An Extracytoplasmic Function Sigma Factor Controls β-Lactamase Gene Expression inBacillus anthracisand OtherBacillus cereusGroup Species

Abstract

The susceptibility of most Bacillus anthracis strains to ␤-lactam antibiotics is intriguing considering that the closely related species Bacillus cereus and Bacillus thuringiensis typically produce ␤-lactamases and the B. anthracis genome harbors two ␤-lactamase genes, bla1 and bla2. We show that ␤-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 ␤-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on ␤-lactamase activity in a prototypical penicillin-susceptible strain. Complementation with sigP and rsiP from the penicillinresistant strain, but not with sigP and rsiP from the penicillin-susceptible strain, conferred constitutive ␤-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 ␤-lactamase activity, suggesting that while B. anthracis contains the genes necessary for sensing ␤-lactam antibiotics, the B. anthracis sigP and rsiP gene products are not sufficient for bla induction. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensisare the best-studied members of the B. cereus group. Extensive genomic studies, including DNA-DNA hybridization, 16S and 23S rRNA sequence comparisons, multilocus sequence typing, multilocus enzyme electrophoresis, and amplified fragment length polymorphism analysis, have revealed a high degree of phylogenetic relatedness among these organisms, leading to the proposal that B. anthracis, B. cereus, and B. thuringiensis may be viewed as a single species (7, 8, 12, 23, 28, 46-48, 51, 56). Despite the remarkably high degree of DNA sequence similarity and synteny among these species, there are notable species-specific phenotypes, many of which are attributed to plasmid content. B. anthracis, the causative agent of anthrax, typically contains two virulence plasmids, pXO1 and pXO2, which harbor genes encoding toxins and capsule biosynthetic enzymes, respectively (57,73). Large, transmissible plasmids associated with the insect pathogen B. thuringiensis usually contain genes encoding insect toxins, which are produced as large parasporal inclusions (40,77). Strains of B. cereus, a ubiquitous soil bacterium and opportunistic pathogen, can harbor a variety of extrachromosomal elements, including plasmids with high levels of similarity to pXO1 (44,52).In addition to plasmid-associated traits, some species-specific phenotypes are due to differential expression of chromosomal genes. B. cereus and B. thuringiensis have certain phospolipase, hem...

Bovine Bacillus anthracis in Cameroon

“…It is worth noting that the start codon for SigP in B. anthracis strains, except in JF3964 and CI, is generally ATG, whereas in B. cereus and B. thuringiensis, it is TTG. We interpret the moderate resistance to penicillin (MIC, 8 g/ml) in strain JF3964 to be due to a partial repression of SigP by the corresponding RsiP, in contrast to what occurs in common B. anthracis strains, which are assumed to exhibit full repression of their SigP by RsiP, and in contrast to B. anthracis strain 32, where a truncated RsiP is unable to repress SigP, leading to very high penicillin resistance (18). Although strain JF3964 has additional characteristics specific to B. cereus, it harbors the virulence genes which are associated with the virulence plasmids pXO1 and pXO2 in B. anthracis.…”

“…Operon sigP-rsiP encodes sigma factor P (SigP) and the repressor of sigma factor P (RsiP), the latter of which affects the expression of the ␤-lactamase genes bla1 and bla2 (18). This was recently demonstrated with B. anthracis strain 32, which contains a truncated repressor gene, rsiP, resulting from one mutation in the sigP-rsiP operon.…”

“…This was recently demonstrated with B. anthracis strain 32, which contains a truncated repressor gene, rsiP, resulting from one mutation in the sigP-rsiP operon. In this strain, the repressor cannot bind to the sigma factor, which enables the latter to interact with RNA polymerase and transcript bla genes, leading to a 6,000-times-higher MIC for penicillin (18). The entire sigP-rsiP operon was amplified from the penicillinresistant strain JF3964 by PCR using FIREPol DNA polymerase I (Solis Biodyne) and primers BaSigPXL (5Ј-GCTTTTTCTATT TTATTATGGCTC-3Ј) and sigmaR (5Ј-TGCTGCTCTCGTTA CATCAGA-3Ј).…”

Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology

“…For example, B. anthracis has two ␤-lactamase genes, bla1 and bla2, on the chromosome, but this species is rarely resistant to ␤-lactam antimicrobials such as penicillin. Phenotypic susceptibility of most strains is due to a mutation(s) in the regulatory genes that prevent induction of ␤-lactamase gene expression (8,9). Another issue associated with the use of genetic analysis to predict antimicrobial resistance is the inability to detect all possible mechanisms of resistance to the antimicrobials of interest.…”

Optical Screening for Rapid Antimicrobial Susceptibility Testing and for Observation of Phenotypic Diversity among Strains of the Genetically Clonal Species Bacillus anthracis

“…B. anthracis is usually susceptible to the antimicrobial agents approved for treatment and postexposure prophylaxis, although naturally occurring resistance to penicillin (PEN) has been documented, and a single isolate with resistance to erythromycin has been reported (11)(12)(13)(14)(15). The introduction of antimicrobial resistance genes and the use of selective pressure for mutations associated with resistance have been described in the literature (16,17).…”