DNase-Sensitive and -Resistant Modes of Biofilm Formation by Listeria monocytogenes.

Listeria monocytogenes is able to form biofilms on various surfaces and this ability is thought to contribute to persistence in the environment and on contact surfaces in the food industry. Extracellular DNA (eDNA) is a component of the biofilm matrix of many bacterial species and was shown to play a role in biofilm establishment of L. monocytogenes. In the present study, the effect of DNaseI treatment on biofilm formation of L. monocytogenes EGD-e was investigated under static and dynamic conditions in normal or diluted complex medium at different temperatures. Biofilm formation was quantified by crystal violet staining or visualized by confocal laser scanning microscopy. Biomass of surface-attached L. monocytogenes varies depending on temperature and dilution of media. Interestingly, L. monocytogenes EGD-e forms DNase-sensitive biofilms in diluted medium whereas in full strength medium DNaseI treatment had no effect. In line with these observations, eDNA is present in the matrix of biofilms grown in diluted but not full strength medium and supernatants of biofilms grown in diluted medium contain chromosomal DNA. The DNase-sensitive phenotype could be clearly linked to reduced ionic strength in the environment since dilution of medium in PBS or saline abolished DNase sensitivity. Several other but not all species of the genus Listeria display DNase-sensitive and -resistant modes of biofilm formation. These results indicate that L. monocytogenes biofilms are DNase-sensitive especially at low ionic strength, which might favor bacterial lysis and release of chromosomal DNA. Since low nutrient concentrations with increased osmotic pressure are conditions frequently found in food processing environments, DNaseI treatment represents an option to prevent or remove Listeria biofilms in industrial settings.

Arabitol metabolism of Corynebacterium glutamicum and its regulation by AtlR.

Expression profiling of Corynebacterium glutamicum in comparison to a derivative deficient in the transcriptional regulator AtlR (previously known as SucR or MtlR) revealed eight genes showing more than 4-fold higher mRNA levels in the mutant. Four of these genes are located in the direct vicinity of the atlR gene, i.e., xylB, rbtT, mtlD, and sixA, annotated as encoding xylulokinase, the ribitol transporter, mannitol 2-dehydrogenase, and phosphohistidine phosphatase, respectively. Transcriptional analysis indicated that atlR and the four genes are organized as atlR-xylB and rbtT-mtlD-sixA operons. Growth experiments with C. glutamicum and C. glutamicum ΔatlR, ΔxylB, ΔrbtT, ΔmtlD, and ΔsixA derivatives with sugar alcohols revealed that (i) wild-type C. glutamicum grows on D-arabitol but not on other sugar alcohols, (ii) growth in the presence of D-arabitol allows subsequent growth on D-mannitol, (iii) D-arabitol is cometabolized with glucose and preferentially utilized over D-mannitol, (iv) RbtT and XylB are involved in D-arabitol but not in D-mannitol metabolism, (v) MtlD is required for D-arabitol and D-mannitol metabolism, and (vi) SixA is not required for growth on any of the substrates tested. Furthermore, we show that MtlD confers D-arabitol and D-mannitol dehydrogenase activities, that the levels of these and also xylulokinase activities are generally high in the C. glutamicum ΔatlR mutant, whereas in the parental strain, they were high when cells were grown in the presence of D-arabitol and very low when cells were grown in its absence. Our results show that the XylB, RbtT, and MtlD proteins allow the growth of C. glutamicum on D-arabitol and that D-arabitol metabolism is subject to arabitol-dependent derepression by AtlR.

Control of adhA and sucR expression by the SucR regulator in Corynebacterium glutamicum.

The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to a complex carbon source-dependent regulation mediated by RamA, RamB and GlxR. In this study we identified SucR as the fourth transcriptional regulator involved in expression control of the adhA gene. SucR specifically binds to the adhA promoter and acts as transcriptional repressor independent of the carbon source used. Furthermore, we found that SucR negatively controls the expression of its own gene. This negative autoregulation is mediated by binding of SucR to at least one of four identified binding sites located in the promoter region of sucR. EMSA experiments and subsequent sequence analysis led to the identification of the SucR consensus binding sequence YYAACAWMAW. This binding motif is different from the binding site (ACTCTAGGGG) recently described for SucR in the promoter region of the sucCD operon. However, we were not able to detect a specific interaction of purified SucR protein with this motif present in the sucCD promoter region.

Complete genome sequence of Bifidobacterium bifidum S17.

Here, we report on the first completely annotated genome sequence of a Bifidobacterium bifidum strain. B. bifidum S17, isolated from feces of a breast-fed infant, was shown to strongly adhere to intestinal epithelial cells and has potent anti-inflammatory activity in vitro and in vivo. The genome sequence will provide new insights into the biology of this potential probiotic organism and allow for the characterization of the molecular mechanisms underlying its beneficial properties.

RamA and RamB are global transcriptional regulators in Corynebacterium glutamicum and control genes for enzymes of the central metabolism.

In Corynebacterium glutamicum, the transcriptional regulators of acetate metabolism RamA (encoded by cg2831) and RamB (encoded by cg0444) play an important role in expression control of genes involved in acetate and ethanol metabolism. Both regulators were speculated to have broader significance in expression control of further genes in the central metabolism of C. glutamicum. Here we investigated the RamA and RamB regulons by genome-wide transcriptome analysis with special emphasis on genes encoding enzymes of the central carbon metabolism. When compared to the parental wild-type, 253 genes and 81 genes showed different mRNA levels in defined RamA- and RamB-deficient C. glutamicum strains, respectively. Among these were genes involved in sugar uptake, glycolysis, gluconeogenesis, acetate, l-lactate or ethanol metabolism. The direct interaction of RamA and RamB proteins with the respective promoter/operator fragments was demonstrated in vitro by electrophoretic mobility shift assays. Taken together, we present evidence for an important role of RamA and RamB in global gene expression control in C. glutamicum.

The transcriptional regulators RamA and RamB are involved in the regulation of glycogen synthesis in Corynebacterium glutamicum.

When grown in glucose-, fructose- or sucrose-containing medium, the amino acid producer Corynebacterium glutamicum transiently accumulates large amounts of glycogen (up to 10% of its dry weight), whereas only a marginal amount of glycogen is formed during growth with acetate. This carbon-source-dependent regulation is at least partially due to transcriptional control of glgC, encoding ADP-glucose pyrophosphorylase, the first enzyme of glycogen synthesis from glucose-1-phosphate. Here, we have analysed a possible regulatory role for the transcriptional regulators RamA and RamB on glycogen content of the cells and on control of expression of glgC and of glgA, which encodes the second enzyme of glycogen synthesis, glycogen synthase. Determination of the glycogen content of RamA- and RamB-deficient C. glutamicum indicated that RamA and RamB influence glycogen synthesis positively and negatively, respectively. In accordance with the identification of putative RamA and RamB binding sites upstream of glgC and glgA, both regulators were found to bind specifically to the glgC-glgA intergenic promoter region. Promoter activity assays in wild-type and RamA- and RamB-deficient strains of C. glutamicum revealed that (i) RamA is a positive regulator of glgC and glgA, (ii) RamB is a negative regulator of glgA and (iii) neither RamA nor RamB alone is responsible for the carbon-source-dependent regulation of glycogen synthesis in C. glutamicum.

Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB.

Corynebacterium glutamicum has been shown to grow with ethanol as the sole or as additional carbon and energy source and accordingly, to possess both alcohol dehydrogenase and acetaldehyde dehydrogenase (ALDH) activities, which are responsible for the two-step ethanol oxidation to acetate. Here we identify and functionally analyze the C. glutamicum ALDH gene (cg3096, ald), its expression and its regulation. Directed inactivation of the chromosomal ald gene led to the absence of detectable ALDH activity and to the inability to grow on or to utilize ethanol, indicating that the ald gene product is essential for ethanol metabolism and that no ALDH isoenzymes are present in C. glutamicum. Transcriptional analysis revealed that ald from C. glutamicum is monocistronic, that ald transcription is initiated 92 nucleotides upstream of the translational start codon ATG and that ald expression is much lower in the presence of glucose in the growth medium. Further analysis revealed that transcription of the ald gene is under control of the transcriptional regulators RamA and RamB. Both these proteins directly bind to the respective promoter region, RamA is essential for expression and RamB exerts a slightly negative effect on ald expression on all carbon sources tested.

L-valine production during growth of pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum in the presence of ethanol or by inactivation of the transcriptional regulator SugR.

Pyruvate dehydrogenase complex-deficient strains of Corynebacterium glutamicum produce L-valine from glucose only after depletion of the acetate required for growth. Here we show that inactivation of the DeoR-type transcriptional regulator SugR or replacement of acetate by ethanol already in course of the growth phase results in efficient L-valine production.

Ethanol catabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.

RamB, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to regulation by RamA and RamB.

In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that RamB represses its own expression by direct interaction with a 13-bp motif in the ramB promoter region. Additionally, ramB expression is subject to carbon source-dependent positive control by RamA.

Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production.

Corynebacterium glutamicum, well known for the industrial production of amino acids, grows aerobically on a variety of mono- and disaccharides and on alcohols and organic acids as single or combined sources of carbon and energy. Members of the genera Corynebacterium and Brevibacterium were here tested for their ability to use the homopolysaccharide starch as a substrate for growth. None of the 24 type strains tested showed growth on or degradation of this substrate, indicating that none of the strains synthesized and secreted starch-degrading enzymes. Introducing the Streptomyces griseus amy gene on an expression vector into the lysine-producer C. glutamicum DM1730, we constructed a C. glutamicum strain synthesizing and secreting alpha-amylase into the culture broth. Although some high-molecular-weight degradation products remained in the culture broth, this recombinant strain effectively used soluble starch as carbon and energy substrate for growth and also for lysine production. Thus, employment of our construct allows avoidance of the cost-intensive enzymatic hydrolysis of the starch, which commercially is used as a substrate in industrial amino acid fermentations.