- Volume 163, Issue 9, 2017

Hypomyces perniciosus has been reported as a destructive pathogen of Agaricus bisporus. Previous research suggested that the pathogenesis may not only be perpetuated by H. perniciosus, but also by bacteria. Clarification of the interaction between A. bisporus and H. perniciosus is a prerequisite for the development of effective control measures against wet bubble disease. Here, the effects of H. perniciosus on A. bisporus mycelia are examined in dual culture on agar media and in open-ended test tubes. During disease development, the putative causal agents and cytology of wet bubble-diseased mushrooms were followed microscopically. The interaction between H. perniciosus and the basidiome of A. bisporus was also studied using dual-cultured H. perniciosus and basidiome tissues. Dual-cultured mycelia from both fungi showed that growth continued even after contact was made, without any observable antagonistic lines or cytoplasmic changes of A. bisporus mycelia. Hypomyces perniciosus could be isolated from diseased basidiomes any time after inoculation, but bacteria were only recovered after the basidiomes of A. bisporus had been killed by H. perniciosus. Dual culture of the basidiome tissue of A. bisporus and H. perniciosus on agar media established that H. perniciosus can independently and rapidly degrade the basidiomes of A. bisporus. We conclude that H. perniciosus has no pathogenic activity on the mycelial stage of A. bisporus, but it can destroy A. bisporus basidiomes in the absence of bacteria. Wet bubble disease is evidently not caused by bacteria, but by the fungus, although bacteria likely participate in the disease after invasion by the fungus.

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

Biofilm accounts for 65–80 % of microbial infections in humans. Considerable evidence links biofilm formation by oral microbiota to oral disease and consequently systemic infections. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species of the oral microbiota and it contributes to biofilm development in the oral cavity. Due to its altered biofilm formation, we investigated a biofilm mutant, ΔSSA_0351, that is deficient in type I signal peptidase (SPase) in this study. Although the growth curve of the ΔSSA_0351 mutant showed no significant difference from that of the wild-type strain SK36, biofilm assays using both microtitre plate assay and confocal laser scanning microscopy (CLSM) confirmed a sharp reduction in biofilm formation in the mutant compared to the wild-type strain and the paralogous mutant ΔSSA_0849. Scanning electron microscopy (SEM) revealed remarkable differences in the cell surface morphologies and chain length of the ΔSSA_0351 mutant compared with those of the wild-type strain. Transcriptomic and proteomic assays using RNA sequencing and mass spectrometry, respectively, were conducted on the ΔSSA_0351 mutant to evaluate the functional impact of SPase on biofilm formation. Subsequently, bioinformatics analysis revealed a number of proteins that were differentially regulated in the ΔSSA_0351 mutant, narrowing down the list of SPase substrates involved in biofilm formation to lactate dehydrogenase (SSA_1221) and a short-chain dehydrogenase (SSA_0291). With further experimentation, this list defined the link between SSA_0351-encoded SPase, cell wall biosynthesis and biofilm formation.

The ggpS gene, which encodes the key enzyme for the synthesis of the compatible solute glucosylglycerol (GG), has a promoter region that overlaps with the upstream-located gene slr1670 in the cyanobacterium Synechocystissp. PCC 6803. Like ggpS, the slr1670 gene is salt-induced and encodes a putative glucosylhydrolase. A mutant strain with a slr1670 deletion was generated and found to be unable to adapt the internal GG concentrations in response to changes in external salinities. Whereas cells of the wild-type reduced the internal pool of GG when exposed to gradual and abrupt hypo-osmotic treatments, or when the compatible solute trehalose was added to the growth medium, the internal GG pool of ∆slr1670 mutant cells remained unchanged. These findings indicated that the protein Slr1670 is involved in GG breakdown. The biochemical activity of this GG-hydrolase enzyme was verified using recombinant Slr1670 protein, which split GG into glucose and glycerol. These results validate that Slr1670, which was named GghA, acts as a GG hydrolase. GghA is involved in GG turnover in fluctuating salinities, and similar proteins are found in the genomes of other GG-synthesizing cyanobacteria.

Pseudomonasaeruginosa uses choline as a source of carbon and nitrogen, and also for the synthesis of glycine betaine, an osmoprotectant under stress conditions such as drought and salinity. The transcription factor GbdR is the specific regulator of choline metabolism and it belongs to the Arac/XylS family of transcriptional regulators. Despite the link between choline catabolism and bacterial pathogenicity, gbdR regulation has not been explored in detail. In the present work, we describe how gbdR transcription can be initiated from a σ54-dependent promoter. gbdR transcription can be activated by NtrC in the absence of a preferential nitrogen source, by CbrB in the absence of a preferential carbon source, and by the integration host factor favouring DNA bending. In addition, we found that BetI negatively regulates gbdR expression in the absence of choline. We identified two overlapping BetI binding sites in the gbdR promoter sequence, providing an additional example of σ54-promoter down-regulation. Based on our findings, we propose a model for gdbR regulation and its impact on choline metabolism.

Small bacteriophage-like particles called gene transfer agents (GTAs) that mediate DNA transfer between cells are produced by a variety of prokaryotes. The model GTA, produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), is controlled by several cellular regulators, and production is induced upon entry into the stationary phase. We report that RcGTA production and gene transfer are stimulated by nutrient depletion. Cells depleted of organic carbon or blocked for amino acid biosynthesis increased RcGTA production and release from cells. Furthermore, cells lacking the sole RelA-SpoT homologue produced decreased levels of RcGTA, and the RNA polymerase omega (ω) subunit was required for appreciable production of RcGTA.

Bordetella pertussis, a human pathogenic bacterium, produces either one or two types of serologically distinct fimbriae, Fim2 and Fim3, as virulence factors. The expression of fim2 and fim3 is regulated by the BvgAS two-component system and the length of poly(C) stretches in Pfim promoters. In the Bvg+ phase, B. pertussis virulence-activated genes (vags) are up-regulated and virulence-repressed genes (vrgs) are down-regulated. Previous studies have shown that fim2 is a vag, but there is no consensus on fim3 regulation. We examined the regulation of fimbrial expression in B. pertussis clinical isolates. Our findings indicate that fim2 is a vag, while fim3 is a vag when Pfim3 poly(C)>13C, and a vrg when poly(C)≤13C. Although increased fim3 expression was observed in the Bvg– phase in isolates with Pfim3 poly(C)≤13C, Fim3 production was not detected, suggesting post-transcriptional regulation of fim3 expression. These findings provide an insight into the regulation of fimbrial expression in B. pertussis.