We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesteroldependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid-to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at ϳ30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.

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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...

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified to be a promising candidate for adjunct therapy. Human-derived bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the gene locus were potent reuterin producers, with 17938 inhibiting growth at a level on par with the level of growth inhibition by vancomycin. Targeted mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of with glycerol was effective against colonization in complex human fecal microbial communities, whereas treatment with either glycerol or alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with elicited changes in the composition and function of the human microbial community that preferentially targets outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of , and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.