β-Lactam Resistance in Azospirillum baldaniorum Sp245 Is Mediated by Lytic Transglycosylase and β-Lactamase and Regulated by a Cascade of RpoE7→RpoH3 Sigma Factors.

Bacterial resistance to β-lactam antibiotics is often mediated by β-lactamases and lytic transglycosylases. Azospirillum baldaniorum Sp245 is a plant-growth-promoting rhizobacterium that shows high levels of resistance to ampicillin. Investigating the molecular basis of ampicillin resistance and its regulation in A. baldaniorum Sp245, we found that a gene encoding lytic transglycosylase (Ltg1) is organized divergently from a gene encoding an extracytoplasmic function (ECF) σ factor (RpoE7) in its genome. Inactivation of rpoE7 in A. baldaniorum Sp245 led to increased ability to form cell-cell aggregates and produce exopolysaccharides and biofilm, suggesting that rpoE7 might contribute to antibiotic resistance. Inactivation of ltg1 in A. baldaniorum Sp245, however, adversely affected its growth, indicating a requirement of Ltg1 for optimal growth. The expression of rpoE7 , as well that of as ltg1 , was positively regulated by RpoE7, and overexpression of RpoE7 conferred ampicillin sensitivity to both the rpoE7 :: km mutant and its parent. In addition, RpoE7 negatively regulated the expression of a gene encoding a β-lactamase ( bla1 ). Out of the 5 paralogs of RpoH encoded in the genome of A. baldaniorum Sp245, RpoH3 played major roles in conferring ampicillin sensitivity and in the downregulation of bla1 . The expression of rpoH3 was positively regulated by RpoE7. Collectively, these observations reveal a novel regulatory cascade of RpoE7-RpoH3 σ factors that negatively regulates ampicillin resistance in A. baldaniorum Sp245 by controlling the expression of a β-lactamase and a lytic transglycosylase. In the absence of a cognate anti-sigma factor, addressing how the activity of RpoE7 is regulated by β-lactams will unravel new mechanisms of regulation of β-lactam resistance in bacteria. IMPORTANCE Antimicrobial resistance is a global health problem that requires a better understanding of the mechanisms that bacteria use to resist antibiotics. Bacteria inhabiting the plant rhizosphere are a potential source of antibiotic resistance, but their mechanisms controlling antibiotic resistance are poorly understood. A. baldaniorum Sp245 is a rhizobacterium that is known for its characteristic resistance to ampicillin. Here, we show that an AmpC-type β-lactamase and a lytic transglycosylase mediate resistance to ampicillin in A. baldaniorum Sp245. While the gene encoding lytic transglycosylase is positively regulated by an ECF σ-factor (RpoE7), a cascade of RpoE7 and RpoH3 σ factors negatively regulates the expression of β-lactamase. This is the first evidence showing involvement of a regulatory cascade of σ factors in the regulation of ampicillin resistance in a rhizobacterium.