ACTJK, A TWO-COMPONENT SYSTEM OF ENSIFER MELILOTI INVOLVED IN ACID TOLERANCE

CAFIERO, JUAN HILARIO; DEL PAPA, MARÍA FLORENCIA; VACCA, CAROLINA; DRAGHI, WALTER OMAR; ALBICORO, FRANCISCO JAVIER; LAGARES, ANTONIO

Congreso; Reunion conjunta SAIB-SAMIGE 2020; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular y Sociedad Argentina de Microbiología General

The legume-symbiont model-soil bacterium Ensifer meliloti associates with legume genera such as Medicago, Melilotus and Trigonella to develop N2-fixing symbioses. These symbiotic associations are very important for agriculture, both economically and ecologically. Furthermore, the distribution of acidic soils in the world and the sensitivity of this microorganism to acidity have encouraged research on how this symbiosis responds to acidity. ActJ and ActK of E. meliloti conform a two-component system (TCS) in which the information is transferred from a histidine residue in the histidine kinase (HK) ActK to an aspartate residue in the response regulator (RR) ActJ. Bioinformatic tools show us that these proteins are contiguously encoded, contain typical domain architectures and amino acid residue conservation. The analysis of gene neighborhood revealed synteny within Rhizobiales order. To experimentally confirm the operon prediction, we performed intergenic PCR reactions on randomly synthetized cDNA in order to analyze which genes were co-transcribed in a unique mRNA. We experimentally confirmed that at pH 7.0 and pH 5.6 actJ, actK and glnE were co-transcribed in a unique mRNA. However, degP1 was co-transcribed with actJ, actK and glnE mainly at pH 5.6. Based on the foregoing observations, we used promoter-EGFP fusions to monitor the gene expression levels at different pHs. Our data showed that degP1 and actJ transcription increase in low pH in a ActJ-dependent manner. In addition, we determine that ActJ had an active role in triggering the acid-tolerance response (ATR) phenotype. In-frame deletion mutants of actJ, actK were previously constructed and they showed a significantly reduced growth rate at low pH in GS minimal medium. To ask whether these proteins do indeed have a conserved phosphorylation site, we construct point mutations to test whether the absence of His250 in ActK and Asp55 in ActJ, both phosphorylatable sites, would impact E. meliloti phenotype. Indeed, while wild-type ActK rescued ∆actK acid growth and while wild-type ActJ rescued ∆actJ acid growth, in trans expression of ActKH250A and ActJD55A did not restore wild-type levels of growth under acid conditions. All these results suggest that the phosphotransfer ability is critical to free-living cell duplication under acid stress and provides evidence that a TCS is operating within ActJ and ActK proteins.