- Volume 158, Issue 4, 2012

Escherichia coli O157 : H7 cells that interact with intestinal epithelial cells in animals and humans do so after passage through the low pH of the stomach. This study compared adherence and its associated virulence gene expression in acid-treated (AT) and non-acid treated (NAT) E. coli O157 : H7 strain 86-24 in vitro and in ligated pig intestine. It was found that in vitro, AT O157 : H7 had significantly decreased adherence accompanied by decreased expression of stcE and toxB but not of the locus of enterocyte effacement (LEE) genes. Expression of gadE, genes involved in quorum sensing, and the global regulators cyaA, hfq, lrp, fis and himA was significantly increased; notably, ureD expression was increased 29-fold compared with NAT O157 : H7. AT O157 : H7 colonized the pig intestine as effectively as NAT O157 : H7 bacteria. Expression of 70 of 72 virulence genes from bacteria recovered from the intestine was similar between AT and NAT O157 : H7, except ureD, pagC and bax, whose level of expression was reduced in the AT bacteria. Genes involved in acid response, regulators gadE, cyaA and hfq, and toxin synthesis genes (stx2A and stx2B) were expressed at significantly reduced levels in the intestine by both AT and NAT strains. Expression in the intestine of the LEE and putative adhesion factors cahA, iha and lpf2 was at levels similar to those in vitro, while ehaA and ureD in NAT O157 : H7 were expressed significantly more highly in vivo than in vitro. These data indicate that AT and NAT O157 : H7 behave differently, and that expression of their virulence genes is regulated differently in vitro from in vivo.

Cereulide, produced by certain Bacillus cereus strains, is a lipophilic cyclic peptide of 1152 Da that binds K+ ions with high specificity and affinity. It is toxic to humans, but its role for the producer organism is not known. We report here that cereulide operates for B. cereus to scavenge potassium when the environment is growth limiting for this ion. Cereulide-producing B. cereus showed higher maximal growth rates (µmax) than cereulide non-producing B. cereus in K+-deficient medium (K+ concentration ~1 mM). The cereulide-producing strains grew faster in K+-deficient than in K+-rich medium with or without added cereulide. Cereulide non-producing B. cereus neither increased µmax in K+-deficient medium compared with K+-rich medium, nor benefited from added cereulide. Cereulide-producing strains outcompeted GFP-labelled Bacillus thuringiensis in potassium-deficient (K+ concentration ~1 mM) but not in potassium-rich (K+ concentration ~30 mM) medium. Exposure to 2 µM cereulide in potassium-free medium lacking an energy source caused, within seconds, a major efflux of cellular K+ from B. cereus not producing cereulide as well as from Bacillus subtilis. Cereulide depleted the cereulide non-producing B. cereus and B. subtilis cells of a major part of their K+ stores, but did not affect cereulide-producing B. cereus strains. Externally added 6–10 µM cereulide triggered the generation of biofilms and pellicles by B. cereus. The results indicate that both endogenous and externally accessible cereulide supports the fitness of cereulide-producing B. cereus in environments where the potassium concentration is low.

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low-molecular-mass thiol found in bacteria such as Bacillus sp., Staphylococcus aureus and Deinococcus radiodurans. Like other low-molecular-mass thiols such as glutathione and mycothiol, BSH is likely to be involved in protection against environmental toxins including thiol-reactive antibiotics. We report here a BSH-dependent detoxification mechanism in S. aureus. When S. aureus Newman strain was treated with monobromobimane and monochlorobimane, the cellular BSH was converted to the fluorescent S-conjugate BS-bimane. A bacillithiol conjugate amidase activity acted upon the BS-bimane to produce Cys-bimane, which was then acetylated by an N-acetyltransferase to generate N-acetyl-Cys-bimane, a mercapturic acid. An S. aureus mutant lacking BSH did not produce mercapturic acid when treated with monobromobimane and monochlorobimane, confirming the involvement of bacillithiol. Furthermore, treatment of S. aureus Newman with rifamycin, the parent compound of the first-line anti-tuberculosis drug, rifampicin, indicated that this thiol-reactive antibiotic is also detoxified in a BSH-dependent manner, since mercapturic acids of rifamycin were observed in the culture medium. These data indicate that toxins and thiol-reactive antibiotics are detoxified to less potent mercapturic acids in a BSH-dependent manner and then exported out of the cell in S. aureus.

Burkholderia phymatum STM815 is a β-rhizobial strain that can effectively nodulate several species of the large legume genus Mimosa. Two Tn5-induced mutants of this strain, KM16-22 and KM51, failed to form root nodules on Mimosa pudica, but still caused root hair deformation, which is one of the early steps of rhizobial infection. Both mutants grew well in a complex medium. However, KM16-22 could not grow on minimal medium unless a sugar and a metabolic intermediate such as pyruvate were provided, and KM51 also could not grow on minimal medium unless a sugar was added. The Tn5-interrupted genes of the mutants showed strong homologies to pgm, which encodes 2,3-biphosphoglycerate-dependent phosphoglycerate mutase (dPGM), and fbp, which encodes fructose 1,6-bisphosphatase (FBPase). Both enzymes are known to be involved in obligate steps in carbohydrate metabolism. Enzyme assays confirmed that KM16-22 and KM51 had indeed lost dPGM and FBPase activity, respectively, whilst the activities of these enzymes were expressed normally in both free-living bacteria and symbiotic bacteroids of the parental strain STM815. Both mutants recovered their enzyme activity after the introduction of wild-type pgm or fbp genes, were subsequently able to use carbohydrate as a carbon source, and were able to form root nodules on M. pudica and to fix nitrogen as efficiently as the parental strain. We conclude that the enzymes dPGM and FBPase are essential for the formation of a symbiosis with the host plant.