Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation

Acid stress resistance of the food-borne human pathogen Bacillus cereus may contribute to its survival in acidic environments, such as encountered in soil, food and the human gastrointestinal tract. The acid stress responses of B. cereus strains ATCC 14579 and ATCC 10987 were analysed in aerobically grown cultures acidified to pH values ranging from pH 5.4 to pH 4.4 with HCI. Comparative phenotype and transcriptome analyses revealed three acid stressinduced responses in this pH range: growth rate reduction, growth arrest and loss of viability. These physiological responses showed to be associated with metabolic shifts and the induction of general stress response mechanisms with a major oxidative component, including upregulation of catalases and superoxide dismutases. Flow cytometry analysis in combination with the hydroxyl (OH center dot) and peroxynitrite (ONOO-)-specific fluorescent probe 3'-(phydroxyphenyl) fluorescein (HPF) showed excessive radicals to be formed in both B. cereus strains in bactericidal conditions only. Our study shows that radicals can indicate acid-induced malfunctioning of cellular processes that lead to cell death

Germinant receptor diversity and germination responses of four strains of the Bacillus cereus group

Four strains of the Bacillus cereus group were compared for their germinant receptor composition and spore germination capacity. Phylogenetic analysis of the germinant receptor encoding operons of the enterotoxic strains B. cereus ATCC 14579 and ATCC 10987, the emetic strain AH187, and the psychrotolerant strain Bacillus weihenstephanensis KBAB4, indicated a core group of five germinant receptor operons to be present in the four strains, with each strain containing one to three additional receptors. Using quantitative PCR, induction of expression during sporulation was confirmed for all identified germinant receptor operons in these strains. Despite the large overlap in receptors, diversity in amino acid-induced germination capacity was observed, with six out of 20 amino acids, serving as germinants for spores of all four strains. Each strain showed unique features: efficient germination of strain KBAB4 spores required non-inducing amounts of inosine as the co-germinant, strain ATCC 10987 spores germinated only efficiently after heat activation. Furthermore, strain ATCC 14579 and AH187 spores germinated without heat activation or inosine, with strain ATCC 14579 spores being triggered by all amino acids except phenylalanine and strain AH187 spores being specifically triggered efficiently only by phenylalanine. Analysis of all germination data did not reveal strict linkages between specific germinants and germinant receptors. Finally, the diversity in nutrient-induced germination capacity was also reflected in the diverse germination responses of heat-activated spores of the four B. cereus strains in food matrices, such as milk, rice water and meat bouillon, indicating that amino acid composition and/or availability of inosine are important germination determinants in foods. Keywords: Ger operon; Food preservation; B. weihenstephanensis; Sporulatio

A wide variety of Clostridium perfringens type A food-borne isolates that carry a chromosomal cpe gene belong to one multilocus sequence typing cluster

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)5 fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods

Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm

Towards a new ring expansion/contraction mechanism useful for the synthesis of [3]Ladderane

De begeleider en/of auteur heeft geen toestemming gegeven tot het openbaar maken van de scriptie. The supervisor and/or the author did not authorize public publication of the thesis.

Fermentation in cyanobacteria

Although cyanobacteria are oxygenic phototrophic organisms, they often thrive in environments that become periodically anoxic. This is particularly the case in the dark when photosynthetic oxygen evolution does not take place. Whereas cyanobacteria generally utilize endogenous storage carbohydrate by aerobic respiration, they must use alternative ways for energy generation under dark anoxic conditions. This aspect of metabolism of cyanobacteria has received little attention but nevertheless in recent years a steadily increasing number of publications have reported the capacity of fermentation in cyanobacteria. This review summarizes these reports and gives a critical consideration of the energetics of dark fermentation in a number of species. There are a variety of different fermentation pathways in cyanobacteria. These include home-and heterolactic acid fermentation, mixed acid fermentation and homoacetate fermentation, Products of fermentation include CO2, H-2, formate, acetate, lactate and ethanol. In all species investigated, fermentation is constitutive. All enzymes of the fermentative pathways are present in photoautotrophically grown cells. Many cyanobacteria are also capable of using elemental sulfur as electron acceptor. In most cases it seems unlikely that sulfur respiration occurs, The main advantage of sulfur reduction seems to be the higher yield of ATP which can be achieved during fermentation. Besides oxygen and elemental sulfur no other electron accepters for chemotrophic metabolism are known so far in cyanobacteria. Calculations show that the yield of ATP during fermentation, although it is low relative to aerobic respiration, exceeds the amount that is likely to be. required for maintenance, which appears to be very low in these cyanobacteria. The possibility of a limited amount oi biosynthesis during anaerobic dark metabolism is discussed. [KEYWORDS: fermentation; cyanobacteria; dark metabolism; Embden-Meyerhof-Parnas pathway; lactate dehydrogenase; lactate fermentation; mixed acid fermentation; sulfur reduction Blue-green-alga; oscillatoria-agardhii gomont; spring microbial mat; light-dark cycle; solar lake sinai; heterotrophic growth; unicellular cyanobacterium; ferredoxin oxidoreductase; anaerobic conditions; anoxygenic photosynthesis]

A comparison of fermentation in the cyanobacterium Microcystis PCC7806 grown under a light/dark cycle and continuous light

The cyanobacterium Microcystis PCC7806, grown under continuous light, fermented endogenously stored glycogen to equimolar amounts of acetate and ethanol when incubated anaerobically in the dark. In addition, H-2, CO2 and some L-lactate were produced. This fermentation pattern differed from that displayed by cells which had been grown under an alternating light/dark (16/8 h) cycle. Such cells produced much more ethanol than acetate, while no lactate was formed. These differences could not be related to the levels of key enzymes of fermentation, which were identical in the two cultures. The cultures grown under continuous light contained twice as much glycogen as the light/dark-grown cells and the former metabolized it at a rate approximately 3 times as fast as the latter culture. Fermentation in the culture grown under continuous light showed low carbon recovery (59-80%) and high oxidation/reduction balance (approximately 1.5). On the basis of calculations of ATP yield it was concluded that this culture was capable of growth driven by fermentation The increase in structural cell material would account for the missing carbon and reduction equivalents. [KEYWORDS: acetate kinase; alcohol dehydrogenase; Cyanobacterium; dark metabolism; Embden-Meyerhof-Parnas pathway; fermentation; hydrogenase; lactate dehydrogenase; light/dark cycle; pyruvate:ferredoxin oxidoreductase Dark; metabolism; nitrogen]