ABSTRACT
Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.
ABSTRACT
Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.
ABSTRACT
Objective To characterize Hfq-dependent phenotypes in stress response and to dissect Hfq-dependent transcription of virulence genes and stress-responsive genes in Vibrio cholera.Methods The hfq null mutant strain (△hfq) and the complemented mutant strain (△hfq/pUC18-hfq) were constructed from the wild-type Vibrio cholera.Comparisons on the motility,biofilm formation,growth under various oxygen-supplying conditions,outer membrane resistance,and sensitivity to oxidative stress were analyzed between the wild type strain and the mutant strains.Reverse-transcript fluorescence quantitative PCR (RT-qPCR) was used to determine the transcriptional levels of target genes in the above mentioned strains.Results △hfq and △hfq/pUC18-hfq strains were successfully constructed.The motility,outer membrane resistance and sensitivity to oxidative stress were reduced,but biofilm formation was enhanced in the hfq null mutant strain.RT-qPCR testified that Hfq had regulation effects on gene transcription for forming falagellum,extracellular polysaccharide,outer membrane protein and oxidative stress in Vibrio cholera.Conclusion As a RNA chaperone,Hfq could affect Vibrio cholera in its biofilm formation,resistance to oxidative stress and antibiotics resistance through regulating the transcription of multiple metabolic genes and virulence genes,which indicates that Hfq,combined with other regulators,may play a key role in the complex regulation of metabolic genes and virulence genes.