carries two distinct accessory resistance regions, a blaIMP-4-carrying class 1 integron In823b, and a truncated Tn3-family unit transposon ΔTn6292-3′ harboring the quinolone resistance gene qnrS1. Massive fragmentation and rearrangement of these accessory genetic contents occur among p1220-IMP and IMP-HZ1 relative to pP378-IMP. blaIMP-4 is also present in the In823b remnants from p1220-IMP and IMP-HZ1, while qnrS1 is located in a Tn6292-derive fragment from pIMP-HZ1 but not found in p1220-IMP. pP378-IMP represents the first fully sequenced IncN-type plasmid from P. aeruginosa.

IMP-1 encoded by a novel Tn402-like class 1 integron in clinical Achromobacter xylosoxidans, China

Achromobacter xylosoxidans strain A22732 is isolated from a pneumonia patient in China and produces carbapenemases OXA-114e and IMP-1, which are encoded by chromosome and plasmid, respectively, and confer resistance to multiple ß-lactam antibiotics including carbapenems. The blaIMP-1 gene together with aacA7 and orfE is captured by a novel Tn402-like class 1 integron in a conjugative IncP-1ß plasmid. In addition to the intrinsic integron promoter PcW, there is still a blaIMP-1 gene cassette-specific promoter. This is the first report of carbapenemase-encoding IncP-1ß plasmid in clinical bacterial isolate.

CRP Acts as a Transcriptional Repressor of the YPO1635-phoPQ-YPO1632 Operon in Yersinia pestis

AbstractYersinia pestis is the causative agent of plague. Both cyclic AMP receptor protein (CRP) and PhoP are involved in regulating virulence-related genes in Y. pestis. The phoPQ loci are transcribed as two distinct operons, YPO1635-phoPQ-YPO1632 and phoPQ-YPO1632. In the present work, the regulation of the first operon by CRP was investigated using primer extension, LacZ fusion, electrophoretic mobility shift assay, and DNase I footprinting assays. The results showed that CRP bound to a DNA region overlapping core promoter −10 element and transcription start of YPO1635 to repress the expression of YPO1635-phoPQ-YPO1632. Taken together with our previous results, complex regulatory interactions of CRP-AMP and PhoP/PhoQ were proposed in Y. pestis, which would contribute to tightly controlled expression of virulence-related genes.

The first report of detecting the blaSIM-2 gene and determining the complete sequence of the SIM-encoding plasmid

An imipenem-resistant Pseudomonas aeruginosa strain HN39 that harbours a blaSIM-2-carrying plasmid pHN39-SIM, was isolated from a patient with craniocerebral infections in China. The SIM-2 protein differs from SIM-1 by a single amino acid substitution Gly198Asp. pHN39-SIM is a novel 282-kb megaplasmid and it possesses the replication and partition systems of an unknown incompatibility group. pHN39-SIM carries a total of ten separate accessory modules especially including a novel 38.8-kb multidrug resistance region. In addition to the known transposable elements ISPst3, a Tn5563a remnant, IS1071, Tn5046, ΔTn4662a and ΔTn512, harboured in these accessory modules are six novel ones ISPa59 to ISPa62, In1208 and Tn6284. The multidrug resistance region is composed of Tn6284 generated from the insertion of an In4-family integron In1208 into Tn5046, and a Tn4662a-derived element with the insertion of ΔTn512 connected with two other genes. In1208 carries not only blaSIM-2 but several additional genes accounting for resistance to erythromycin, chloramphenicol, rifampicin, streptomycin, quaternary ammonium compounds, sulphonamides and mercury.