In Bacillus subtilis the physiological consequences of depriving lipoteichoic acid and wall teichoic acid of D-alanine ester were analysed using insertional inactivation of the genes of the dlt operon. Mutant strains which lacked positively charged o-alanine ester in teichoic acids bound more positively charged cytochrome c than other strains. These mutant strains also showed enhanced autolysis and a higher susceptibility to methicillin, which was expressed as accelerated wall lysis, a faster loss of viability and a slower recovery in the postantibiotic phase. The effects of methicillin could be suppressed by simultaneous addition of magnesium ions at low concentrations. The degradation of whole bacteria by bone-marrow-derived macrophages was not influenced by the surface charge and alanylation of the teichoic acids had no protective effect.

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f Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 ؋ 10 7 spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques. Microbial contamination on surfaces is a recurring problem within health, pharmaceutical, and food industry sectors (1, 2, 3). Thus, decontamination is a crucial step to ensure the sterility of food processing equipment, minimize spread of pathogens, and prevent the transmission of nosocomial infections (4). Common decontamination and disinfection procedures that are widely used for microbial inactivation include high temperatures, chemicals, or ionizing radiation (reviewed in reference 5). In order to ensure the efficiency and to validate the continuous functionality of a disinfection or sterilization procedure, biological testing standards are required. Bacterial spores are frequently used as a biological indicator (BI) of sterility, primarily because bacterial spores exhibit elevated resistance to chemical and physical methods of sterilization (6-11). Hence, a process that achieves full spore inactivation ensures complete elimination of other contaminating microorganisms.Variations in the performance of a BI have been reported repeatedly (12, 13). Besides variations in the intrinsic resistance properties of the microorganisms conferred, for instance, by variations in genetic traits or alteration of sporulation conditions (14), extrinsic factors also may affect the performance of BIs and, subsequently, the accurate determination of spore resistance and inactivation. For example, the sterilization results may be altered by poor choices of the carrier material for spore deposition (13,15) and, in particular, the BI manufacturing procedure (16). The method by which spores are mounted on carriers also is extremely impo...

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In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (“ripening”) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.