Suicide Plasmid Research Articles

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.Methods and Principal FindingsTo facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.ConclusionsWe have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Attenuated virulence of pigment-producing mutant of Aeromonas veronii bv. sobria in HeLa cells and Nile tilapia (Oreochromis niloticus)

Aeromonas species are potential water/foodborne pathogens, whereas Aeromonas veronii bv. sobria is one of the most virulent species to human and fish. Most current experimental evidence has publicized that suicide plasmid dependent IS1-element untargeted integration into A. veronii bv. sobria ATCC 9071T strain was recently used to generate brown pigment-producing and spontaneous pelleting (BP+SP+) mutant. Current study was conducted to compare virulence of wild-type ATCC 9071T strain and its BP+SP+ mutant with respect to cytotoxicity in HeLa cells and lethality in Nile tilapia. It was found that the cytotoxicity of wild-type ATCC 9071T strain to HeLa cells has reached 75% versus 50% for the cytotoxicity of BP+SP+ mutant. Further, the median lethal dose (LD50) of wild-type ATCC 9071T strain in Nile tilapia was 8.25 Log10 colony-forming units (CFU)/ml, compared to 9.16Log10CFU/ml for the LD50 of BP+SP+ mutant. Thus, current study supports the notion that non pigment-producing Aeromonas strains are more virulent than pigment-producing ones.

A safety type genetically engineered bacterium with red fluorescence which can be used to degrade organophosphorus pesticides

To effectively biodegrade organophosphorus pesticides residue in environment, we constructed a genetically engineered bacterium (GEB) which can not only emit red fluorescence but also degrade organophosphorus pesticides residue, and this GEB can commit suicide when required. Two genes with different functions were placed under the control of different promoters. One was the dual gene expression vector pL-DsRed–pL-OPH in which genes coding for DsRed and organophosphorus hydrolase were independently placed downstream of two pL promoters. These genes could be expressed freely as long as the GEB was alive. The other was the conditional suicide plasmid pDS containing two suicide cassettes designed to induce bacteria to commit suicide when they detect arabinose. The lethal gene used in the suicide plasmid was the nuclease gene of Serratia marcescens without the leader-coding sequence. This was put under the control of the T7 promoter. Applying this type of secure GEB could potentially be a less hazardous environmental strategy in degrading pesticides and contamination.

Transposition of IS elements induced by electroporation of suicide plasmid in Acidithiobacillus caldus

Transposition insertional mutagenesis of the insertion sequences (IS elements) was discovered for the first time in Acidithiobacillus caldus (A. caldus), when A. caldus MTH-04 hsdM (type I restriction-modification system M-subunit) mutant was constructed by electroporation of a suicide plasmid. The IS element, specifically inserting into hsdM gene, was analyzed, identified, and named ISAtc2. The transposition frequency of ISAtc2 was ranged from 4% to 7%, and no reverse mutation occurred in the mutants after 50 generations of proliferation without selective pressure. These results revealed that transposition of IS elements on A. caldus chromosome could regulate the gene expression and metabolic pathways by gene inactivation, gene loss and gene acquisition. Therefore, the transposition of IS elements in A. caldus may be an important and unique regulation mechanism for adaptation to the living condition.

Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni

To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization. The plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice. The fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05). The function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Metabolism of Salmonella enterica serovar typhi influenced by RpoE and RpoS under hyperosmosis

To study the role of RpoE and RpoS on the influence of the metabolism and growth of bacterial under hyperosmotic stress. The rpoS/rpoE double deletion mutant of Salmonella enterica serovar typhi (S. typhi) was prepared by homologous recombination through the suicide plasmid mediated. The recombination was visualized by PCR. Growth curves were drawn by using photometric value A600 as the ordinate and cultivation time as abscissa. The survival abilities of bacterial were compared under hyperosmotic stress. Statistical differences of early logarithmic growth stage (4 h) and laters logarithmic growth stage (12 h) were analyzed by one-way ANOVA. The expression difference of metabolism related genes of wild-type and mutant strains of S. Typhi incubated under hyperosmotic stress were investigated by Salmonella genomic DNA microarray. Real-time quantitative PCR (qRT-PCR) was performed to validate the results of microarray assay in some selected genes. The rpoS/rpoE double deletion mutant of S. Typhi was successfully generated. The analysis of growth curve showed that the 4-hour and 12-hour A600 values were separately 0.503 ± 0.018 and 2.060 ± 0.112 in rpoS deletion mutant strains, 0.293 ± 0.053 and 1.933 ± 0.115 in rpoE deletion mutant strains, and 0.051 ± 0.007 and 0.963 ± 0.111 in rpoS/rpoE double deletion mutant strains; all of which were lower than the values of wild-type strains, who were 0.725 ± 0.097 and 2.496 ± 0.171, respectively. The difference were statistically significant (P < 0.05). The genomic DNA microarray revealed that 42 genes relevant with bacterial metabolism were influenced by RpoE and RpoS. Results of qRT-PCR showed that the expression values of rpsE, rbsK, nusG and etuB in rpoS deletion mutant strains were (1.86 ± 0.14)×10(6), (1.37 ± 0.11)×10(6), (2.72 ± 0.58)×10(6) and (8.27 ± 1.01)×10(6) copies/µg, respectively; while those in rpoE deletion mutant strains were (2.19 ± 0.17)×10(6), (1.51 ± 0.12)×10(6), (2.73 ± 0.57)×10(6) and (9.63 ± 1.42)×10(6) copies/µg, respectively. Compared with the values in wild-type strains, which were separately (1.94 ± 0.10)×10(6), (1.52 ± 0.11)×10(6), (2.39 ± 0.52)×10(6) and (10.83 ± 1.52)×10(6) copies/µg, the differences was not statistical significance (P > 0.05). However, compared with the values in rpoS/rpoE double mutant strains, which were separately (5.64 ± 0.59)×10(6), (4.17 ± 0.40)×10(6), (9.44 ± 1.22)×10(6) and (2.95 ± 0.88)×10(6) copies/µg, the difference was significant (P < 0.05). RpoE and RpoS could influence the expression of lots of metabolism genes. Together, they regulated the metabolism and growth of S. Typhi under hyperosmotic stress.

Suicide Plasmid-Dependent IS1-Element Untargeted Integration into Aeromonas veronii bv. sobria Generates Brown Pigment-Producing and Spontaneous Pelleting Mutant

Foodborne Gram-negative pathogens belonging to the genus Aeromonas are variable in harboring insertion sequence (IS) elements that play an important role in the generation of dysfunctional relatives of known genes. Using suicide plasmids carrying an IS1-element, untargeted integration is a common problem during experimental trials to generate specific mutations by homologous recombination. In this work, different strains of Aeromonas veronii bv. sobria (AeG1 and ATCC 9071T), A. hydrophila ATCC 19570, and A. sobria ATCC 43979T are examined for acquisition of IS1-element from pYAK1 suicide plasmid. It was found that untargeted integration of IS1-element is encountered only in ATCC 9071T strain. Such untargeted integration generates a novel brown pigment-producing and spontaneous pelleting (BP(+)SP(+)) mutant. Furthermore, BP(+)SP(+) mutant strain secretes significantly higher quantity of PilF homologous protein than the wild-type strain and displays an enhanced protein tyrosine phosphorylation activity. Thus, current work shows that Aeromonas spp. strains are variable in their susceptibility for suicide plasmid-dependent IS1-element untargeted integration as well as the susceptible strain is changed to mimic pigment-producing and spontaneous pelleting strains that are naturally occurring among heterogeneous group of foodborne aeromonads.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Bacteriochlorophyll f: properties of chlorosomes containing the “forbidden chlorophyll”

The chlorosomes of green sulfur bacteria (GSB) are mainly assembled from one of three types of bacteriochlorophylls (BChls), BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl fF. The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Qy absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 40% faster than the bchU mutant at low irradiance (10 μmol photons m−2 s-1). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally.

Fis Is Essential for the Stability of Linear Plasmid pBSSB1 and Affects the Motility of Salmonella enterica Serovar Typhi

pBSSB1 is a 27 kb non-bacteriophage-related linear plasmid first found in Salmonella enterica serovar Typhi (S. Typhi), but the mechanism underlying the replication of pBSSB1 is currently unknown. Previous reports showed that the factor for inversion stimulation (Fis) encoded by fis can affect the replication, transcription and other processes through binding DNA. Here, a fis deletion mutant of S. Typhi (Δfis) was prepared through the homologous recombination mediated by suicide plasmid and the loss of pBSSB1 in Δfis was observed surprisingly by pulsed field gel electrophoresis (PFGE). Subsequently, the loss of pBSSB1 was verified by PCR and Southern blot. In addition, the motility of Δfis was deficient and the flagellin of Δfis could not be detected by 2-dimensional polyacrylamide gel electrophoresis. All these results show that Fis is essential for the stability of pBSSB1 and affects the motility of S. Typhi.

Two Systems for Targeted Gene Deletion in Coxiella burnetii

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. C. burnetii's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori-based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5' and 3' flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5' or 3' target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. ΔdotA and ΔdotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of C. burnetii genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.

A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4–borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches.

Augmented production of alginate oligosaccharides by the Pseudomonas mendocina NK-01 mutant

Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHAMCL) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHAMCL synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight (MW). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHAMCL and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

BackgroundBacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.Methodology/Principal FindingsWe have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.Conclusions/SignificanceThe results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.

Green fluorescent protein (GFP)-labeling of enterobacteria associated with fruit flies (Diptera: Tephritidae) and persistence in their natural hostRhagoletis completaCresson.

Fruit flies (Diptera: Tephritidae) are a highly successful, widespread group of insects that cause economic damage in agriculture. Data available so far on the composition of the bacterial community associated with their digestive tract indicate that members of Enterobacteriaceae are the species most often isolated. Bacteria naturally occurring in insect guts may be engineered and used to study the spatial and functional interactions of microbes within the insect system and offer one route to meet the demand for novel insect pest management strategies. With this aim we introduced by conjugation the gfp gene carried by the suicide plasmid pTn5gfpmut1 into Klebsiella oxytoca and Raoultella (formerly Klebsiella ) spp. strains isolated from the oesophageal bulb of the fruit flies Ceratitis capitata (Wiedemann) and Rhagoletis completa Cresson, respectively. The GFP-encoding gene was stably maintained in two tested transgenic strains, both originally isolated from R. completa. In one case, GFP-labeled bacterial cells were used to feed larvae and adults of the original host. Genetically modified bacteria were able to colonize the gut of larvae and persisted through all larval instars to pupal stage.

Construction and identification of mutation of pgm gene attenuate the virulent of Brucella melitensis strain 16

Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines. Key words: Brucella vaccine; pgm genes; Gene fusion; Mutagenesis, insertional

Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica

The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (ΔpyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ΔpyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.

CheA/CheY signaling systemresponsible for chemotaxis in vitro and colonization in vivo of Campylobacter jejuni

Objective To determine the effects of Che A and CheY proteins ofCampylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization invivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector andexpression strain, respectively, prokaryotic expression systems of cheA and cheY genes ofC. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the targetrecombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG andrCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript-II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-outmutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotacticability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting thebacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY afterDOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. Thedifference of colonization ability between cheA- mutant and wild-type of C. jejuni in micewere checked and then compared. Results The constructed prokaryotic expression systemscould efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed thecheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotacticability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis ofwild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levelsof CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). Thecolonization ability in murine jejunum of cheA- mutant was also lower than that of thewild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system(Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activatedby dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro andcolonization in vivo of C. jejuni, and this protein can be used as a target for developingnovel anti- C. jejuni drugs.

Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

Role of Helicobacter pylori cheA gene in chemotaxis in vitro and colonizationin vivo

Objective To determine the effect of cheA gene of Helicobacter pylori in the bacterial chemotaxis in vitro and colonization in vivo. Methods The entire cheA and cheY genes were amplified and cloned from genomic DNA of H. pylori NCTC11637 strain. Subsequently, the prokaryotic expression systems of cheA and cheY genes were generated and the target recombinant proteins rCheA and rCheY were extracted by Ni-NTA affinity chromatography. Rabbits were immunized with either rCheA or rCheY for obtaining antisera, and rCheA-IgG and rCheY-IgG in the antisera were prepared using ammonium sulfate precipitation plus DEAE-52 column chromatography. A suicide plasmid of cheA gene was constructed and then a cheA gene knock-out mutant ( cheA - ) was generated based on homologous recombinant exchange using the suicide plasmid. The cheA- mutant was identified using PCR and sequencing. The phosphorylation levels of CheA and CheY molecules of cheA - and wild-type strain were determined by using rCheA-IgG and rCheY-IgG anchoring the target proteins and protein phosphorylation detection kit. The differences of chemotaxis in vitro and colonization in vivo between cheA- mutant and wild-type strain were compared using chemotactic model and BALB/c infection model of H. pylori. Results The cheA gene knock-out in genome of cheA- mutant was confirmed by the results of PCR and sequencing. After treated with 0. 001-0. 1 mol/L HCI for 10 min, the phosphorylation levels of CheA and CheY molecules of wild-type strain were rapidly descended from ( 59.6 ±11.5) μmol and (55.5 ± 10.2) μmol to ( 10.8 ± 2.6) and (5. 5 ± 1.2) μmol (P ＜ 0.05 ), while the phosphorylation of CheY molecule of cheA - mutant was no markedly changed with a persistent lower level ( P ＞0.05). The diameters [(10-20) ± (2-3) mm] of chemotactic aggregative rings of cheA- mutant were significantly less than those [(16-24) ± (2-3)mm] of wild-type strain (P ＜0.05). The positive isolation rate (90%) of H. pylori in gastric biopsy specimens of mice that infected with wild-type strain was remarkably higher than that (40%) of mice that infected with cheA- mutant (P ＜0.05). The result of fluorescence quantitative was also showed that the numbers (6.3 × 103 ±2.1 × 103 copies/mg) of H. pylori in gastric biopsy specimens of wild-type strain infected mice were significantly larger than those (8.3 × 101 ±3. 1 × 101 copies/mg) in gastric biopsy specimens ofcheA- mutant infected mice (P＜0.05). Conclusion The cheA gene of H. pylori has an important role in the bacterial chemotaxis in vitro and colonization in vivo. Key words: Helicobacter pylori ; cheA gene ; cheY gene ; Gene knock-out ; Chemotaxis ;