Regulation of Expression of β‐lactamase (ampC) and Lytic Transglycosylase by a Cascade of RpoE7‐>RpoH3 Sigma Factors Regulates Ampicillin Resistance in Azospirillum brasilense Sp245

Abstract

Bacteria colonizing rhizosphere of plants face an intense competition for the nutrients present in the root exudates. This competition also involves production of antibiotics as well as of the enzymes that prevent inhibitory effects of antibiotics. Extra‐cytoplasmic function (ECF) sigma factors are known to initiate the transcriptions of genes involved in the adaptation against several stresses including cell envelope stress caused by antibiotics. Azospirillum brasilense is a plant growth promoting rhizobacterium, which colonizes the rhizosphere of a large number of non‐legume crops. The genome of A.brasilense Sp245 encodes 23 different sigma factors; one σ54 (RpoN), one σ70 (RpoD) and 21 alternative sigma factors of σ70 family. Out of 21 alternative sigma factors, 5 belong to RpoH, 10 to RpoE (ECF), and 6 to FecI type of σ factors. When the gene encoding RpoE7 was inactivated, the rpoE7::km mutant showed resistance to higher concentration of β‐lactam antibiotics compared to its parent. Inactivation of rpoE7 led to a decrease in the activity of its own promoter as well as of its divergently organized gene encoding a lytic‐transglycosylase. Transcription start site of rpoE7 was identified by 5’RACE, and studies with lacZ fusions with the promoters of rpoE7 and lytic transglycosylase genes indicated that both the promoters were regulated by RpoE7. Since β‐lactamases are known to be involved in conferring resistance to β‐lactams, we examined the expression of β‐lactamase paralogs in the parent and rpoE7::km mutant, and observed upregulation of one β‐lactamase (AmpC) in rpoE7::km compared to the wild type. The promoter activity of ampC and its regulator ampR were also increased in the rpoE7::km mutant. We also noted that RpoE7 regulated the expression of AmpC indirectly via RpoH3 sigma factor. Altogether, these data suggest that ampicillin resistance in A. brasilense is regulated by a regulatory cascade of RpoE7 and RpoH3.Support or Funding InformationParul Pandey, Ashutosh Prakash Dubey, Shivangi Mishra and Vijay Shankar Singh are Thankful to ICAR, CSIR, and UGC respectively for the research grant.