RNA-binding Protein Hfq-mediated Regulatory Mechanisms Underlying Carbon Catabolite Repression And Optimal Nitrogenase Activity In Pseudomonas Stutzeri A1501

The RNA-binding protein Hfq is phylogenetically widespread and plays key roles in biofilm formation,QS,carbon catabolic repression（CCR）,and nitrogen fixation.In Pseudomonas,Hfq promotes the pairing of small RNAs（s RNA）to target m RNAs that can lead to positive regulation of the m RNA or binding to the 5’UTR by blocking ribosome entry m RNA that can lead to negative regulation of the m RNA.In recent study,the RNA chaperone Hfq is identified as the principal post-transcriptional regulator of CCR in Pseudomonas,binding to CA-motif sequences within the ribosome binding site of the m RNA,and to repress translation,Crc plays an ancillary role,as full-fledged repression of CCR-regulated m RNAs in vivo required its presence.The noncoding RNA（nc RNA）such as Crc Z acts as a decoy to abrogate Hfq-mediated translational repression of catabolic genes.The root-associated bacterium Pseudomonas stutzeri A1501 is a rare example of Pseudomonas strain with nitrogen fixation ability,it can survive in the soil,and colonize to the surface of the rice root.P.stutzeri A1501 is a metabolically versatile organism with the ability to utilize numerous carbon sources.Until now,preliminary phenotypic on the biological function of it has been studied.However it’s roles in CCR,colonization to host root and the targets to nitrogen fixation remain largely unknown.My dissertation focused on the regulatory mechanism underlying carbon catabolite repression and nitrogen fixation by RNA chaperone Hfq in P.stutzeri A1501.The main research results are described as follows:1.P.stutzeri A1501 grows well in succinate,lactate,or citrate as the single carbon sources(OD₆₀₀=1.2),and grows weakly in glucose(OD₆₀₀=0.3);A1501 exhibits diauxic growth in a medium containing both glucose and any one of succinate,lactate,or citrate,the utilization of these carbon substrate pairs is hierarchical.The growth curve is shown together with the measured concentrations of glucose remaining in the medium.Succinate is consumed during the first growth phase but glucose is not.TheΔcrc Z/Y double mutants were unable to utilize glucose and citrate,indicating that citrate is a non-preferred carbon source.Here we show that root-associated diazotrophic P.stutzeri A1501preferentially catabolizes succinate,followed by the less favourable substrate citrate,and ultimately glucose.2.Microscale thermophoresis（MST）was used to analyze the ability of Hfq to recognize predicted CA motifs in the 5’untranslated region of the target genes,including the glt R,opr B,and zwf genes responsible for glucose utilization,and the cit N gene encoding citrate transporter.The binding kd values was 629.3 n M,416.67 n M,and 243.95 n M,respectively.Crc cannot bind to the target RNA along but can enhance the binding ability of Hfq to the target,and its binding kd values were 36.02 n M,37.2 n M,and 27.6 n M,respectively.The above results provide key molecular evidence for revealing the mechanism of A1501 carbon source utilization priority.3.Crc Y/Crc Z competitively sequester Hfq/Crc away from its m RNA targets.During diauxic growth on succinate and glucose,the absolute copy number of Crc Z increased sharply with the consumption of succinate and reaches a peak（9.6×10⁵ Copies/ng RNA）at the first lag phase.The absolute copies number of Crc Z and Crc Y vary from succinate to glucose,which were expressed at a low level when grow in succinate and lactate,at an intermediate level when grow in citrate,but a high level in glucose.MST further confirmed that Hfq can bind Crc Y/Z with high affinity in vitro.Taken together,combined with the data thatΔcrc Z/Y double mutant cannot utilize non-preferred carbon sources,suggest that Crc Z/Y can release its inhibition of the utilization of non-preferred carbon sources by competitively binding to Hfq,and plays key role in this process.4.The transcriptome results showed that Hfq,as a global regulator,participated in multiple metabolic regulations of A1501 under CCR conditions and nitrogen fixation conditions,and the differentially changed genes mainly included carbon and nitrogen metabolism,extracellular polysaccharide synthesis,chemotaxis,flagellar synthesis,etc.and rhizosphere colonization-related genes,especially nitrogen fixation-related genes nif A and nif HDK,were up-regulated by 2-4 times;exopolysaccharide synthesis genes（PST＿249-PST＿2501）were down-regulated by 5-50 times,This results indicating that Hfq,especially in the rhizosphere,may play an important regulatory role in nitrogen fixation and exopolysaccharide production.5.Under rhizosphere conditions,theΔhfq nitrogenase activity was only 25%compared with wild type.MST results indicated that Hfq directly binds to the nif H coding region CA motif.In the presence of rifampicin,the half-life of nif H m RNA in the WT strain was 11 min,but it decreased to 7 min for theΔhfq mutant,indicating that Hfq enhanced the stability of nif H m RNA by binding to nif H m RNA,thereby enhancing the nitrogen-fixing activity of A1501.Root surface colonization and exopolysaccharide assay showed that the addition of glucose increased the root surface colonization ability of A1501 by 73%,and the exopolysaccharide formation ability increased by 36%.Deletion of Hfq reduced the A1501 exopolysaccharide production capacity by 80%,and the rhizosphere colonization capacity was only 20%compared to the wild type.Compared with the wild type,the relative expression of the psl-like gene inΔhfq was down-regulated（5-50 times）,and the above results indicated that Hfq positively regulates the synthesis of exopolysaccharide,thereby enhancing the rhizosphere colonization ability of A1501.Taken together,in P.stutzeri A1501,Hfq not only participates in the sequential process of preferential metabolism of carbon sources as a major repressor but also participates in the process of rhizosphere colonization as an activator,especially in the regulation of nitrogen fixation gene expression directly at the post-transcriptional level,which is the first report.The results laid an important theoretical basis for revealing the molecular mechanism of efficient carbon source utilization,rhizosphere competition,and interaction with host crops by nitrogen-fixing microorganisms.