Identification And Characterization Of Sigma Factors In Lysobacter Enzymogenes OH11

Lysobacter enzymogenes belongs to the Lysobacter genus of the Xanthomodaceae family,which is an important source of gram-negative bacteria for biological control.This kind of bacteria exhinites broad-spectrum antimicrobial activities to multiple plant pathogenic fungi and oomycetes by producing various extracellular enzymes and small-molecule antibacterial natural products.Strain OH11 has been isolated from our laboratory that produces the secondary metabolite HSAF(Heat Stable Antifugnal Factor)as a broad-specturm antifungal antibiotic with a new structure and mode of action.Apart from the HSAF,strain OH11 can also form a T4P(Type IV pili)-driven twitching motility(TM)as an important mechanism for its adherence,colonization and infection in the fungal hyphae.Although our laboratory has elucidated the HSAF biosynthetic pathway and identified several key genes of TM in L.enzymogenes,however,the corresponding underlying regulatory mechanisms remain largely unknown at this bacterium.Sigma factor(?)is a prokaryotic RNA polymerase subunit in bacteria,which can help RNA polymerase specifically,recognizes and binds to promoter in the template strand.In other words,sigma factors greatly improve the binding force between polymerase and the promoter region of DNA,resulting in activating or inhibiting the initial transcription of the target gene.The objective of this study is to explore the potential contribution of ? factors to HSAF biosynthesis and TM formation by using strain OH11 as the model bacterium.In the present work,a computational analysis led to identification of a total of 28 ?factors in the complete genome of strain OH11,including 27 ?70(RpoD)family proteins and 1 a54(RpoN)family protein.By using homologous recombination approach,the 27 a factor coding genes were individually in-frame deleted,and each corresponding mutant was generated.In the following contents,we found that none of the 27 a factors had an effect on the HSAF biosynthesis,because all the test a-factor mutants produced the wild-type HSAF yield quantificated by HPLC analyses.However,we show that only the RpoN,of the 28 ?factors,was involved in regulating TM,because the rpoN mutant was unable to form mobile cells at the margin of its colony that is an indicator of T4P-mediated TM in L.enzymogenes.In agreement with this finding,the electron microscopy shows that the wild-type OH11 produced multiple T4Ps on the cell surface,whereas the rpoN mutant completely lose such an ability.All the phenotypic deficiency of the rpoN mutant was rescued by the in trans complementation.In addition,under the conditions tested in this study,the other 27 ?-factor mutants produced the wild-type level in forming mobile cells at the margin of each respective colony,suggesting all of them were not involved in regulating TM formation in L.enzymogenes.Finally,we also show that the RpoN was involved in regulating TM in L.enzymogenes strain C3,a well-characterized isolate of USA.Mutation of rpoN led to a complete loss of TM,as similar with the case from strain OH11,revealing the regulation of TM by RpoN appears to be a conservative mechanism in different strains of L.enzymogenes.PilA has been identified as the major structural protein to form T4P,and the pilA mutant completely lost T4P and TM.We speculate that RpoN regulated TM formation by influencing PilA.As a supportive evidence,we show that compared to the wild-type OH11,the transcription of pilA was almost completely shut down in the rpoN mutant.Being consistent with this result,overexpression of pilA rescued the deficiency of the the rpoN mutants in produing TM.Finally,the bacterial one-hybrid and EMSA(gel retardation electrophoresis)results show that the RpoN did not directly bind to the pilA promoter region,raising the possibility that the RpoN regulates pilA transcription through an indirect mechanism.