Wide Range Of Bacterial Species Research Articles

CRISPR Interference-Based Functional Small RNA Genomics.

Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at-or migration along-predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.

Bacillus Species with Dye-remediation Potential – A Mini Review

Dyes are either natural or synthetic pigments used as colour for different items including textile materials, leather, cosmetics, plastic, paper, printing ink, food, human hair or paintings. The persisting colour and toxic compounds contained in most dyes leads to serious environmental pollution which is unsuitable for the survival of many ecologically important organisms. A wide range of bacterial species have been reported to effectively remediate dyes of various types and classes. This paper reviews bacterial species from the genus Bacillus with the potentiality to remediate synthetic dyes based on published literatures. Bacillus species remediate dyes basically through biosorption and enzymatic active. The most commonly used Bacillus species are Bacillus subtilis, B. cereus, B. megaterium, B. fusiformis, B. odysseyi, B. mycoides, B. paramycoides, B. pseudomycoides, B. flexus, B. cohnii, B. licheniformis, B. spizizenii, B. algicola, B. vallismortis, B. vietnamensis, B. stratosphericus, B. halodurans, B. albus, B. aryabhattai and B. velezensis. Other researches have also proven that mixed cultures of Bacillus species and species from other microbial genera display significant efficiency in the remediation of various dyes. These species produce enzymes and compounds that aid in the breakdown of dyes, thus, reducing their impact to the environment. The use of the aforementioned Bacillus species in the remediation of other dye related environmental pollutants is highly recommended.

Identification of genes involved in enhanced membrane vesicle formation in Pseudomonas aeruginosa biofilms: surface sensing facilitates vesiculation.

Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.

Genome-Wide Association Studies (GWAS) Approaches for the Detection of Genetic Variants Associated with Antibiotic Resistance: A Systematic Review.

Antibiotic resistance is a significant threat to public health worldwide. Genome-wide association studies (GWAS) have emerged as a powerful tool to identify genetic variants associated with this antibiotic resistance. By analyzing large datasets of bacterial genomes, GWAS can provide valuable insights into the resistance mechanisms and facilitate the discovery of new drug targets. The present study aimed to undertake a systematic review of different GWAS approaches used for detecting genetic variants associated with antibiotic resistance. We comprehensively searched the PubMed and Scopus databases to identify relevant studies published from 2013 to February 2023. A total of 40 studies met our inclusion criteria. These studies explored a wide range of bacterial species, antibiotics, and study designs. Notably, most of the studies were centered around human pathogens such as Mycobacterium tuberculosis, Escherichia coli, Neisseria gonorrhoeae, and Staphylococcus aureus. The review seeks to explore the several GWAS approaches utilized to investigate the genetic mechanisms associated with antibiotic resistance. Furthermore, it examines the contributions of GWAS approaches in identifying resistance-associated genetic variants through binary and continuous phenotypes. Overall, GWAS holds great potential to enhance our understanding of bacterial resistance and improve strategies to combat infectious diseases.

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa.

Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

Blautia coccoides JCM1395T Achieved Intratumoral Growth with Minimal Inflammation: Evidence for Live Bacterial Therapeutic Potential by an Optimized Sample Preparation and Colony PCR Method.

We demonstrate that Blautia coccoides JCM1395T has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, Blautia coccoides JCM1395T and Bacteroides vulgatus JCM5826T were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that Blautia coccoides JCM1395T efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to Bifidobacterium sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.

Evaluation of efficacy and safety of lascufloxacin for nursing and healthcare associated pneumonia: single-arm, open-label clinical trial: A study protocol.

Lascufloxacin hydrochloride (LSFX) is a quinolone antibiotic that inhibits DNA gyrase and topoisomerase IV of bacteria, it is anticipated to minimize antibiotic resistance in bacteria. It exhibits antibacterial activity against a relatively wide range of bacterial species, including anaerobic bacteria, and its efficacy and safety against community-acquired pneumonia have been shown; however, its efficacy and safety against nursing and healthcare associated pneumonia (NHCAP) have not been verified. Here, a single-arm, open-label, uncontrolled study was conducted in which LSFX was administered to patients with NHCAP at 24 facilities. The research subjects (77 cases) were orally administered 75 mg of LSFX once a day for 7 days. The primary endpoint was the clinical efficacy at the time of test of cure (TOC) (TOC; 5-10 days after the end of LSFX administration), while the secondary endpoints were the efficacy at the time of end of treatment, early clinical efficacy, microbiological efficacy at the time of TOC and end of treatment, and safety evaluation of LSFX. NHCAP is a common pneumonia in clinical settings and a notable pneumonia whose mortality is high compared to community-acquired pneumonia. The present study showed the efficacy and safety of LSFX against NHCAP, which could lead to a larger number of therapeutic options for NHCAP.

Upconversion nanoparticle-based aptasensor for rapid and ultrasensitive detection of Staphylococcus aureus by low-speed centrifugation.

Opportunistic foodborne pathogens such as Staphylococcus aureus (S. aureus) can cause a wide variety of threats to public health. There is an urgent clinical need for a fast, simple, low-cost, and sensitive method. Here, we designed a fluorescence-based aptamer biosensor (aptasensor) for S. aureus detection using core-shell structured upconversion nanoparticles (CS-UCNPs) as a beacon. A S. aureus-specific aptamer was modified on the surface of CS-UCNPs for binding pathogens. The S. aureus bound to CS-UCNPs can then be isolated from the detection system by simple low-speed centrifugation. Thus, an aptasensor was successfully established for the detection of S. aureus. The fluorescence intensity of CS-UCNPs correlated with the concentration of S. aureus within the range of 6.36 × 102 to 6.36 × 108 CFU mL-1, resulting in the detected limit of S. aureus being 60 CFU mL-1. The aptasensor performed well in real food samples (milk) with a detection limit of 146 CFU mL-1 for S. aureus. Furthermore, we applied our aptasensor in chicken muscles for S. aureus detection, and compared it with the plate count gold standard method. There was no significant difference between our aptasensor and the plate count method within the detected limit, while the time for the aptasensor (0.58 h) was shorter than that of the plate count method (3-4 d). Therefore, we succeeded in the design of a simple, sensitive and fast CS-UCNPs aptasensor for S. aureus detection. This aptasensor system would have the potential for the detection of a wide range of bacterial species by switching the corresponding aptamer.

Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Bacterial killing activity and lysozymes: A stable defence mechanism in stallion seminal plasma?

During insemination, bacterial contamination of the ejaculate can lead to reduced sperm quality and transmission of pathogens to the female, thus should be avoided. The semen of a variety of animal taxa possess antimicrobial properties against a wide range of bacterial species through antimicrobial molecules, such as lysozyme, but their variance and the factors influencing it are unknown for most species. In this study, the antibacterial defence (bacterial killing activity (BKA) against Escherichia (E.) coli and Staphylococcus (S.) aureus as well as lysozyme concentration) was studied in seminal fluid from two consecutive ejaculates of 18 stallions. All ejaculates showed BKA against the tested bacteria, which correlated between the two consecutive ejaculates (rS =0.526, p=.025 for E. coli and rS =0.656, p=.003 for S. aureus) and appeared to be stable over the tested period. The lysozyme concentration (LC) showed no significant correlation between the consecutive ejaculates (rS =0.161, p=.681). However, LC had a positive correlation to the ratio of apoptotic spermatozoa within the ejaculates (rS =0.426, p=.019). In contrast to other livestock (e.g., boar, bull), the BKA in stallion semen did not correlate significantly with the age of the animal nor sperm quality characteristics.

Characterization of the resistome in Lactobacillus genomic sequences from the human gut

The gut is a complex environment inhabited by a wide range of bacterial species. Lactobacillus species constitute a significant proportion of this environment and, due to their mobile genetic elements such as plasmids and transposons, are more likely to acquire and transfer antibiotic resistance genes through horizontal gene transfer (HGT). The current study obtained and analysed 321 genome assemblies to determine the prevalence of intrinsic and acquired antibiotic resistance genes (ARGs) among Lactobacillus species colonizing the human gastrointestinal tract. A total of four high-frequency resistance genes were identified, including dfra42 (42%), poxtA (17.4%), lmrB (12%), and BJP-1 (7.7%); aside from dfra42, which is an intrinsic resistance gene, the other genes are acquired resistance genes. PoxtA was found in several different species, mainly in L. paracasei, whereas BJP-1 and lmrB were found in only one species, L. rhamnosus. IS5-like elements family transposase flanked 11% and 8% of detected lmrB and BJP-1, respectively, while a variety of insertion sequences surrounded 22% of identified poxtA. Furthermore, to the best of our knowledge, this is the first report of BJP-1 in lactobacilli that would suggest it has transferred from soil microbiota to humans. According to the 'One Health' perspective, early detection of a new reservoir would control the global spread of the antibiotic-resistant bacterial species among the three environments, which include humans, the environment, and animals. Finally, the study's findings may then highlight the possibility of lactobacilli acquiring or transmitting resistance to other species within or outside the human intestine.

Unbridled Integrons: A Matter of Host Factors.

Integrons are powerful recombination systems found in bacteria, which act as platforms capable of capturing, stockpiling, excising and reordering mobile elements called cassettes. These dynamic genetic machineries confer a very high potential of adaptation to their host and have quickly found themselves at the forefront of antibiotic resistance, allowing for the quick emergence of multi-resistant phenotypes in a wide range of bacterial species. Part of the success of the integron is explained by its ability to integrate various environmental and biological signals in order to allow the host to respond to these optimally. In this review, we highlight the substantial interconnectivity that exists between integrons and their hosts and its importance to face changing environments. We list the factors influencing the expression of the cassettes, the expression of the integrase, and the various recombination reactions catalyzed by the integrase. The combination of all these host factors allows for a very tight regulation of the system at the cost of a limited ability to spread by horizontal gene transfer and function in remotely related hosts. Hence, we underline the important consequences these factors have on the evolution of integrons. Indeed, we propose that sedentary chromosomal integrons that were less connected or connected via more universal factors are those that have been more successful upon mobilization in mobile genetic structures, in contrast to those that were connected to species-specific host factors. Thus, the level of specificity of the involved host factors network may have been decisive for the transition from chromosomal integrons to the mobile integrons, which are now widespread. As such, integrons represent a perfect example of the conflicting relationship between the ability to control a biological system and its potential for transferability.

Chromatic Bacteria v.2-A Himar1 Transposon-Based Delivery Vector to Extend the Host Range of a Toolbox to Fluorescently Tag Bacteria

A recent publication described the construction and utility of a comprehensive “Chromatic Bacteria” toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a series of Himar1 transposon vectors was constructed to mediate insertion of fluorescent protein and antibiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacterial species. To test the suitability of the new Himar1 Chromatic Bacteria plasmid derivatives, conjugations were attempted on recently isolated non-model organisms. Although we were unsuccessful in delivering the plasmids into Gram-positive bacterial isolates, we successfully modified previously recalcitrant isolates to the first set of the Chromatic Bacteria toolbox, such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria.

Tatajuba: exploring the distribution of homopolymer tracts.

Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialized bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and help predict their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source licence GNU GPLv3.

Promotech: a general tool for bacterial promoter recognition

Promoters are genomic regions where the transcription machinery binds to initiate the transcription of specific genes. Computational tools for identifying bacterial promoters have been around for decades. However, most of these tools were designed to recognize promoters in one or few bacterial species. Here, we present Promotech, a machine-learning-based method for promoter recognition in a wide range of bacterial species. We compare Promotech’s performance with the performance of five other promoter prediction methods. Promotech outperforms these other programs in terms of area under the precision-recall curve (AUPRC) or precision at the same level of recall. Promotech is available at https://github.com/BioinformaticsLabAtMUN/PromoTech.

A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria.

Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.

16S rRNA Gene-Based Profiling of the Microbial Community in an Acid Mine Drainage Fe Precipitate at Libiola Mine (Liguria, Italy)

Acid mine drainage (AMD) is a common environmental problem in many sulphide mines worldwide, and it is widely accepted that the microbial community plays a major role in keeping the process of acid generation active. The aim of this work is to describe, for the first time, the microbial community thriving in goethite and jarosite Fe precipitates from the AMD of the Libiola mine. The observed association is dominated by Proteobacteria (>50%), followed by Bacteroidetes (22.75%), Actinobacteria (7.13%), Acidobacteria (5.79%), Firmicutes (2.56%), and Nitrospirae (1.88%). Primary producers seem to be limited to macroalgae, with chemiolithotrophic strains being almost absent. A phylogenetic analysis of bacterial sequences highlighted the presence of heterotrophic bacteria, including genera actively involved in the AMD Fe cycle and genera (such as Cytophaga and Flavobacterium) that are able to reduce cellulose. The Fe precipitates constitute a microaerobic and complex environment in which many ecological niches are present, as proved by the wide range of bacterial species observed. This study is the first attempt to quantitatively characterize the microbial community of the studied area and constitutes a starting point to learn more about the microorganisms thriving in the AMD of the Libiola mine, as well as their potential applications.

Development and validation of a liquid chromatography high-resolution mass spectrometry orbitrap method for the sensitive quantification of amoxicillin, piperacillin, tazobactam and meropenem in human faeces

Beta-lactam antibiotics are of vital importance for the treatment of infections in a broad range of patients. Although most systemically administered antibiotics will be excreted renally, a fraction will reach the gastro-intestinal tract, affecting the intestinal microbiome by eradicating a wide range of bacterial species while facilitating the growth of antimicrobial-resistant species. A better understanding of the kinetics of beta-lactam antibiotics in the gastro-intestinal tract is essential to study their role in the development of antibiotic resistance in bacteria and to help develop future therapies to prevent damage to, or restore, the intestinal microbiome.Analysis of beta-lactam antibiotics in faeces is particularly challenging due to the heterogeneous nature of the matrix, rapid degradation of some beta-lactam antibiotics in faeces and very strong ion suppression when using mass spectrometry. Sample preparation was optimized using a sequential strategy of experimental designs. It resulted in lyophilization, a MOPS buffer system and the addition of the beta-lactamase inhibitor avibactam to minimize degradation of antibiotics allowing sensitive quantification. The developed liquid chromatography method with high-resolution mass spectrometric detection was successfully validated according to bioanalytical EMA guidelines and had a linear range of 1–200 μg g−1 lyophilized faeces for amoxicillin, piperacillin and meropenem; and 0.5–100 μg g−1 lyophilized faeces for tazobactam. Despite the highly complex and heterogeneous composition of faeces, the accuracy (0.1–15%) and precision (1.7–12.1%) were in line with those obtained for quantification methods of beta-lactam antibiotics in plasma, the golden standard matrix for therapeutic drug monitoring. The applicability of the method was illustrated by successful quantification of piperacillin and tazobactam in faeces from an intensive care unit patient receiving piperacillin/tazobactam in a continuous intravenous infusion. Both piperacillin and tazobactam were still present six days after discontinuation of the therapy.

The Bacillus subtilis monothiol bacilliredoxin BrxC (YtxJ) and the Bdr (YpdA) disulfide reductase reduce S-bacillithiolated proteins

The bacterial cytosol is generally a reducing environment with protein cysteine residues maintained in their thiol form. The low molecular weight thiol bacillithiol (BSH) serves as a general thiol reductant, analogous to glutathione, in a wide range of bacterial species. Proteins modified by disulfide bond formation with BSH (S-bacillithiolation) are reduced by the action of bacilliredoxins, BrxA and BrxB. Here, the YtxJ protein is identified as a monothiol bacilliredoxin, renamed BrxC, and is implicated in BSH removal from oxidized cytosolic proteins, including the glyceraldehyde 3-phosphate dehydrogenases GapA and GapB. BrxC can also debacillithiolate the mixed disulfide form of the bacilliredoxin BrxB. Bdr is a thioredoxin reductase-like flavoprotein with bacillithiol-disulfide (BSSB) reductase activity. Here, Bdr is shown to additionally function as a bacilliredoxin reductase. Bdr and BrxB function cooperatively to debacillithiolate OhrR, a transcription factor regulated by S-bacillithiolation on its sole cysteine residue. Collectively, these results expand our understanding of the BSH redox network comprised of three bacilliredoxins and a BSSB reductase that serve to counter the widespread protein S-bacillithiolation that results from conditions of disulfide stress.

Genome-Wide Identification of Resveratrol Intrinsic Resistance Determinants in Staphylococcus aureus.

Resveratrol has been extensively studied due to its potential health benefits in multiple diseases, for example, cancer, obesity and cardiovascular diseases. Besides these properties, resveratrol displays inhibitory activity against a wide range of bacterial species; however, the cellular effects of resveratrol in bacteria remain incompletely understood, especially in the human pathogen, Staphylococcus aureus. In this study, we aimed to identify intrinsic resistance genes that aid S. aureus in tolerating the activity of resveratrol. We screened the Nebraska Transposon Mutant Library, consisting of 1920 mutants with inactivation of non-essential genes in S. aureus JE2, for increased susceptibly to resveratrol. On agar plates containing 0.5× the minimum inhibitory concentration (MIC), 17 transposon mutants failed to grow. Of these, four mutants showed a two-fold reduction in MIC, being the clpP protease mutant and three mutants with deficiencies in the electron transport chain (menD, hemB, aroC). The remaining 13 mutants did not show a reduction in MIC, but were confirmed by spot-assays to have increased susceptibility to resveratrol. Several genes were associated with DNA damage repair (recJ, xerC and xseA). Treatment of S. aureus JE2 with sub-inhibitory concentrations of resveratrol did not affect the expression of recJ, xerC and xseA, but increased expression of the SOS–stress response genes lexA and recA, suggesting that resveratrol interferes with DNA integrity in S. aureus. Expression of error-prone DNA polymerases are part of the SOS–stress response and we could show that sub-inhibitory concentrations of resveratrol increased overall mutation frequency as measured by formation of rifampicin resistant mutants. Our data show that DNA repair systems are important determinants aiding S. aureus to overcome the inhibitory activity of resveratrol. Activation of the SOS response by resveratrol could potentially facilitate the development of resistance towards conventional antibiotics in S. aureus.