F+RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+RNA phages varied, and may bias extrapolation of their initial proportion in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+RNA phages are used for microbial source tracking.

Olive brine represents a stressful environment due to high NaCl concentration, presence of phenolic compounds known as antimicrobials, and low availability of nutrients. Thus, only a few strains of lactic acid bacteria (LAB) are adapted to grow and ferment table olives. To identify the mechanisms by which these few strains are able to grow in olive brine, Lactobacillus pentosus C11, a particularly resistant strain isolated from naturally fermented table olives, was mutagenized by random transposition using the Pjunc-TpaseIS1223 system (Licandro-Seraut H, Brinster S, van de Guchte M, Scornec H, Maguin E, Sansonetti P, Cavin JF, Serror P. 2012. Appl. Environ. Microbiol. 78:5417-5423). A library of 6000 mutants was generated and screened for the adaptation and subsequent growth in a medium, named BSM (Brine Screening Medium), which displays the stressful conditions encountered in olive brine. Five transposition mutants impaired in growth on BSM were identified. Transposition occurred in two open reading frames and in three transcription terminators affecting stability of transcripts. Thus, several essential genes for adaptation and growth of L. pentosus C11 in olive brine were identified.

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high performance thin layer chromatography (HPTLC). This metagenome extract thin layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) but also other flavonoid-modifying activities. The developed screening method is highly sensitive and the amount of 4 ng modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate, and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferases (UGT) genes. The metagenome-derived gtfC gene encoded for a 52 kDa protein and the deduced amino acid sequence was weakly similar to putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones and stilbenes but accepted also isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated MW of 46 kDa. The deduced amino acid sequence was highly similar to MGTs from Bacillus thuringiensis. Recombinant MgtB transferred sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones and flavanones. Moreover MgtB exhibited high activity on larger flavonoid molecules as tiliroside.

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (acyl-CoA oxidase from Micrococcus luteus and fadB and fadM from E. coli) led to the production of 50 to 65 mg/l of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/l under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

UV light only penetrates liquid food surfaces to a very short depth, thereby limiting its industrial application in food pasteurization. One promising alternative is the combination of UV light with mild heat (UV-H), which has been demonstrated to produce a synergistic bactericidal effect. The aim of this paper is to elucidate the mechanism of synergistic cellular inactivation resulting from the simultaneous application of UV light and heat. The lethality of UV-H treatments remained constant below approximately 45 °C, while lethality increased exponentially as the temperature increased. The percentage of synergism reached a maximum (40.3%) at 55 °C. Neither the flow regime nor changes in the delivered dose by UV lamps contributed to the observed synergism. UV-H inactivation curves of the parental E. coli strain obtained in a caffeic acid-selective recovery medium followed a similar profile to those obtained with uvrA(-) mutant cells in a non-selective medium. Thermal fluidification of membranes and synergistic lethal effects started around 40-45 °C. Chemical membrane fluidification with benzyl alcohol decreased the UV resistance of the parental strain but not of the uvrA(-) . These results suggest that the synergistic lethal effect of UV-H treatments is due to the inhibition of DNA excision repair resulting from the membrane fluidification caused by simultaneous heating.

During chemolithoautotrophic thiosulfate oxidation the phylogenetically-diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis and Thiomicrospira crunogena rendered steady enrichment of (34)S in the end product sulfate, with overall fractionation ranging between -4.6‰ and +5.8‰. Fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of (34)S enrichment. In case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, over which tetrathionate-intermediate formation is completed and sulfate production starts. Almost identical (34)S enrichment rates observed during the peak sulfate-producing stage of all the three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates as well as cysteine S-sulfonates in P. pantotrophus) in the actively sulfate-producing cells of all three species. Inducible expression of soxB during tetrathionate oxidation as well as the second leg of thiosulfate oxidation by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably however, no other Sox protein except SoxB could be detected upon MALDI-MS/MS analysis of all such T. kashmirensis proteins which appeared to be thiosulfate-inducible in 2D gel electrophoresis. Instead, several other redox proteins were found to be at-least twofold over-expressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.

During the past few decades numerous plasmid vectors have been developed for cloning, gene expression analysis and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery and ligation, but increasingly, procedures are developed to assemble large synthetic DNAs. In this study we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least up to 75 kb) into one or more specific sites (gene for tRNA-glycine) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host forcing the integration of the construct. Upon integration the system is again silenced. Two variants with different genetic features were produced, one in form of a cloning vector in E. coli, and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and BAC DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

Aminopeptidase-N (APN1) and Alkaline phosphatase (ALP) proteins located at the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis (Bt) subsp kurstaki. In this work we analyzed the role of ALP and APN1 on the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab binds preferentially to ALP during first instars while binding to APN was observed after the 3(rd) instar of larval development. Cry1Ac binds to APN throughout all larval development with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in E. coli. Surface Plasmon Resonance binding analysis showed that recombinant ALP binds to Cry1Ac with sixteen-fold lower affinity than to Cry1Aa or to Cry1Ab toxins. Down-regulation of APN1 and ALP expression by RNA interference using specific double-stranded RNA, correlated with a corresponding reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP or APN1 silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity while Cry1Ac relied principally on APN1. These results indicated ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

Cyanophycin, a polyamide of cyanobacterial or non-cyanobacterial origin and consisting of aspartate, arginine plus lysine was synthesized in different recombinant strains of E. coli expressing cphA from Synechocystis sp. PCC 6308, PCC 6803, Anabaena sp. PCC 7120 or Acinetobacter calcoaceticus ADP1. The molar aspartate:arginine:lysine ratio of a water-soluble form isolated from a recombinant strain expressing CphA6308 was 1:0.5:0.5 possessing a lysine content as high as never mentioned before. The water-insoluble form consisted instead of mainly aspartate and arginine and with a lower portion of lysine accounting to only 5 mol% at the maximum. It could be confirmed that synthesis of soluble CGP is independent of the origin of cphA. Soluble CGP isolated from all recombinant strains contained a least 17 mol% lysine. The total CGP portion of cell dry matter (CDM) synthesized by CphA6308 from recombinant E. coli was about 30 % (wt/wt) including 23 % (wt/wt) of soluble CGP using terrific broth complex medium for cultivation at 30 °C for 72 h. Enhanced production of soluble CGP instead of its insoluble form is interesting for further application and makes recombinant E. coli more attractive as suitable source for production of polyaspartic acid or dipeptides. In addition, a new low-cost, time-saving, effective and common isolation procedure for mainly soluble CGP, suitable for large-scale application, was established in this study.

Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas, or expose animals to pathogens in their new environment. Mycobacterium avium subspecies paratuberculosis (MAP), a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the MAP infection status of wood bison herds in Canada, and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n=267) in nine herds using direct PCR for three MAP-specific genetic targets which range in copy number within the MAP genome. Restriction enzyme analysis (REA) and sequencing of IS1311 was performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support MAP growth from feces and tissues of direct PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of MAP from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different MAP strain types exist in Canadian wood bison. The presence of different MAP strains among wood bison herds should be considered when planning future translocations.