Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani, Rajasthan, India.
Microbiol Spectr. 2022 Jun 29;10(3):e0020222. doi: 10.1128/spectrum.00202-22. Epub 2022 Jun 9.
The CRISPR-Cas mediated regulation of biofilm by Salmonella enterica serovar Typhimurium was investigated by deleting CRISPR-Cas components , , , and We determined that the system positively regulates surface biofilm while inhibiting pellicle biofilm formation. Results of real-time PCR suggest that the flagellar (, ) and curli () genes were repressed in knockout strains, causing reduced surface biofilm. The mutants displayed altered pellicle biofilm architecture. They exhibited bacterial multilayers and a denser extracellular matrix with enhanced cellulose and less curli, ergo weaker pellicles than those of the wild type. The cellulose secretion was more in the knockout strains due to the upregulation of , which is necessary for cellulose export. We hypothesized that the secreted cellulose quickly integrates into the pellicle, leading to enhanced pellicular cellulose in the knockout strains. We determined that is upregulated in the knockout strains, thereby inhibiting the expression of and, hence, also of and . The conflicting upregulation of , the last gene of the operon, could be caused by independent regulation by the CRISPR-Cas system owing to a partial match between the CRISPR spacers and gene. The cAMP-regulated protein (CRP)-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of governing the availability of the sigma factor σ that further regulates class 3 flagellar genes (, , and ). Additionally, the variations in the lipopolysaccharide (LPS) profile and expression of LPS-related genes (, , and ) in knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle biofilm. In addition to being implicated in bacterial immunity and genome editing, the CRISPR-Cas system has recently been demonstrated to regulate endogenous gene expression and biofilm formation. While the function of individual genes in controlling Salmonella biofilm has been explored, the regulatory role of CRISPR arrays in biofilm is less studied. Moreover, studies have focused on the effects of the CRISPR-Cas system on surface-associated biofilms, and comprehensive studies on the impact of the system on pellicle biofilm remain an unexplored niche. We demonstrate that the CRISPR array and genes modulate the expression of various biofilm genes in Salmonella, whereby surface and pellicle biofilm formation is distinctively regulated.
我们通过删除 CRISPR-Cas 成分 、 、 、 和 来研究鼠伤寒沙门氏菌中 CRISPR-Cas 介导的生物膜调控。结果表明，该系统正向调节表面生物膜，同时抑制菌膜生物膜的形成。实时 PCR 的结果表明，鞭毛（ 、 ）和卷曲（ ）基因在敲除株中受到抑制，导致表面生物膜减少。突变体表现出改变的菌膜生物膜结构。它们显示出细菌多层结构和更密集的细胞外基质，增强了纤维素和较少的卷曲，因此比野生型菌膜更弱。由于 上调，敲除株的纤维素分泌更多，这对于纤维素外排是必要的。我们假设分泌的纤维素很快整合到菌膜中，导致敲除株中菌膜纤维素增加。我们确定 在上调，从而抑制 、 和 、 的表达。操纵子最后一个基因 的冲突上调可能是由于 CRISPR-Cas 系统的独立调控引起的，因为 CRISPR 间隔子和 基因之间存在部分匹配。CRISPR-Cas 系统可能通过调控 来调节操纵子的最后一个基因，从而绕过敲除株中 cAMP 调节的鞭毛基因的 CRP 调节，该基因控制着 sigma 因子 σ 的可用性，进一步调节第三类鞭毛基因（ 、 、和 ）。此外，敲除株中脂多糖（LPS）谱和 LPS 相关基因（ 、 、和 ）的表达变化也可能导致菌膜结构的改变。总的来说，我们确定 CRISPR-Cas 系统差异调节表面附着和菌膜生物膜的形成。除了参与细菌免疫和基因组编辑外，CRISPR-Cas 系统最近还被证明可以调节内源性基因表达和生物膜形成。虽然已经探索了单个 基因在控制沙门氏菌生物膜中的作用，但 CRISPR 阵列在生物膜中的调节作用研究较少。此外，研究集中在 CRISPR-Cas 系统对表面相关生物膜的影响上，而系统对菌膜生物膜的全面研究仍然是一个未探索的领域。我们证明，CRISPR 阵列和 基因调节沙门氏菌中各种生物膜基因的表达，从而独特地调节表面和菌膜生物膜的形成。
