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

Abstract

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.

Keywords: ECF σ factor; RpoE7; RpoH; antibiotic resistance; bacteria; lytic transglycosylase; transcription regulation; β-lactamase.

FIG 1

(A) Genomic organization of the gene encoding rpoE7 in A. baldaniorum Sp245, showing a divergently oriented lytic transglycosylase (Ltg) and a PepSY_2 domain protein-encoding gene located downstream of rpoE7. (B) Culture tubes of A. baldaniorum Sp245 and the rpoE7::km mutant during the exponential phase at 22 h show difference in their ability to flocculate. (C) Scanning electron microscopy (SEM) images of A. baldaniorum Sp245 and the rpoE7::km mutant (magnification, ×5,000. (D) Comparison of biofilm-forming abilities of A. baldaniorum Sp245 and the rpoE7::km mutant. (E) Comparison of exopolysaccharide production by A. baldaniorum Sp245 and the rpoE7::km mutant.

FIG 2

(A) Growth curves of A. baldaniorum Sp245, the ltg1::km mutant, and the ltg1::km complemented strain in minimal malate medium. Each point of the curve shows the mean of three replicates obtained from three different experiments, and error bars indicate standard deviation (SD) at each point. (B) Effect of overproduction of Ltg1 of A. baldaniorum Sp245 on the growth of Escherichia coli DH5α in LB broth at 37°C with and without isopropyl-β-d-thiogalactopyranoside (IPTG) induction of the plasmid pAK007 and pAK008 expressing ltg1 from pMMB206 and pAK032, respectively. I, induced; UI, uninduced. (C) Relative expression of the 6 lytic transglycosylase paralogs of A. baldaniorum Sp245 by quantitative reverse transcription-PCR (RT-PCR) by using threshold cycle values obtained from RNA samples from A. baldaniorum Sp245 and its rpoE7::km mutant. Each bar shows the mean and standard deviation of values obtained from the three replicates.

FIG 3

Effect of expression of rpoE7 and ltg1 on ampicillin sensitivity of the rpoE7::km mutant of A. baldaniorum Sp245 on an Minimal Malate Agar (MMA) plate supplemented with 450 μg ampicillin and triphenyl tetrazolium chloride (TTC; 50 mg/mL) at 10-fold dilutions (10⁻¹, 10⁻², and 10⁻³⁾ of the culture.

FIG 4

(A) Chromatogram showing transcription start site of rpoE7 determined by 5′ rapid amplification of cDNA ends (RACE) with a “G” (encircled in red) as a transcriptional start point. (B) Nucleotide sequence upstream of rpoE7 showing −10 and −35 hexamers of the promoter are underlined. Upstream region of ltg1 gene showing predicted possible −35 and −10 elements (underlined). (C) Comparison of the β-galactosidase activities of rpoE7::lacZ and ltg1::lacZ fusions in A. baldaniorum Sp245 and its rpoE7::km mutant. 1, Sp245 (rpoE7:lacZ); 2, rpoE7::km(rpoE7::lacZ); 3, Sp245 (ltg1::lacZ); 4, rpoE7::km(ltg1::lacZ). Error bars show standard deviations (SD) for triplicates of three independent experiments. (D) Comparison of the β-galactosidase activities of the rpoE7::lacZ fusion in A. baldaniorum Sp245 under different abiotic stresses. Error bars show standard deviations (SD) for triplicates of three independent experiments.

FIG 5

(A) Relative expression of the 5 β-lactamase paralogs of A. baldaniorum Sp245 by quantitative RT-PCR using threshold cycle values obtained from RNA samples of A. baldaniorum Sp245 and its rpoE7::km mutant. Each bar shows the mean and standard deviation of values obtained from the three replicates. Error bars show standard deviations (SD) for triplicates of three independent experiments. (B) Comparison of the β-galactosidase activities from bla1::lacZ in A. baldaniorum Sp245 (no. 1), the rpoE7::km mutant (no. 2), and the rpoE7::km complemented strain (no. 3).

FIG 6

(A) LB agar plate (plate a) depicting ampicillin resistance in E coli strain DH5α overexpressing bla1 (pAK002::bla1↑) of A. baldaniorum Sp245. E coli DH5α lacking bla1 (pAK002) (plate b) is sensitive to ampicillin (450 μg/mL). (B) Plate c shows normal growth of Sp7 and bla1::km mutant without antibiotic. MMA plates showing resistance of A. baldaniorum Sp245 (plate d) and sensitivity of the bla1::km mutant (plate d) to ampicillin. Plate e shows resistance of the bla1::km mutant to kanamycin.

FIG 7

(A) Ampicillin sensitivity of A. baldaniorum Sp245 carrying broad-host-range vector pAK032 and A. baldaniorum Sp245 derivatives harboring different rpoH paralogs cloned in pAK032 in MM medium amended with ampicillin (450 μg/mL) and TTC (50 mg/mL) at 10-fold dilutions (10⁻¹, 10⁻², and 10⁻³) of the culture. (B) Comparison of the β-galactosidase activities from bla1::lacZ in A. baldaniorum Sp245; 2, Sp245 (RpoH1↑); 3, Sp245 (RpoH2↑); 4, Sp245 (RpoH3↑); 5, Sp245 (RpoH4↑); 6, Sp245 (RpoH5↑). Error bars show standard deviations (SD) for triplicates of the three independent experiments. (C) Effect of the overexpression of rpoE7 on the expression of the rpoH3::lacZ fusion and of rpoH3 overexpression on the expression of the rpoE7::lacZ fusion in A. baldaniorum Sp245. Error bars show standard deviations (SD) for triplicates of the three independent experiments.

FIG 8

Proposed scheme of alternative sigma factors regulating the expression of the bla1 gene conferring ampicillin resistance in A. baldaniorum Sp245. RpoE7, along with the core enzyme, activates the promoters of ltg1 and rpoE7. Then, RpoE7 activates the expression of RpoH3, which, in turn negatively regulates the expression of AmpC type β-lactamase. Thus, a cascade of RpoE7-RpoH3 negatively regulates bla1 expression and ampicillin resistance in A. baldaniorum Sp245.