| Lysobacter enzymogenes OH11 is a kind of no flagella Gram-negative bacteria which isolated from the rhizosphere soil of pepper.It uses type ? pilus mediated twitching motility to close to plant pathogenic fungi and Oomycetes.Meanwhile,produces abundant lytic enzymes and heat-stable antifungal factor(HSAF)to inhibit the growth of pathogen for its biological control.In pathogenic bacteria,ClpXP is a highly conserved protein degradation system that has a significant effect on the quality,physiology,metabolism,and virulence.T6SS is a short-range contact-dependent weapon,which can inject a variety of toxic effects into prokaryotic and eukaryotic cells to help the pathogenic bacteria to gain competitive advantage and maintain pathogenicity.Pyrimidines is an important nucleotide,which is involves in the growth and metabolism of most cellular processes in almost all living organisms.The de novo synthesis of pyrimidines is a central metabolic pathway that comprises the activity of six highly conserved enzymatic reactions.The purpose of this study is to explore the function and mechanism of these three systems in biocontrol bacteria with OH 11 as working model and HSAF as the indicator phenotype.The main results are as follows:1.Via a BLASTP search using E.coli ClpX and ClpP as queries,we identified the respective homologs in the L.enzymogenes OH11 genome,wherein Le0048 and Le0049 exhibited a 75%and 77%identity to the E.coli ClpX and ClpP,respectively.Therefore,we named Le0048 and Le0049 as ClpX and ClpP,respectively.We generated an inframe clpP deletion mutant from strain OH 11 to disrupt the ClpXP function and found that ClpP controls the HSAF biosynthesis and twitching motility.Proteomics data showed that the protein levels of several genes related to HSAF biosynthesis and pilus-related genes required for twitching motility changed significantly,which was consistent with the phenotypes of ClpP regulated twitching motility and HSAF.Meanwhile,that extracellular proteases were also significantly affected by ClpP,which was double confirmed by the extracellular protease detection experiment.Together,these results show that the protease degradation system is involved in the formation of twitching motility,HSAF biosynthesis and extracellular protease activity in Lysobacter enzymogenes OH11,thus revealing the important function of ClpXP in the biocontrol bacteria.2.Inner tube protein Hcp is the key component of T6SS assembly and maintain functional.According to COG database,found one protein in OH11 that has the same COG number with Hcp(COG3157),so we named this protein as Hcp.We found that in no HSAF biosynthesis and secretion condition,the nutrient-rich medium(LB),T6SS assembled,enabling Hcp to be secreted via TssM.However,in HSAF biosynthesis and secretion condition,the nutrient-poor medium(10%TSB),T6SS assembly remains inactive,but the inner tube protein Hcp is synthesized and intracellularly accumulate.We further explored the function of intracellular expression of Hcp under this condition and found that the ability of biosynthesize HSAF decreased significantly after knocking out Hcp.For mechanism study,we used co-IP/MS(immunocoprecipitation/mass spectrometry)identified 30 candidate proteins(including transcription factor Clp)which interact with Hcp in vivo.Recently,our team has shown that c-di-GMP receptor protein Clp can directly bind to the two sites in the promoter region(PA and PB)of HSAF biosynthesis gene cluster,thus activating HSAF biosynthesis gene expression.But the intracellular second messenger c-di-GMP inhibits the ability of Clp binds to PA,thus inhibits the HSAF biosynthesis gene cluster expression.We used bacterial two hybrid system(B2H),pull-down and MST showed that Hcp interacts with the cNMP domain of transcription factor Clp required for activating HSAF biosynthesis operon expression.Hcp binding with Clp does not affect the ability of Clp binding to PA and PB,and the stability of Clp.However,Hcp competes with c-di-GMP in binding with Clp to dissociate the Clp-c-di-GMP complex.The increased concentration of free Clp leads to higher operon expression and an elevated amount of secreted HSAF.3.The de novo synthesis of pyrimidines is a central metabolic pathway that comprises six highly conserved enzymatic reactions,including six enzymes,carA/carB and pyrB-F.By bioinformatics analysis,found that OH11 has the homologs of carA/carB?pvrB?pyrD and pyrE instead of pyrC and pyrF.However.Le0752 has the same functional domain with PyrF(OMPdecase domain),a key enzyme in the de novo synthesis of pyrimidine.We generated an inframe Le0752 deletion mutant and found that Le0752 controls HSAF biosynthesis vie growth.Biochemical experiments and complementation of Le0752 with classical pyrF from Xanthomonas oryzae pv.oryzae PXO99A via chromosomal integration confirmed that Le0752 encodes the active orotidine-5'-phosphate decarboxylase.Therefore,the findings indicate that Le0752 is indeed a pyrF homolog.Interestingly,Le0752 was detected as an extracellular protein fraction via western blotting,confirmed that it secreted by L.enzymogenes OH11.Comparative genomic analysis showed that Le0752 and Le4373,Le1850,Le4375,Le2995,Le3030 only exist in Lysobacter genus.In order to study the other 5 genes function,we generated individual deletion mutants and subsequently quantified the biocontrol related phenotypes,such as HSAF levels and twitching motility in each of the resulting strains.However,no significant affection were find.In this study,we used Lysobacter emzymogenes OH11 as working model,reveals some functions and mechanisms of regulation HSAF biosynthesis of ClpXP degradation system,the corn component of T6SS(Hcp)and the key enzyme of pyrimidine biosynthesis pathway(Le0752)in L.enzymogenes OH11.Based on this,expanded the diversity of these systems and found some new mechanisms in biological control bacteria.Elaborate understanding of such conservative systems regarding their roles and mechanisms in regulating HSAF would enable to achieve the goal of high-yield of the latter that would aid in the management of crop diseases. |
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