Cloning And Characterization Of The RNA Chaperone HFQ In Lysobacter Enzymogenes

Lysobacter enzymogenes is a ubiquitous plant-associated and environmental-friendly bacterium emerging as a novel biological control agent.Our lab isolated L. enzymogenes OH 11 from cayenne-root soil. It is characterizaed with a high G+C content and gliding motility and has been shown to produce extracellular lytic enzymes including chitinase, protease, cellulase and β-1,3-glucanase. In addition, Lysobacter enzymogenes has good antagonism activity for many plant pathogenic fungi, oomycetes and bacterium, mainly because it can produce secondary antimicrobial substance:The HSAF is a heat-stable and broad-spectrum antifungal and anti-oomycete compound with a novel model of action, the cyclic lipodepsipeptide WAP8294A2, which exhibits anti-gram-positive bacteria. But the regulatory mechanisms of HSAF and WAP-8294A2 biosynthesis is largely unknown in L. enzymogenes. Hfq is a highly conserved RNA-binding protein and a key factor of post-transcriptional regulation, and also a global regulator of gene expression in bacteria. Hfq plays an important role in diversity life activities such as growth, tolerance H2O2 and virulence of some pathogens, etc. The objectives of this work are to clear the function of Hfq in Lysobacter enzymogenes.In present study, with the genomic information of Xanthomonas and Escherichia coli in NCBI database we cloned a Hfq protein. In the L. enzymogenes strain OH11, Hfq protein consists of 91 amino acids that has 89% identity with the Hfq protein from Xanthomonas campestris pv. campestris str. ATCC 33913 and 75% identity with the Hfq protein from Escherichia coli str. K-12 substr. MG1655. Besides that, hfq is flanked upstream by gene miaA and downstream by gene hflX, hflK and hflC, which is the same in the E.coli that hfq is in the miaA-hfq-hflX-hflK-hflC cluster.To begin to understand the role of Hfq in the L. enzymogenes strain OH11, we constructed an hfq mutant Ahfq and its complementation strain △hfq(hfq)△a/p based on mark-free genome replacement method. We found that loss of hfq reduced growth rate and was more sensitive to SDS, which harms the cell membrane and kills the cells, loss of hfq influenced the colony morphology but did not affect the surface motility. These results showed that hfq regulated the growth of the bacteria, environmental response and colony morphology. Compared with wild type, the mutation of hfq increased the yield of HSAF and WAP-8294A2, antagonistic activity against Rhizoctonia solani and Fusarium graminearum also increased. These results revealed that hfq participated in the regulation the biosynthesis of secondary metabolism substance HSAF and WAP-8294A2. What’s more, △hfq could not degrade the chitin in the medium, and the mutation loss the ability of chitin degradation is related to the synthesis of chitinase A significant reduced and the loss of ability to secrete. Overall, we preliminary studied the hfq gene in L. enzymogenes play a key role in the regulation of several important biological function, including growth, environmental response, colony morphology, antibiotic biosynthesis, chitinase production/secretion, antifungal activity.