- Volume 160, Issue 4, 2014

Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA).

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

In this study, we characterized FgIlv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acid (BCAA, including leucine, isoleucine and valine) biosynthetic pathway that exists in a variety of organisms from bacteria to fungi and higher plants, but not in mammals. The FgILV5 deletion mutant ΔFgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs. When grown on potato-dextrose agar plates, ΔFgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in ΔFgIlv5-4. Exogenous addition of 1 mM isoleucine and valine was able to rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that ΔFgIlv5-4 was more sensitive to osmotic and oxidative stresses than the wild-type strain. In addition, virulence of ΔFgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol production was detected in ΔFgIlv5-4 in wheat kernels. The results of this study indicate that FgIlv5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F. graminearum.

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP–DnaA protein.

EIIANtr is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIANtr regulates K+ homeostasis through interaction with the K+ transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIANtr influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIANtr increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIANtr, has the opposite effect. To clarify the role of EIIANtr in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIANtr from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIANtr interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIANtr with KdpD appears to be absent in R. eutropha, although R. eutropha EIIANtr can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary ‘ancient’ task of EIIANtr that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIANtr might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.

The facultative anaerobic bacterium Listeria monocytogenes encounters microaerophilic or anaerobic conditions in various environments, e.g. in soil, in decaying plant material, in food products and in the host gut. To elucidate the adaptation of Listeria monocytogenes to variations in oxygen tension, global transcription analyses using DNA microarrays were performed. In total, 139 genes were found to be transcribed differently during aerobic and anaerobic growth; 111 genes were downregulated and 28 genes were upregulated anaerobically. The oxygen-dependent transcription of central metabolic genes is in agreement with results from earlier physiological studies. Of those genes more strongly expressed under lower oxygen tension, 20 were knocked out individually. Growth analysis of these knock out mutants did not indicate an essential function for the respective genes during anaerobiosis. However, even if not essential, transcriptional induction of several genes might optimize the bacterial fitness of Listeria monocytogenes in anaerobic niches, e.g. during colonization of the gut. For example, expression of the anaerobically upregulated gene lmo0355, encoding a fumarate reductase α chain, supported growth on 10 mM fumarate under anaerobic but not under aerobic growth conditions. Genes essential for anaerobic growth were identified by screening a mutant library. Eleven out of 1360 investigated mutants were sensitive to anaerobiosis. All 11 mutants were interrupted in the atp locus. These results were further confirmed by phenotypic analysis of respective in-frame deletion and complementation mutants, suggesting that the generation of a proton motive force via F1F0-ATPase is essential for anaerobic proliferation of Listeria monocytogenes.

Dickeya dadantii is a phytopathogenic enterobacterium that causes soft rot disease in a wide range of plant species. Maceration, an apparent symptom of the disease, is the result of the synthesis and secretion of a set of plant cell wall-degrading enzymes (PCWDEs), but many additional factors are required for full virulence. Among these, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in the synthesis of PCWDEs. PecS is one of the major regulators of virulence in D. dadantii, acting mainly as a repressor of various cellular functions including virulence, motility and synthesis of PCWDEs. The present study shows that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs, indicating that PecS cannot be de-repressed in strains devoid of OPGs.

In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform.

This study characterized the two-component regulatory systems encoded by bfrKRT and cemAKR, and assessed their influence on biofilm formation by Lactobacillus reuteri 100-23. A method for deletion of multiple genes was employed to disrupt the genetic loci of two-component systems. The operons bfrKRT and cemAKR showed complementary organization. Genes bfrKRT encode a histidine kinase, a response regulator and an ATP-binding cassette-type transporter with a bacteriocin-processing peptidase domain, respectively. Genes cemAKR code for a signal peptide, a histidine kinase and a response regulator, respectively. Deletion of single or multiple genes in the operons bfrKRT and cemAKR did not affect cell morphology, growth or the sensitivity to various stressors. However, gene disruption affected biofilm formation; this effect was dependent on the carbon source. Deletion of bfrK or cemA increased sucrose-dependent biofilm formation in vitro. Glucose-dependent biofilm formation was particularly increased by deletion of cemK. The expression of cemK and cemR was altered by deletion of bfrK, indicating cross-talk between these two regulatory systems. These results may contribute to our understanding of the genetic factors related to the biofilm formation and competitiveness of L. reuteri in intestinal ecosystems.

Yarrowia lipolytica, an ascomycete with biotechnological potential, is able to form either yeast cells or hyphae and pseudohyphae in response to environmental conditions. This study shows that the morphology of Y. lipolytica, cultivated in batch cultures on hydrophilic (glucose and glycerol) and hydrophobic (olive oil) media, was not affected by the nature of the carbon source, nor by the nature or the concentration of the nitrogen source. By contrast, dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. Specifically, when growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used. Experimental data obtained from a continuous culture of Y. lipolytica on glycerol, being used as carbon and energy source, demonstrated that the mycelium-to-yeast form transition occurs when DOC increases from 0.1 to 1.5 mg l−1. DOC also affected the yeast physiology, as the activity of enzymes implicated in lipid biosynthesis (i.e. ATP-citrate lyase, malic enzyme) was upregulated at high DOC whereas the activity of enzymes implicated in glycerol assimilation (such as glycerol dehydrogenase and kinase) remained fundamentally unaffected in the cell-free extract.

Streptococcus mutans encounters an array of sugar moieties within the oral cavity due to a varied human diet. One such sugar is β-d-glucose 1-phosphate (βDG1P), which must be converted to glucose 6-phosphate (G6P) before further metabolism to lactic acid. The conversion of βDG1P to G6P is mediated by β-phosphoglucomutase, which has not been previously observed in any oral streptococci, but has been extensively characterized and the gene designated pgmB in Lactococcus lactis. An orthologue was identified in S. mutans, SMU.1747c, and deletion of the gene resulted in the inability of the deletion strain to convert βDG1P to G6P, indicating that SMU.1747c is a β-phosphoglucomutase and should be designated pgmB. In this study, we sought to characterize how deletion of pgmB affected known virulence factors of S. mutans, specifically acid tolerance. The ΔpgmB strain showed a decreased ability to survive acid challenge. Additionally, the strain lacking β-phosphoglucomutase had a diminished glycolytic profile compared with the parental strain. Deletion of pgmB had a negative impact on the virulence of S. mutans in the Galleria mellonella (greater wax worm) animal model. Our results indicate that pgmB plays a role at the juncture of carbohydrate metabolism and virulence.