Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

N-Acetyl-Cysteinylated Streptophenazines from Streptomyces.

Here, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by the soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example of how to leverage metabolomics for the discovery of new natural products and point out the often-neglected contribution of house-keeping cellular functions to natural product diversification.

Lichenysin Production by Bacillus licheniformis Food Isolates and Toxicity to Human Cells.

Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains (high producers B4094 and B4123, and type strain DSM13 T ) was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4-20 times lower in milk than in LB broth (maximum 36 µg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11-C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 µg/ml for Caco-2 human intestinal epithelial cells and 16.8 µg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested.