scholarly journals The Acquisition of the scr Gene Cluster Encoding Sucrose Metabolization Enzymes Enables Strains of Vibrio parahaemolyticus and Vibrio vulnificus to Utilize Sucrose as Carbon Source

2021 ◽  
Vol 12 ◽  
Author(s):  
Jens Andre Hammerl ◽  
Cornelia Göllner ◽  
Claudia Jäckel ◽  
Fatima Swidan ◽  
Helena Gutmann ◽  
...  
Keyword(s):  
Carbon Source ◽  
Vibrio Parahaemolyticus ◽  
Genomic Island ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
E Coli ◽  
Gene Coding ◽  
Chromosome Ii ◽  
Pcr Targeting ◽  
Sucrose Utilization

Most strains of Vibrio parahaemolyticus are unable to utilize sucrose as carbon source, though few exceptions exist. We investigated a sucrose-positive V. parahaemolyticus strain by whole-genome sequencing (WGS) and confirmed the presences of a genomic island containing sucrose utilization genes. A 4.7 kb DNA cluster consisting of three genes: scrA encoding a sucrose uptake protein, scrK encoding a fructokinase, and scrB coding for a sucrose-6-phosphate hydrolase, was PCR amplified and inserted into the Vibrio/Escherichia coli shuttle vector pVv3. Two recombinant plasmids, only differing in the orientation of the insert with respect to the pVv3-lacZα-fragment, conferred the E. coli K12 transformants the ability to utilize sucrose. The introduction of the two plasmids into sucrose-negative V. parahaemolyticus and V. vulnificus strains also results in a change of the sucrose utilization phenotype from negative to positive. By performing a multiplex PCR targeting scrA, scrK, and scrB, 43 scr-positive V. parahaemolyticus isolates from our collection of retail strains were detected and confirmed to be able to use sucrose as carbon source. Strains unable to utilize the disaccharide were negative by PCR for the scr genes. For in-depth characterization, 17 sucrose-positive V. parahaemolyticus were subjected to WGS. A genomic island with a nucleotide identity of >95% containing scrA, scrB, scrK and three additional coding sequences (CDS) were identified in all strains. The additional genes were predicted as a gene coding for a transcriptional regulator (scrR), a porin encoding gene and a CDS of unknown function. Sequence comparison indicated that the genomic island was located in the same region of the chromosome II in all analyzed V. parahaemolyticus strains. Structural comparison of the genomes with sequences of the sucrose utilizing species V. alginolyticus revealed the same genomic island, which indicates a possible distribution of this genetic structure by horizontal gene transfer. The comparison of all genome sequences based on SNP differences reveals that the presence of sucrose utilizing genes is found in genetically diverse V. parahaemolyticus strains and is not restricted to a subset of closely related strains.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

scholarly journals Cold Shock Induces Chromosomal qnr in Vibrio Species and Plasmid-Mediated qnrS1 in Escherichia coli

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Hee-Chang Jang ◽  
Yin Wang ◽  
Chunhui Chen ◽  
Laura Vinué ◽  
George A. Jacoby ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Community ◽  
Vibrio Parahaemolyticus ◽  
Cold Shock ◽  
Vibrio Vulnificus ◽  
Wild Type ◽  
Induced Expression ◽  
Content Type ◽  
E Coli ◽  
Dna Damaging Agents

ABSTRACT qnr genes are found in aquatic bacteria and were present in the bacterial community before the introduction of synthetic quinolones. Their natural functions are unknown. We evaluated expression of chromosomal qnr in Vibrio species in response to environmental stresses and DNA-damaging agents. Subinhibitory concentrations of quinolones, but not other DNA-damaging agents, increased expression of chromosomal qnr by more than five times in Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio mytili. Cold shock also induced expression of qnr in V. parahaemolyticus, V. vulnificus, and V. mytili, as well as expression of qnrS1 in Escherichia coli. qnrS1 induction by cold shock was not altered in ΔihfA or ΔihfB mutants or in a strain overexpressing dnaA, all of which otherwise directly modulate qnrS1 induction by ciprofloxacin. In contrast, the level of qnrS1 induction by cold shock was reduced in a ΔcspA mutant in the cold shock regulon compared to the wild type. In conclusion, cold shock and quinolones induce expression of chromosomal qnr in Vibrio species and of the related qnrS1 gene in E. coli.

scholarly journals Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− from E. coli O157:H7

2005 ◽  
Vol 71 (8) ◽  
pp. 4875-4878 ◽  
Author(s):  
Andreas Janka ◽  
Georg Becker ◽  
Anne-Katharina Sonntag ◽  
Martina Bielaszewska ◽  
Ulrich Dobrindt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Island ◽  
Enterohemorrhagic Escherichia Coli ◽  
Resistance Locus ◽  
Escherichia Coli O157 ◽  
Related Sequence ◽  
E Coli ◽  
Pcr Targeting

ABSTRACT A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H− from EHEC O157:H7.

scholarly journals A Markerless Gene Deletion System in Streptococcus suis by Using the Copper-Inducible Vibrio parahaemolyticus YoeB Toxin as a Counterselectable Marker

2021 ◽  
Vol 9 (5) ◽  
pp. 1095
Author(s):  
Chengkun Zheng ◽  
Man Wei ◽  
Jun Qiu ◽  
Jinquan Li
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Gene Deletion ◽  
Efflux Pump ◽  
Shuttle Vector ◽  
Streptococcus Suis ◽  
Liquid Media ◽  
Zoonotic Pathogen ◽  
E Coli ◽  
Reverse Transcription Quantitative Pcr ◽  
Severe Infections

Streptococcus suis is an important zoonotic pathogen causing severe infections in swine and humans. Induction of the Vibrio parahaemolyticus YoeB toxin in Escherichia coli resulted in cell death, leading to the speculation that YoeBVp can be a counterselectable marker. Herein, the counterselection potential of YoeBVp was assessed in S. suis. The yoeBVp gene was placed under the copper-induced promoter PcopA. The PcopA-yoeBVp construct was cloned into the S. suis-E. coli shuttle vector pSET2 and introduced into S. suis to assess the effect of YoeBVp expression on S. suis growth. Reverse transcription quantitative PCR showed that copper induced yoeBVp expression. Growth curve analyses and spot dilution assays showed that YoeBVp expression inhibited S. suis growth both in liquid media and on agar plates, revealing that YoeBVp has the potential to be a counterselectable marker for S. suis. A SCIY cassette comprising the spectinomycin-resistance gene and copper-induced yoeBVp was constructed. Using the SCIY cassette and peptide-induced competence, a novel two-step markerless gene deletion method was established for S. suis. Moreover, using the ΔperR mutant generated by this method, we demonstrated that PmtA, a ferrous iron and cobalt efflux pump in S. suis, was negatively regulated by the PerR regulator.

scholarly journals Sialic Acid Catabolism and Transport Gene Clusters Are Lineage Specific in Vibrio vulnificus

2012 ◽  
Vol 78 (9) ◽  
pp. 3407-3415 ◽  
Author(s):  
Jean-Bernard Lubin ◽  
Joseph J. Kingston ◽  
Nityananda Chowdhury ◽  
E. Fidelma Boyd
Keyword(s):  
Sialic Acid ◽  
Carbon Source ◽  
Vibrio Vulnificus ◽  
Pathogenicity Island ◽  
Gene Clusters ◽  
Acid Uptake ◽  
Acid Transport ◽  
Content Type ◽  
Natural Isolates ◽  
Chromosome Ii

ABSTRACTSialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that severalVibriospecies carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. InVibrio choleraeon chromosome I, these genes are carried on theVibriopathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequencedVibrio vulnificusclinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present withinV. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I ofV. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region inV. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation insiaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific inV. vulnificusand that the TRAP transporter is essential for sialic acid uptake.

scholarly journals Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

2007 ◽  
Vol 56 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Dobryan M. Tracz ◽  
Paul G. Backhouse ◽  
Adam B. Olson ◽  
Joanne K. McCrea ◽  
Julie A. Walsh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Vibrio Vulnificus ◽  
Comparative Sequence Analysis ◽  
Molecular Techniques ◽  
Array Technology ◽  
Species Specific ◽  
Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

scholarly journals Immunomagnetic Separation and Coagglutination of Vibrio parahaemolyticus with Anti-Flagellar Protein Monoclonal Antibody

2008 ◽  
Vol 15 (10) ◽  
pp. 1541-1546 ◽  
Author(s):  
S. Datta ◽  
M. E. Janes ◽  
J. G. Simonson
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Cell Suspension ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Immunomagnetic Separation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Isolation ◽  
Flagellar Protein ◽  
Phosphate Buffered Saline

ABSTRACT Mice were immunized by injection of Vibrio parahaemolyticus ATCC 17802 polar flagellin in order to produce monoclonal antibodies (mAbs). mAbs were analyzed by anti-H enzyme-linked immunosorbent assay using V. parahaemolyticus polar flagellar cores. The mAb exhibiting the highest anti-H titer was coated onto Cowan I Staphylococcus aureus cells at a concentration of 75 μg/ml cell suspension and used for slide coagglutination. Of 41 isolates identified genetically as V. parahaemolyticus, 100% coagglutinated with the anti-H mAb within 30 s, and the mAb did not react with 30 isolates identified as Vibrio vulnificus. A strong coagglutination reaction with V. parahaemolyticus ATCC 17802 was still observed when the S. aureus cells were armed with as little as 15 μg of mAb/ml S. aureus cell suspension. At this concentration, the mAb cross-reacted with three other Vibrio species, suggesting that they share an identical H antigen or antigens. The anti-H mAb was then used to optimize an immunomagnetic separation protocol which exhibited from 35% to about 45% binding of 102 to 103 V. parahaemolyticus cells in phosphate-buffered saline. The mAb would be useful for the rapid and selective isolation, concentration, and detection of V. parahaemolyticus cells from environmental sources.

scholarly journals Macrolide Resistance Gene mreA ofStreptococcus agalactiae Encodes a Flavokinase

2001 ◽  
Vol 45 (8) ◽  
pp. 2280-2286 ◽  
Author(s):  
Gervais Clarebout ◽  
Corinne Villers ◽  
Roland Leclercq
Keyword(s):  
High Performance ◽  
Shuttle Vector ◽  
Sodium Dodecyl ◽  
Macrolide Resistance ◽  
Upstream Region ◽  
Flavin Mononucleotide ◽  
Active Efflux ◽  
Fourfold Increase ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned inEscherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreArevealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

scholarly journals Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

2004 ◽  
Vol 47 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Paramasamy Gunasekaran
Keyword(s):  
Copy Number ◽  
Zymomonas Mobilis ◽  
Northern Blot Analysis ◽  
Shuttle Vector ◽  
Transcript Levels ◽  
E Coli ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Sacb Gene ◽  
In Cells

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB.

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