Characterization of Basal Transcriptomes Identifies Potential Metabolic and Virulence-Associated Adaptations Among Diverse Nontyphoidal Salmonella enterica Serovars

The zoonotic pathogen Salmonella enterica includes >2,600 serovars, which differ in the range of hosts they infect and the severity of disease they cause. To further elucidate the mechanisms behind these differences, we performed transcriptomic comparisons of nontyphoidal Salmonella (NTS) serovars with the model for NTS pathogenesis, S. Typhimurium. Specifically, we used RNA-seq to characterize the understudied NTS serovars S. Javiana and S. Cerro, representing a serovar frequently attributed to human infection via contact with amphibians and reptiles, and a serovar primarily associated with cattle, respectively. Whole-genome sequence (WGS) data were utilized to ensure that strains characterized with RNA-seq were representative of their respective serovars. RNA extracted from representative strains of each serovar grown to late exponential phase in Luria-Bertani (LB) broth showed that transcript abundances of core genes were significantly higher (p<0.001) than those of accessory genes for all three serovars. Inter-serovar comparisons identified that transcript abundances of genes in Salmonella Pathogenicity Island (SPI) 1 were significantly higher in both S. Javiana and S. Typhimurium compared to S. Cerro. Together, our data highlight potential transcriptional mechanisms that may facilitate S. Cerro and S. Javiana survival in and adaptation to their respective hosts and impact their ability to cause disease in others. Furthermore, our analyses demonstrate the utility of omics approaches in advancing our understanding of the diversity of metabolic and virulence mechanisms of different NTS serovars.

The Biocatalytic Production of 3-Hydroxypropionaldehyde and Evaluation of Its Stability

3-Hydroxypropionaldehyde (3-HPA, reuterin) is a broad-spectrum natural antimicrobial agent used in the food industry and other fields. The low yield from the industrial production of 3-HPA using Lactobacillus reuteri and the spontaneous conversion of 3-HPA to acrolein have limited its more widespread use. We isolated L. reuteri BR201 as a biocatalyst for 3-HPA production and confirmed the effect of each factor in the two-step procedure for 3-HPA bioconversion. After initial cultivation for 8 h (late exponential phase), this isolate produced 378 mM of 3-HPA in 1 h at a concentration of OD600 nm 100, 30 °C, and an initial glycerol concentration of 500 mM. This is the highest reported biocatalytic yield of 3-HPA from a glycerol aqueous solution without additives. We confirmed that 4 mM of 3-HPA had antimicrobial activity against five pathogens. The degradation of 3-HPA to acrolein was greater at high temperatures, and there was little degradation when 3-HPA was maintained at 4 °C for 4 weeks. Our results may be useful for future applications of 3-HPA.

The Differential Proteome of the ProbioticLactobacillus acidophilusNCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Twoβ-Glycoside Hydrolases

Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacteriumLactobacillus acidophilusNCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4–7) and the alkaline (pH 6–11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

Bacillus Licheniformis Coated Bioparticles for Hydrogen Peroxide Degradation

The potential use of Bacillus licheniformis coated bioparticles for hydrogen peroxide (H2O2) degradation was assessed in this study. Bioparticles were made by mixing zeolite, activated carbon and cement in ratio 20:5:6 for attachment of biofilm. The efficiency of H2O2 degradation was examined in the presence and absence of biofilm (control) on bioparticles. Optimisation of biofilm development (7 and 10 days) and reusability were also investigated for H2O2degradation. Actively growing bacterial suspension (late exponential phase) of B.licheniformis was used in development of pure culture biofilm. The 7–day biofilm coated bioparticles system successfully achieved complete H2O2 degradation within an hour (highest rate = 1.17 % H2O2 degraded per minute) while the control showed no significant H2O2 degradation. After repeated use of biofilm coated bioparticles, the rate of H2O2 degradation declined to 0.654 % H2O2degraded per minute, and second use, the rate of H2O2 degradation was 0.166 % H2O2 degraded per minute. Field Emission Scanning Electron Microscope (FESEM) images of the biofilm coated bioparticles showed the attachment of cells and formation of extracellular polymeric substances (EPS), whereas the control showed no biofilm formed.

Temporal Expression of Staphylococcal Enterotoxin H in Comparison with Accessory Gene Regulator–Dependent and –Independent Enterotoxins

Using sandwich enzyme-linked immunosorbent assay (ELISA), the production of staphylococcal enterotoxin (SE) H was determined in 22 Staphylococcus aureus isolates bearing the seh gene. Samples of supernatants were taken at four time points corresponding to exponential phase (optical density at 600 nm [OD600] 0.3 to 0.6), late exponential phase (OD600 2 to 4), early stationary phase (OD600 4 to 6), and late stationary phase (OD600 7 to 12). In four isolates, SEH was detectable at a very low level at the first time point. In 18 isolates, the earliest SEH production was detected in the late exponential phase. For all isolates, there was an increase of SEH concentration with time. Western blot analysis revealed that SEH production, similar to SEA, started in the early exponential phase (OD600 ~ 0.5). Isolates with high SEH productivity, as measured by ELISA, demonstrated a higher seh transcription as well. sec transcription was induced in the stationary phase. An induction in the sea transcript was observed during mid- to late exponential phase. Expression profile of seh was similar to that of sea. We showed that the seh expression profile is similar to that of Agr-independent sea and not to that of Agr-dependent sec genes. SEH can be effectively expressed at low bacterial counts, meaning that even in an environment not favorable for S. aureus growth, seh-bearing strains can pose a risk for food safety.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

The Streptococcus mutans Cid and Lrg systems modulate virulence traits in response to multiple environmental signals

The tight control of autolysis by Streptococcus mutans is critical for proper virulence gene expression and biofilm formation. A pair of dicistronic operons, SMU.575/574 (lrgAB) and SMU.1701/1700 (designated cidAB), encode putative membrane proteins that share structural features with the bacteriophage-encoded holin family of proteins, which modulate host cell lysis during lytic infection. Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance. Expression of lrgAB was repressed during early exponential phase and was induced over 1000-fold as cells entered late exponential phase, whereas cidAB expression declined from early to late exponential phase. A two-component system encoded immediately upstream of lrgAB (LytST) was required for activation of lrgAB expression, but not for cid expression. In addition to availability of oxygen, glucose levels were revealed to affect lrg and cid transcription differentially and significantly, probably through CcpA (carbon catabolite protein A). Collectively, these findings demonstrate that the Cid/Lrg system can affect several virulence traits of S. mutans, and its expression is controlled by two major environmental signals, oxygen and glucose. Moreover, cid/lrg expression is tightly regulated by LytST and CcpA.

An Unexpected Effect of Tetracycline Concentration: Growth Phase-Associated Excision of the Bacteroides Mobilizable Transposon NBU1

ABSTRACT Early studies of the Bacteroides mobilizable transposon NBU1 established that excision, the first step in NBU1 transfer, requires exposure of the cells to tetracycline. More recently, we found that excision is also associated with growth phase; even after exposure to tetracycline, excision is detectable only after the cells enter late exponential phase. The tetracycline effect is mediated by a two-component regulatory system, RteA and RteB, which is provided in trans by an integrated self-transmissible element, CTnDOT. The rteA and rteB genes are part of a three-gene operon that also contains the tetracycline resistance gene tetQ. We report here that neither transcription nor translation of the tetQ-rteA-rteB operon is affected by growth phase. Moreover, RteA is not required for the growth phase effect, because a mutant form of RteB that does not require phosphorylation by RteA did not make excision independent of growth phase. Two conditions made NBU1 excision independent of growth phase. One was reducing the tetracycline concentration from an inhibitory concentration (1 μg/ml) to a subinhibitory level (0.05 μg/ml). Independence of growth phase also occurred when rteA and rteB were placed under the control of a heterologous maltose-inducible promoter, P susA . Our results suggest that at low concentrations of tetracycline, ribosomes are capable of translating enough RteA and RteB for excision to occur. At higher tetracycline concentrations, however, TetQ is needed to protect enough ribosomes to allow the translation of excision genes, and this protection takes time to develop. Thus, subinhibitory concentrations of tetracycline may increase the probability of gene transfer because, in contrast to inhibitory concentrations, excision can occur at all phases of growth.