Identification Of Na~+/H~+Antiporter And Its Transcriptional Regulation In Myxococcus Xanthus DK1622

Salt stress is one of the most common survival challenges for bacteria. Excessive sodium ions alter the structure of biological macromolecules and intracellular osmotic pressure, resulting in inhibition of metabolism and cell dehydration. During the long-term process of evolution, microorganisms have developed strategies for adaptation to salt stress. For example, some bacteriacan accumulate KC1or other compatible solutesas osmotic protective agents. Besides, bacteria can efflux excess sodium ions out of cells to avoid poisoning. Na+/H+antiporters （NHAs）, first reported in1974in Escherichia coli, are major Na+-excreting system in bacteria. NHAs belong to secondary sodium pump system, and widely distribute in a variety of life forms, such as bacteria, archaea, fungi, plants, animals, mammals and even humans. As transmembrane proteins, NHAs export sodium ions out of cells through transmembrane proton electrochemical gradient engendered by primary proton pumps like ATPase （F0F1） and respiratory chain, and at the same time transport H+into cellsto maintain a relatively low concentration of sodium ions. The major functions of NHAs are:a) create transmembrane Na+electrochemical gradient in cells; b) release Na+or Li+to eliminate toxicity; c) keep intracellular pH homeostasis under alkaline conditions; d) regulate cell volume.Currently, the most intensively studied NHA is NhaA in E. coli （Ec-NhaA）. NhaA family exists in many bacteria and archaea, and its Na+transport activity is highly pH-dependent. The transcription of Ec-NhaA is regulated by Ec-NhaR, which belongs to LysR family, a class of positive regulators in prokaryotes. Ec-NhaR can bond topromoter sequence of Ec-NhaA gene to inhibit its transcription. However, Na+can alter the conformation of Ec-NhaR protein, resulting instimulation of Ec-NhaA transcription Thus, Ec-NhaR regulates the expression of Ec-NhaA by acting as both the sensor and the tranducer of Na+signal.Myxococcus xanthus is a Gram-negative soil bacteria with a complex and fascinatinglife cycle, including many multicellular behaviors, such as density-dependence of cell growth, predation, fruiting body development and differentiationresponse to starvation. Previously, our groupfound2genes encoding putative Na+/H+antiporter NhaA in M. xanthus DK1622and constructed gene knock-out mutants△MXAN₃898（YL1015） and△MXAN₆055（YL1012）. Preliminary results indicated that the growth of YL1012under high Na+or Li+decreased significantly, implying that MXAN₆055may play an important role in response to salt stress of M. xanthus DK1622. In this study, we further studied the biochemistry property of MXAN₆055gene product as possible NhaA. We also studied the transcriptional regulation mechanism of MXAN₆055preliminarily.To investigate whether MXAN₆055had an effect on growth of M. xanthus DK1622under salt stress conditions, we have constructed and analyzed a series of mutants related to this gene. The significant decrease of growth of MXAN₆055knock-out mutant YL1012under salt stress conditions showed the possible relation between MXAN₆055and the response of M. xanthus DK1622to salt stress. In order to certificatethat this property is caused by the deletion of MXAN₆055, gene was complemented into YL1012and its growth under salt stress conditionswas recovered, confirming that the deficiency of growth under salt stress is lead by MXAN₆055. However, overexpression of MXAN₆055didn’t result in significant increase of salt-tolerant growth, suggesting that regulation of transcriptional level or higher levelof MXAN₆055gene may exist.Using an antiporter-deficient E. coli strain KNabc and functional complementation, this study further verified the NhaA activity of MXAN₆055protein. Three major NHAs genes of E. coli KNabc （nhaA, nhaB and chaA） have all been knocked-out and thus sensitive to Na+. The presence of MXAN₆055enabled E. coli KNabc to survive in the presence of600mM NaC1or100mM LiCl, and the survivability was pH-dependent:MXAN₆055showed the highest activity in pH7.0-8.0. Everted membrane vesicles （EMVs） assay indicated that MXAN₆055had Na+transport activity in both pH7.0and8.0. Due to low activity of EMVs, we didn’t detect Li+transport activity of MXAN₆055. EMVs assay in E. coli KNabc revealed that MXAN₆055protein had Na+（Li+） tolerance and NhaA activity. This paper also tried to identify Na+/H+transport activity of MXAN₆055in homogenous strain M. xanthus DK1622. However, because of the particularity of myxobacteria cells, we failed to get active EMVs in M. xanthus DK1622.Finally, this work studied the transcriptional regulation mechanism of MXAN₆055. We found a gene MXAN₀784encoding LysR family transcriptional regulator in M. xanthus DK1622genome, and its identity with Ec-NhaR was34%. This paper analyzed the growth of MXAN₀784deletion mutant （YL1021） and MXAN₀784overexpression mutant （YL1054） under salt stress conditions. Subsequently, this paper detected the expression level of MXAN₆055in YL1021and YL1054by qRT-PCR. Results showed that both the growth under salt stress conditions and MXAN6055expression level decreasedin YL1021while increased in YL1054, suggesting an up-regulatory effect of MXAN₀784on expression of MXAN₆055. This paper also validated the specific binding ability between MXAN₀784protein and upstream non-coding sequence of MXAN6055in vitro by band shift assay.