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We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display.

Salmonella enterica serovar Typhimurium possesses a stimulon of genes that are differentially regulated in response to conditions of low fluid shear force that increase bacterial virulence and alter other phenotypes. In this study, we show that a previously uncharacterized member of this stimulon, ydcI or STM1625, encodes a highly conserved DNA binding protein with related homologs present in a range of Gram-negative bacterial genera. Gene expression analysis shows that ydcI is expressed in different bacterial genera and is involved in its autoregulation in S. Typhimurium. We demonstrate that purified YdcI protein specifically binds a DNA probe consisting of its own promoter sequence. We constructed an S. Typhimurium ⌬ydcI mutant strain and show that this strain is more sensitive to both organic and inorganic acid stress than is an isogenic WT strain, and this defect is complemented in trans. Moreover, our data indicate that ydcI is part of the rpoS regulon related to stress resistance. The S. Typhimurium ⌬ydcI mutant was able to invade cultured cells to the same degree as the WT strain, but a strain in which ydcI expression is induced invaded cells at a level 2.8 times higher than that of the WT. In addition, induction of ydcI expression in S. Typhimurium resulted in the formation of a biofilm in stationary-phase cultures. These data indicate the ydcI gene encodes a conserved DNA binding protein involved with aspects of prokaryotic biology related to stress resistance and possibly virulence.Bacterial growth environments characterized by low fluid shear force have been shown to induce a multitude of phenotypic responses, including altered acid, oxidative, thermal, and osmotic stress resistance (7,33,36,41,(52)(53)(54)(55), increased biofilm formation (6, 33, 52), altered protein secretion (14, 15), altered cell surface lipid and polysaccharide profiles (6, 7, 55), and increased survival in cellular and animal hosts (39,(52)(53)(54). Notably, the virulence of Salmonella enterica serovar Typhimurium is increased by low fluid shear growth conditions as measured using murine infection assays and tissue culture models (39,(52)(53)(54). Low fluid shear force (defined here as approximately Ͻ0.01 to 0.2 dynes/cm 2 ) is characterized by a lowturbulence, low-agitation environment, as opposed to high fluid shear (defined here as approximately from 5 to Ͼ50 dynes/cm 2 ) where liquid moves with higher velocity over the cellular surface (3-5, 21, 23, 25, 36). Low fluid shear growth environments include spaceflight, ground-based suspension culture models such as the rotating-wall vessel (RWV) bioreactor, and the spaces between cellular microvilli, the last of which is encountered by numerous pathogens during the natural course of infection (21,23,25,30,40,41). Previous work has shown that bacteria grown in low fluid shear environments induce a molecular response which includes genome-wide changes in gene expression (the low fluid shear stimulon) (6,7,39,48,52,53,55). Since growth under low fluid shear conditions is able to ind...

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Tracheal explants from rats exposed to 0.8 ppm (1.9 mg per m3) of ozone 8 hours per day for 1 to 90 days were incubated in culture with glucosamine labeled with carbon-14 or hydrogen-3. Compared with tracheas from control rats exposed to filtered air, the explants demonstrated a decreased rate of glycoprotein secretion for exposure intervals of as long as one week, followed by a rebound to an increased rate of glycoprotein secretion for at least 12 weeks of continued exposure to ozone. Detailed study of the behavior of labeled glycoproteins from the culture medium on chromatography on columns of BioGel A-150m demonstrated that the ratio of the low to high molecular weight peaks increased when there was an increased rate of glycoprotein secretion. This is the first report of a direct biochemical effect induced by ozone on airway metabolism.

Antibiotic-resistant Gram-negative bacteria are emergent pathogens, causing millions of infections worldwide. While there are several classes of antibiotics that are effective against Gram-positive bacteria, the outer membrane (OM) of Gram-negative bacteria excludes high-molecular-weight hydrophobic antibiotics, making these species intrinsically resistant to several classes of antibiotics, including polyketides, aminocoumarins, and macrolides. The overuse of antibiotics such as β-lactams has also promoted the spread of resistance genes throughout Gram-negative bacteria, including the production of extended spectrum β-lactamases (ESBLs). The combination of innate and acquired resistance makes it extremely challenging to identify antibiotics that are effective against Gram-negative bacteria. In this study, we have demonstrated the synergistic effect of outer membrane-permeable cationic polyurethanes with rifampicin, a polyketide that would otherwise be excluded by the OM, on different strains of E. coli, including a clinically isolated uropathogenic multidrug-resistant (MDR) E. coli. Rifampicin combined with a low-dose treatment of a cationic polyurethane reduced the MIC in E. coli of rifampicin by up to 64-fold. The compositions of cationic polyurethanes were designed to have low hemolysis and low cell cytotoxicity while maintaining high antibacterial activity. Our results demonstrate the potential to rescue the large number of available OM-excluded antibiotics to target normally resistant Gram-negative bacteria via synergistic action with these cationic polyurethanes, acting as a novel antibiotic adjuvant class.

Haem-dependent catalase is an antioxidant enzyme that degrades H2O2, producing H2O and O2, and is common in aerobes. Catalase is present in some strictly anaerobic methane-producing archaea (methanogens), but the importance of catalase to the antioxidant system of methanogens is poorly understood. We report here that a survey of the sequenced genomes of methanogens revealed that the majority of species lack genes encoding catalase. Moreover, Methanosarcina acetivorans is a methanogen capable of synthesizing haem and encodes haem-dependent catalase in its genome; yet, Methanosarcina acetivorans cells lack detectable catalase activity. However, inducible expression of the haem-dependent catalase from Escherichia coli (EcKatG) in the chromosome of Methanosarcina acetivorans resulted in a 100-fold increase in the endogenous catalase activity compared with uninduced cells. The increased catalase activity conferred a 10-fold increase in the resistance of EcKatG-induced cells to H2O2 compared with uninduced cells. The EcKatG-induced cells were also able to grow when exposed to levels of H2O2 that inhibited or killed uninduced cells. However, despite the significant increase in catalase activity, growth studies revealed that EcKatG-induced cells did not exhibit increased tolerance to O2 compared with uninduced cells. These results support the lack of catalase in the majority of methanogens, since methanogens are more likely to encounter O2 rather than high concentrations of H2O2 in the natural environment. Catalase appears to be a minor component of the antioxidant system in methanogens, even those that are aerotolerant, including Methanosarcina acetivorans. Importantly, the experimental approach used here demonstrated the feasibility of engineering beneficial traits, such as H2O2 tolerance, in methanogens.

Methanobrevibacter smithii is a common inhabitant of the human gut. Here, we present a draft genome sequence of M. smithii isolate WWM1085, obtained from a human stool sample. This sequence will improve our understanding of the genetic diversity of this human-associated methanogen.