Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist’s toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.

Aims:  To establish a novel cell surface display system that would enable the display of target proteins on Lactobacillus plantarum. Methods and Results:  BlastP analysis of the amino acids sequence data revealed that the N‐terminus of the putative muropeptidase MurO from L. plantarum contained two putative lysin motif (LysM) repeat regions, implying that the MurO was involved in bacterial cell wall binding. To investigate the potential of MurO for surface display, green fluorescent protein (GFP) was fused to MurO at its C‐terminus and the resulting fusion protein was expressed in Escherichia coli. After being mixed with L. plantarum cells in vitro, GFP was successfully displayed on the surfaces of L. plantarum cells. Increases in the fluorescence intensities of chemically pretreated L. plantarum cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for MurO. SDS sensitivity assay showed that the GFP fluorescence intensity was reduced after being treated with SDS. To demonstrate the applicability of the MurO‐mediated surface display system, β‐galactosidase from Bifidobacterium bifidium, in place of GFP, was functionally displayed on the surface of L. plantarum cells via MurO. Conclusions:  The MurO was a novel anchor protein for constructing a surface display system for L. plantarum. Significance and Impact of Study:  The success in surface display of GFP and β‐galactosidase opened up the feasibility of employing the cell wall anchor of MurO for surface display in L. plantarum.

The capability of cold-adaptation is a prerequisite of microorganisms that survive in an environment with frequent fluctuations in temperature. As a global causative agent of vibriosis in marine fish farming, Vibrio anguillarum can efficiently grow and proliferate under cold-stress conditions, which is 15°C lower than the optimal growth temperatures (25-30°C). Our data showed that V. anguillarum was able to synthesize ectoine de novo and that ectoine was essential for its growth under cold stress. Using H nuclear magnetic resonance spectroscopy and mutants lacking ectABC and proVWX (ectoine synthesis and transporter system genes, respectively), we confirmed that accumulation of this compatible solute occurs strictly at low temperatures and that the expression of ectA and proV is highly activated in the stationary growth phase. However, the synthesis of ectoine was repressed by exogenous choline (precursor of glycine betaine), suggesting that ectoine is an alternative compatible solute as a cold-stress protectant in V. anguillarum. Based on these results, we present possible scenarios of the synthesis and uptake of ectoine, which will facilitate the understanding of the molecular mechanism of V. anguillarum adaptation to cold environments and help improve freezing-dry processes for the V. anguillarum live vaccine.

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Traditional soybean paste from Shandong Liangshan and Tianyuan Jiangyuan commercial soybean paste were chosen for analysis and comparison of their bacterial and fungal dynamics using denaturing gel gradient electrophoresis and 16S rRNA gene clone libraries. The bacterial diversity results showed that more than 20 types of bacteria were present in traditional Shandong soybean paste during its fermentation process, whereas only six types of bacteria were present in the commercial soybean paste. The predominant bacteria in the Shandong soybean paste were most closely related to Leuconostoc spp., an uncultured bacterium, Lactococcus lactis, Bacillus licheniformis, Bacillus spp., and Citrobacter freundii. The predominant bacteria in the Tianyuan Jiangyuan soybean paste were most closely related to an uncultured bacterium, Bacillus licheniformis, and an uncultured Leuconostoc spp. The fungal diversity results showed that 10 types of fungi were present in the Shandong soybean paste during the fermentation process, with the predominant fungi being most closely related to Geotrichum spp., an uncultured fungal clone, Aspergillus oryzae, and yeast species. The predominant fungus in the commercial soybean paste was Aspergillus oryzae.