Bioinformatics analysis of gene bhsA and its role in Ca2+ -treated Escherichia coli.

One of the commonly employed methods in molecular biology is to utilize calcium chloride to treat Escherichia coli for the preparation of competent cells to facilitate foreign gene expression. However, the molecular mechanisms underlying Ca2+ mediation of competent cell formation and identification of the key genes involved in the process remain unclear. In previous studies, the combined analysis of transcriptomics and proteomics revealed bhsA as one of the crucial genes. The gene ontology functional annotation of bhsA identified it as a member of the YhcN family encoding an outer membrane protein that confers resistance to various stresses. The IPR0108542 domain found within the protein plays a significant role in stress response and biofilm formation in E. coli. Analysis of the STRING database showed that the proteins interacting with bhsA are primarily involved in biofilm formation and stress resistance. Using the RED homologous recombination method, a bhsA deletion strain was constructed to verify its role in E. coli genetic transformation. Although the mutant strain showed no significant differences in morphology or growth trend when compared to the wild-type strain, its transformation efficiency decreased by 1.14- and 1.64-fold with plasmids pUC19 and pET-32a. Furthermore, the 1-N-phenylnaphthylamine assay indicated a 1.71-fold reduction in cell membrane permeability in the mutant strain.

Review of knockout technology approaches in bacterial drug resistance research.

Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.

Identification of Key Genes during Ca2+-Induced Genetic Transformation in Escherichia coli by Combining Multi-Omics and Gene Knockout Techniques.

The molecular mechanism of the Ca2+-mediated formation of competent cells in Escherichia coli remains unclear. In this study, transcriptome and proteomics techniques were used to screen genes in response to Ca2+ treatment. A total of 333 differentially expressed genes (317 upregulated and 16 downregulated) and 145 differentially expressed proteins (54 upregulated and 91 downregulated) were obtained. These genes and proteins are mainly enriched in cell membrane components, transmembrane transport, and stress response-related functional terms. Fifteen genes with these functions, including yiaW, ygiZ, and osmB, are speculated to play a key role in the cellular response to Ca2+. Three single-gene deletion strains were constructed with the Red homologous recombination method to verify its function in genetic transformation. The transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. None of the three gene deletion strains changed in size, which is one of the main elements of microscopic morphology, but they exhibited different membrane permeabilities and transformation efficiencies. This study demonstrates that Ca2+-mediated competence formation in E. coli is not a simple physicochemical process and may involve the regulation of genes in response to Ca2+. This study lays the foundation for further in-depth analyses of the molecular mechanism of Ca2+-mediated transformation. IMPORTANCE Using transcriptome and proteome techniques and association analysis, we identified several key genes involved in the formation of Ca2+-mediated E. coli DH5α competent cells. We used Red homologous recombination technology to construct three single-gene deletion strains and found that the transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. These results proved that the genetic transformation process is not only a physicochemical process but also a reaction process involving multiple genes. These results suggest ways to improve the horizontal gene transfer mechanism of foodborne microorganisms and provide new ideas for ensuring the safety of food preservation and processing.

Zn2+-dependent enhancement of Atrazine biodegradation by Klebsiella variicola FH-1.

Degradation efficiency of Atrazine by Klebsiella variicola FH-1 is improved by the addition of Zn2+. Both the chromosome and plasmid genomes of strain FH-1 were sequenced and annotated to identify genes involved in the degradation of Atrazine. Four open reading frames (ORFs) 1040, 2582, 3597, and 4043 encoding Zn2+-dependent hydrolases were knocked out to verify their predicted functions in the degradation of Atrazine. In the presence of Zn2+, the biodegradation efficiency of Atrazine by knockout mutant ∆ORF 3597 was 13.7% lower than that of wild type (WT) of strain FH-1 but still 9.4% higher than that of WT without Zn2+. These results indicated that ORF 3597 played a synergistic role but may not be the sole factor involved in the degradation of Atrazine. The enzymatic activities of pydC encoded by ORF 3597 were further characterized in the degradation of Atrazine. Results of fluorescence staining and flow cytometry analyses showed that the survival of bacterial cells and cell membrane permeability were increased in the presence of Zn2+ at different concentrations. Our study provided a scientific foundation for further investigation of the biological mechanisms of improving the degradation of Atrazine by strain FH-1 with the presence of Zn2+.

Comparative Whole Genome Analysis of Escherichia coli O157:H7 Isolates From Feedlot Cattle to Identify Genotypes Associated With the Presence and Absence of stx Genes.

A comparative whole genome analysis was performed on three newly sequenced Escherichia coli O157:H7 strains with different stx profiles, previously isolated from feedlot cattle [C1-010 (stx1-, stx2c+), C1-057 (stx-), and C1-067 (stx1+, stx2a+)], as well as five foodborne outbreak strains and six stx-negative strains from NCBI. Phylogenomic analysis demonstrated that the stx2c-carrying C1-010 and stx-negative C1-057 strains were grouped with the six NCBI stx-negative E. coli O157:H7 strains in Cluster 1, whereas the stx2a-carrying C1-067 and five foodborne outbreak strains were clustered together in Cluster 2. Based on different clusters, we selected the three newly sequenced strains, one stx2a-carrying strain, and the six NCBI stx-negative strains and identify their prophages at the stx insertion sites. All stx-carrying prophages contained both the three Red recombination genes (exo, bet, gam) and their repressor cI. On the other hand, the majority of the stx-negative prophages carried only the three Red recombination genes, but their repressor cI was absent. In the absence of the repressor cI, the consistent expression of the Red recombination genes in prophages might result in more frequent gene exchanges, potentially increasing the probability of the acquisition of stx genes. We further investigated each of the 10 selected E. coli O157:H7 strains for their respective unique metabolic pathway genes. Seven unique metabolic pathway genes in the two stx2a-carrying strains and one in the single stx2c-carrying and seven stx-negative strains were found to be associated with an upstream insertion sequence 629 within a conserved region among these strains. The presence of more unique metabolic pathway genes in stx2a-carrying E. coli O157:H7 strains may potentially increase their competitiveness in complex environments, such as feedlot cattle. For the stx2c-carrying and stx-negative E. coli O157:H7 strains, the fact that they were grouped into the same phylogenomic cluster and had the same unique metabolic pathway genes suggested that they may also share closely related evolutionary pathways. As a consequence, gene exchange between them is more likely to occur. Results from this study could potentially serve as a basis to help develop strategies to reduce the prevalence of pathogenic E. coli O157:H7 in livestock and downstream food production environments.

Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Salmonella enterica Serovar Typhimurium.

To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or λ red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on the Salmonella enterica serovar Typhimurium chromosome. The method combines an efficient seamless assembly system in vitro, red homologous recombination in vivo, and counterselection marker sacB. To test this method, with the seamless assembly system, various modification fragments for target genes cpxR, cpxA, and acrB were rapidly and efficiently constructed in vitro. sacBKan cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of Salmonella Typhimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace the sacBKan cassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 × FLAG tagging of the target genes. This method can be used in any bacterial species that supports sacB gene activity and λ red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

Effect of relA knockout on heterologous resistance of colistin to Acinetobacter baumannii / 上海交通大学学报（医学版）

Objective: To observe the effect of the (p)ppGpp synthase gene (relA) on the heteroresistance of colistin against Acinetobacter baumannii. Methods: The relA gene in Acinetobacter baumannii ATCC19606 was knocked out by Red homologous recombination technique. The biofilm formation of Acinetobacter baumannii was observed by crystal violet staining. The change of heterogeneous colonies of Acinetobacter baumannii under the action of colistin was detected by population analysis profiles (PAP) and the heterogeneity was calculated. The killing curve was used to detect the formation of persistent bacteria in Acinetobacter baumannii under the action of colistin. Results: The relA gene in Acinetobacter baumannii was successfully knocked out, and the relA knockout strain ATCC19606-ΔrelA was obtained. After relA gene knockout, the biofilm formation of Acinetobacter baumannii decreased significantly. After relA gene knockout, Acinetobacter baumannii significantly reduced the heterogeneous colonies and persistent bacteria formation under the action of colistin. Conclusion: The bacterial stringent reaction (p) ppGpp synthase relA may be an important factor affecting the heteroresistance of Acinetobacter baumannii to colistin.

Construction of sRNA-deficient and-overexpressing strains of Yersinia pestis / 军事医学

Objective To construct small RNA deletion and overexpression strains with a length of less than 100 nt in Yersinia pestis.Methods Deletion mutants of the target sRNAs were constructed by increasing the length of homologous regions.Meanwhile, the high copy plasmid pBAD/HisA was modified into an inducible transcriptional vector as an sRNA-overexpression plasmid by using QuikChange lightning site-directed mutagenesis kit .The presence , size, and transcription-al initiation sites of the indicated sRNA were predicted by transcriptome sequencing , primer extension , and previous stud-ies.The full-length DNA fragments of target sRNAs were transformed into the transcriptional vector .The overexpressing strains of sRNAs were identified by Northern Blot .Results and Conclusion Four sRNAs deletion mutants of sR01, sR02, sR03 and HmsA and three sRNAs overexpression mutants MicF , HmsA and CpxQ were successfully constructed .A method of construction of sRNA deficient and overexpressing strains of Y.pestis has been quickly and efficiently established by λ-Red homologous recombination technology and QuikChange ? lightning site-directed mutagenesis kit.

Construction of blaNDM-1gene deletion mutant of Enterobacter cloacae and analysis of its biological characteristics / 中华微生物学和免疫学杂志

Objective To construct a blaNDM-1gene deletion mutant of Enterobacter cloacae and to analyze its biological characteristics. Methods The blaNDM-1gene deletion mutant was constructed by using Red homologous recombination technology and verified by PCR and RT-qPCR. Antimicrobial susceptibility profiles,growth curves and in vitro competition abilities of the original strain and the blaNDM-1gene deletion mutant were analyzed. Results PCR,DNA sequencing and RT-qPCR showed that the blaNDM-1gene dele-tion mutant was successfully constructed. Antimicrobial susceptibility test showed that the original strain was resistant to imipenem,meropenem and ertapenem, while the blaNDM-1gene deletion mutant was sensitive to all. The original strain and the blaNDM-1gene deletion mutant had similar growth curves in Luria-Bertani liq-uid medium. In vitro competition experiment revealed that the competitive index of them was 0.69. Conclu-sion Red homologous recombination technology can be used to knockout the blaNDM-1gene of Enterobacter cloacae,which is associated with antimicrobial resistance and competitiveness.

Direct cloning and transplanting of large DNA fragments from Escherichia coli chromosome.

We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts: cma (which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.

Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant / 军事医学

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .