| Carbon Catabolite Repression(CCR)is an important and complex global regulatory response that allows bacteria to selectively assimilate a preferred compound that provides the most efficient growth,inhibiting the uptake of other non-preferred compounds when the cells are exposed to a mixture of several potential carbon sources.The regulatory systems and the molecular mechanism responsible for CCR are quite different in distinct bacterial species.In Pseudomonas,the main regulatory system of CCR is exerted by the Crc protein,combined with the two-component system CbrA/B and ncRNA,which modulate Crc availability.The catabolite repression control protein(Crc)is known to act as a repressor of global regulator and appears to be the central player mediating CCR in Pseudomonas genus.When preferred carbon source exists,Crc inhibits the expression of genes required for the assimilation of nonpreferred carbon sources by attaching to the specific site of target mRNA.When preferred compound is exhausted,ncRNA binds to Crc protein and relieved the repression effect.However,the catabolite regulating mechanism of Crc is controversial and wheather the nitrogenase genes are regulated by Crc protein has not been reported.Pseudomonas stutzeri A1501,which is isolated from the rice rhizosphere containing the abundant carbon sources and barren nitrogen compounds,is a nitrogen-fixing root-associated model bacteria.As compared to other Pseudomonas,A1501 possesses a set of Crc-centric CCR system allocated in the core genome,but the regulatory mechanism is not clear.In current study,we investigated the biological function of Crc in modulating the carbon catabolite,nitrogen-fixation and rhizosphere colonization in P.stutzeri A1501,meanwhile we explored the mechanism of Crc protein.The main research results are described as follows:1.CCR phenomenon mediated by Crc protein.The results of growth potential assay showed A1501 grow faster in LB medium or in the minimal medium with sodium lactate or succinic acid as the sole carbon source than in the minimal medium with glucose or benzoate as the sole carbon source.It indicated that sodium lactate and succinic acid are preferred carbon sources for A1501,while glucose and benzoate are less preferred compounds.Comparative rates of benzoate and glucose utilization in A1501 and its crc mutant strain were detected by HPLC.When wild type and crc mutant were grown on LB medium containing benzoate for 6 h,the uptake rate of benzoate by wild type was 8.111 ?mol/OD/h while,it was 23.278 ?mol/OD/h by crc mutant.When cells grow in minimal medium supplemented with lactate and glucose as the carbon sources for 12 h,A1501 consumed 27.776 ?mol glucose per OD per h and the crc mutant expended 73.418 ?mol glucose per OD per h.Therefore,a typical CCR phenotype was observed in A1501 and the Crc protein was proposed to be the central regulator mediating CCR based on the largely impaired inhibition of benzoate and glucose assimilation in ?crc.2.Expression pattern of crc gene and the effect of crc gene deletion on the expression of less preferred carbon source catabolic genes.Compared with wild type,the translation level of BenR transcriptional activator and the transcription level of benzoate degradation genes benA and benK were significantly upregulated in ?crc when cells were cultured in LB medium with benzoate.The results of qRT-PCR exhibited that the expression level of edd and zwf genes coding for the key enzyme in glucose metabolism and its regulator GltR were significantly elevated in ?crc compared to wild type A1501 under conditions that both lactate and glucose exists.It suggests that Crc may inhibits the catabolism of less preferred carbon source by suppressing not only expression of the transcriptional activator,but also that of genes coding for the key enzyme in the presence of preferred compound.Meanwhile,when cells grown with benzoate as the carbon source,the transcriptional level of crc in A1501 was lowest compared to the cells cultured in minimal medium supplementary with lactate,succinic acid or glucose.However,compared with the cells in LB or minimal medium with lactate,the transcriptional level of ncRNA CrcZ and CrcY were strongly induced when A1501 use glucose or benzoate as the carbon source.Dual-crcZ/Y double mutant is compromised in the ability to utilize glucose.The above results showed that CCR system modulates the expression pattern of the carbon metabolic networks response to the trophic signals and the intracellular pool of free Crc protein is adjusted by ncRNA CrcZ/Y.3.Effect of Crc protein on nitrogen metabolism and competitive colonization related physiology process.The nitrogen activity of ?crc was only half of that in wild type when using glucose as the sole carbon source.The transcriptional and translational level of nitrogen-fixing genes nifHDK were reduced sharply in crc mutant determined by qRT-PCR and Western blotting.These results manifested that the absence of Crc protein causes the impaired nitrogen fixing ability of A1501.Moreover,when nitrate exists as the sole nitrogen source,the growth potential of crc mutant was substantially lower than wild type under anaerobic condition.And ?crc was in capable of growing with nitrate as nitrogen source.In addition,as compared with wild type,the root surface colonization ability dropped by 50% in ?crc.Further study identified the marked decline in motility,oxidative stress defense and osmotic stress defense caused by the deletion of crc.The above results suggest that Crc protein acts as a global regulator in A1501 and it may plays an important positive regulatory role in several aspects of cell biology,including nitrogen metabolism,root colonization and motility,apart from only participating in the CCR.4.The mechanism of Crc protein in regulating related genes.Interrogation of RNA sequences revealed that the putative Crc-binding sites(AANAANAA)exist in benzoate degradation genes,glucose catabolite genes,nitrogen-fixation genes,denitrification genes,motility genes and the oxidation resistance related genes.While,the location of conserved binding sites are different.The CA motifs are located in the vicinity of translational start site of carbon catabolite genes which were negative controlled by Crc protein.Additional A-rich motifs were found in the DNA region of nitrogen metabolic genes or other targets,positive regulated by Crc.The interaction between Crc protein and RNA targets was not detected in vitro by microscale thermophoresis(MST).Whereas,Crc protein and Hfq protein can form a co-complex with RNA containing a CA motif in vitro.The Kd-value of Hfq with crcZ,benA and nifH respectively increased from 14.2 nmol/L,36.9 nmol/L and 365.0 nmol/L in the absence of Crc to 7.9 nmol/L,12.6 nmol/L and 66.5 nmol/L in the presence of Crc respectively.Taken together,these experimental results indicated that Crc protein could modulate the metabolic pathway activity related to the benzoate degradation,glucose metabolism and nitrogen fixation via binding to the corresponding target mRNA in the presence of Hfq.In conclusion,Crc protein of A1501 exerts a crucial regulatory role in CCR and it represses expression of target RNA through specific binding to the conserved sequence AANAANAA together with Hfq,thus coordinating the activity of target metabolic pathway,to ensure perpetuating efficient use of nutrition to meet the demand of growth and nitrogen fixation in nutritionally complex and changing rhizosphere environment.In the meantime,Crc protein positively modulates,perhaps directly,the process of nitrogen fixation,denitrification,motility and colonization.A model of Crc in modulating the carbon catabolite repression and various physiological processes in P.stutzeri A1501 was proposed.It would be helpful to understand how A1501 maintains the highly efficient metabolism and acquires the advantages in fiercely competitive rhizosphere.The results of this thesis laid a theoretical foundation for further research on Carbon-Nitrogen coupling and the global regulatory mechanism in Pseudomonas. |
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