| Lysobacter species are gram-negative bacteria that are emerging as new sources of antibiotics and antifungal compounds,including HSAF(Heat Stable Antifungal Factor)、which has been identified as a broad-spectrum antifungal compound from L.enzymogenes with a new mode of action.LesR,a LuxR solo,was recently shown to regulate the HSAF biosynthesis and cell aggregation via unidentified mechanisms in L.enzymogenes OH11.The TBDR(JonB-Dependent Receptor)-dependent substrate transport is an active process that requires energy input from the proton motive force across the cytoplasmic membranes、which requires the ligand-loaded TBDRs interact with the TonB protein complex consisting of three inner membrane proteins(TonB/ExbB/ExbD).Nevertheless,TBDR has never been reported to play a role in regulating bacterial antibiotic biosynthesis to our knowledge.A previous study in our Iab have performed a comparative proteomic analysis,and found that LesR controlled the expression of 33 proteins belonging to 10 functional groups,including 9 proteins belonging to TBDR family.A part of the objectives of the present study is to explore the potential regulation of 9 TBDRs in the biosynthesis of HSAF,because the fundamental role of bacterial TBDR in nutrient uptake is highly associated with the nutrient-dependent property of HSAF production.Furthermore,exploring the contribution of 9 TBDRs to cell aggregation is also included in this work.A detailed sequence analysis of these 9 TBDRs showed that each TBDR possessed two conserved domains;a C-terminal membrane-embedded β-barrel domain(ligand_ated_channel)and an N-terminal plug domain(plug domainl～150-200 residues).To explore the role of these 9 TBDRs,we individually in-frame deleted each of them by a homologous recombination approach.The results of HSAF quantification show that knockout of the gene TBDR2,4,8 or 9 had no effect on the HSAF yield.However,inactivation of TBDR7 almost abolished the HSAF production,whereas mutation of TBDR1 significantly enhanced HSAF biosynthesis.Double mutation of TBDR1 and TBDR7 made the HSAF production be approximately equal to wild type,suggesting that TBDR1 and TBDR7 played an opposite role in regulating the HSAF biosynthesis in L.enzymogenes.In the growth detection,we found that mutation of TBDR7 did not alter the growth capability of the wild-type OH11,suggesting that TBDR7 did not involve in bacterial growth.We also determined the transcription of hsaf pks/nrps,the key gene for HSAF biosynthesis between the TBDR7 mutant and wild type.The results show that the transcription of hsaf pks/nrps was almost completely shut down in the TBDR7 mutant in comparison to the wild-type strain.Furthermore,domain deletion and site-directed mutagenesis further showed that the TonB-box region of TBDR7 was critical for its regulation on the biosynthesis of HSAF.In addition,we also investigated the contribution of TBDR in LesR-mediated cell aggregation.We found that 6 TBDR mutants exhibited wild-type level in cell aggregation,suggesting all the 6 TBDR proteins did not participate in cell aggregation in the wild-type background.Overexpression of lesR in the TBDR1 mutant showed no cell aggregation,while overexpression of lesR in other 5 TBDR mutants still accelerated cell aggregation,as similar with its overexpression in the wild-type OH11.These results revealed that TBDR1 played a vital role in the LesR-mediated cell aggregation.Using bacterial one-hybrid system,the direct protein-DNA interaction between LesR and the promoter of TBDR1 and TBDR7 were not detected,indicating that LesR most probably used an indirect manner to control the expression of TBDR1 and TBDR7 in L.enzymogenes.Collectively,our results provide a new insight into the LesR-mediated regulation of the HSAF biosynthesis and cell aggregation.Furthermore,our results also provide the first example showing that TBDR plays a key role in regulating antibiotic biosynthesis,a function which has never been reported for TBDRs in bacteria. |
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