An extracytoplasmic function sigma factor controls beta-lactamase gene expression in Bacillus anthracis and other Bacillus cereus group species

Abstract

The susceptibility of most Bacillus anthracis strains to beta-lactam antibiotics is intriguing considering that the closely related species Bacillus cereus and Bacillus thuringiensis typically produce beta-lactamases and the B. anthracis genome harbors two beta-lactamase genes, bla1 and bla2. We show that beta-lactamase activity associated with B. anthracis is affected by two genes, sigP (BA2502) and rsiP (BA2503), predicted to encode an extracytoplasmic function sigma factor and an anti-sigma factor, respectively. Deletion of the sigP-rsiP locus abolished beta-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on beta-lactamase activity in a prototypical penicillin-susceptible strain. Complementation with sigP and rsiP from the penicillin-resistant strain, but not with sigP and rsiP from the penicillin-susceptible strain, conferred constitutive beta-lactamase activity in both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsiP in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsiP homologues are required for inducible penicillin resistance in these species. Expression of the B. cereus or B. thuringiensis sigP and rsiP genes in a B. anthracis sigP-rsiP-null mutant confers inducible production of beta-lactamase activity, suggesting that while B. anthracis contains the genes necessary for sensing beta-lactam antibiotics, the B. anthracis sigP and rsiP gene products are not sufficient for bla induction.

FIG. 1.

Schematic representation of the bla1 locus. The sigP (BA2502) and rsiP (BA2503) genes are located approximately 5 kb upstream of the bla1 gene. Asterisks indicate the locations of the sequence differences between the sigP-rsiP gene pairs from the penicillin-resistant strain B. anthracis 32 and the prototypical penicillin-susceptible strain 9131.

FIG. 2.

β-Lactamase production by B. anthracis strains in the presence and absence of sigP-rsiP gene pairs. β-Lactamase activities were determined using culture supernatants from parent strain 9131 (Pen^s) and the corresponding ΔsigP ΔrsiP mutant UT334, parent strain UT308 (Pen^r) and the corresponding ΔsigP ΔrsiP mutant UT335, and ΔsigP ΔrsiP mutants complemented with pUTE692 (sigP and rsiP from the Pen^s strain), pUTE693 (sigP and rsiP from the Pen^r strain), and pHT304 (empty vector). Black bars indicate a Pen^s strain (9131) background, and gray bars indicate a Pen^r strain (UT308) background. The error bars represent standard deviations. Bkgrd, background.

FIG. 3.

Conserved sequences in the bla1, bla2, and sigP promoter regions. The putative −35 and −10 sequences are indicated by boldface type. The conserved G residue and AAC motif of the putative −35 region are underlined in the consensus sequence. Putative transcriptional start sites of bla1 and sigP, as determined from the results of 5′-end mapping, are indicated by boldface type and underlining.

FIG. 4.

Fluorescence microscopy of strains UT334 (ΔsigP ΔrsiP) and UT335 (ΔsigP ΔrsiP) harboring GFP translational fusions, observed using light and a fluorescein isothiocyanate filter. The fusions contained sigP genes from a Pen^s or Pen^r (asterisk) background and the 5′ end of the rsiP gene from Pen^s and Pen^r (asterisk) backgrounds. Plus and minus signs indicate that the strains are β-lactamase positive and β-lactamase negative, respectively.

FIG. 5.

β-Lactamase activities of B. anthracis strains containing the sigP-rsiP gene pairs from B. anthracis, B. cereus, and B. thuringiensis grown in the presence and absence of ampicillin. The β-lactamase activities of the sigP-rsiP-null strain UT334 complemented in trans with the sigP-rsiP gene pairs from penicillin-susceptible B. anthracis (pUTE692), penicillin-resistant B. anthracis (pUTE693), B. cereus (pUTE728), and B. thuringiensis (pUTE729) and with the empty vector (pHT304) were compared to the β-lactamase activity of the parent penicillin-resistant strain, UT308. Supernatants from the indicated strains were sampled 4 h after induction with 0.1 μg ampicillin/ml (gray bars) or water (black bars). The error bars represent standard deviations. Bkgrd, background; Ba, B. anthracis; Bc, B. cereus; Bt, B. thuringiensis.