Small RNA GcvB Regulates Oxidative Stress Response of Escherichia coli

Small non-translated regulatory RNAs control plenty of bacterial vital activities. The small RNA GcvB has been extensively studied, indicating the multifaceted roles of GcvB beyond amino acid metabolism. However, few reported GcvB-dependent regulation in minimal medium. Here, by applying a high-resolution RNA-seq assay, we compared the transcriptomes of a wild-type Escherichia coli K-12 strain and its gcvB deletion derivative grown in minimal medium and identified putative targets responding to GcvB, including flu, a determinant gene of auto-aggregation. The following molecular studies and the enhanced auto-aggregation ability of the gcvB knockout strain further substantiated the induced expression of these genes. Intriguingly, the reduced expression of OxyR (the oxidative stress regulator) in the gcvB knockout strain was identified to account for the increased expression of flu. Additionally, GcvB was characterized to up-regulate the expression of OxyR at the translational level. Accordingly, compared to the wild type, the GcvB deletion strain was more sensitive to oxidative stress and lost some its ability to eliminate endogenous reactive oxygen species. Taken together, we reveal that GcvB regulates oxidative stress response by up-regulating OxyR expression. Our findings provide an insight into the diversity of GcvB regulation and add an additional layer to the regulation of OxyR.

Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

Background The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. Conclusions In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.

The Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth Environment

Results from previous investigations into spontaneous rifampicin-resistance (Rif R ) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations may depend on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of ‘jackpot’ mutations which may have arisen early in the growth of a population. Here we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of Rif R mutations in two distinct media: LB, a complete laboratory medium and SMM Asn , a minimal medium utilizing L-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined that the mutation rate to Rif R to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rif R mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%) whereas SMM Asn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMM Asn than in LB medium. We therefore conclude that both the spectrum of Rif R mutations in the B. subtilis rpoB gene and the fitness of resulting mutants to be influenced by the growth environment. Importance The rpoB gene encodes the beta subunit of RNA polymerase, and mutations in rpoB are key determinants of resistance to the clinically important antibiotic rifampicin. We show here that the spectrum of mutations in Bacillus subtilis rpoB depends on the medium in which the cells are cultivated. The results show that not only does the growth environment play a role in natural selection and fitness, but it also influences the probability of mutation at particular bases within the target gene.

A Rapid Method for Performing a Multivariate Optimization of Phage Production Using the RCCD Approach

Bacteriophages can be used in various applications, from the classical approach as substitutes for antibiotics (phage therapy) to new biotechnological uses, i.e., as a protein delivery vehicle, a diagnostic tool for specific strains of bacteria (phage typing), or environmental bioremediation. The demand for bacteriophage production increases daily, and studies that improve these production processes are necessary. This study evaluated the production of a T4-like bacteriophage vB_EcoM-UFV09 (an E. coli-infecting phage with high potential for reducing environmental biofilms) in seven types of culture media (Luria–Bertani broth and the M9 minimal medium with six different carbon sources) employing four cultivation variables (temperature, incubation time, agitation, and multiplicity of infection). For this purpose, the rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for starting to scale up the production process. We used the RCCD to set up the experimental design by combining the cultivation parameters in a specific and systematic way. Despite the high number of conditions evaluated, the results showed that when specific conditions were utilized, viral production was effective even when using a minimal medium, such as M9/glucose, which is less expensive and can significantly reduce costs during large-scale phage production.

Biodegradable properties of organophosphorus insecticides by the potential probiotic Lactobacillus plantarum WCP931 with a degrading gene (opdC)

An organophosphorus (OP) insecticide-mineralizing strain, Lactobacillus plantarum WCP931, harboring a new OP hydrolase (opdC) gene, was isolated during kimchi (Korean traditional food) fermentation. Strain WCP931 exhibited a significant survival rate of 51 to 96% under artificial gastric acid conditions at pH 2 to 3 after 3 h. The opdC gene, consisting of 831 bp encoding 276 amino acids, was cloned from strain WCP907. Recombinant Escherischia coli harboring the opdC gene depleted 77% chlorpyrifos (CP) in M9 minimal medium after 6 days of incubation. The OpdC enzyme represents a novel member of the GHSQG family of esterolytic enzymes or a new Opd group. The OpdC molecular mass was estimated to be approximately 31 kDa by SDS-PAGE and showed maximum activity at pH 6 and 35 °C. The mutated OpdC (Ser116 → Ala116) enzyme had no activity towards OP insecticides and ρ-nitrophenol-β-butyrate. Importantly, the relative activity of OpdC protein against chlorpyrifos, coumafos, diazinon, fenamifos, methyl parathion, and parathion was higher than that against cadosafos, dyfonate, and ethoprofos insecticides. These results suggested the involvement of OpdC in the biodegradation of OP insecticide-contaminated cabbage during fermentation. The new OpdC enzyme expands the heterogeneity of the lactic acid bacterial Opd enzyme group in nature.

Identification of a transcription factor, PunR, that regulates the purine and purine nucleoside transporter punC in E. coli

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

Characterization of Komagataeibacter Isolate Reveals New Prospects on Waste Stream Valorization for Bacterial Cellulose Production

Komagataeibacter spp. have been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose producing bacteria capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in minimal medium containing glucose (3.3±0.3 g/L), pure glycerol (2.2±0.1 g/L) and crude glycerol (2.1±0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in minimal medium supplemented with acetate, BC production was not observed. However, in presence of readily utilizable substrate, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Involvement of a FAD-linked oxidase RSc0454 for expression of the type III secretion system and pathogenicity in Ralstonia solanacearum

Ralstonia solanacearum RSc0454 is predicated as a FAD-linked oxidase based on protein homologies, while containing distinct domains of LDH and SDH. Current study demonstrates RSc0454 exhibits LDH activity and is essential for pathogenicity. Here, we characterized involvement of RSc0454 on bacterial growth and expression of the T3SS in R. solanacearum. RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate, but not lactate, substantially restored some phenotypes of RSc0454 mutants, including faint growth in plants, diminished growth in minimal medium, and lost pathogenicity. The T3SS Expression is directly controlled by a master regulator HrpB, and HrpG and PrhG positively regulate hrpB expression in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, but through novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells and supplementary NADH partially restored diminished growth of RSc0454 mutant in minimal medium at presence of succinate at some moderate concentrations, indicating that unbalanced redox in RSc0454 mutant might be responsible for its no growth in minimal medium. All taken together, these results provide novel insights into understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum.

Activity of Pyrazinamide against Mycobacterium tuberculosis at Neutral pH in PZA-S1 Minimal Medium

Susceptibility testing of tuberculosis (TB) drugs on Mycobacterium tuberculosis is essential for the rapid detection of strains resistant to the drugs, providing the patient with effective treatment, and preventing the spread of drug-resistant TB strains. Pyrazinamide (PZA) is one of the first-line agents used for the treatment of TB. However, current phenotypic PZA susceptibility testing is unreliable due to its performance in acidic pH conditions. The aims of this study were to develop minimal media to determine the activity of PZA at a neutral pH at 37 °C to avoid problems caused by an acidic pH, which is currently used in PZA susceptibility tests, and to identify PZA-resistant M. tuberculosis in media with reproducibility and accuracy. Different minimal media were used to determine the activity of PZA using the broth microdilution method with M. tuberculosis H37Ra as the reference strain. The PZA-S1 minimal medium was proposed as the most suitable medium. PZA inhibited the growth of M. tuberculosis in PZA-S1 at a neutral pH of 6.8, which is the optimal pH for M. tuberculosis growth. Moreover, PZA showed activity at a neutral pH on a PZA-S1 agar plate when using the disk diffusion method. PZA-resistant M. tuberculosis could be identified at a neutral pH in PZA-S1 minimal medium. This study establishes valuable information regarding the testing of PZA’s susceptibility in relation to M. tuberculosis at a neutral pH of 6.8 with reliability and accuracy in clinical settings.