Bacterial Survival Strategies Research Articles

Rational design of a Nano-Antibiotic chitosan hydrogel for the bacterial infection Therapy: In vitro & ex vivo Assessments

In modern times, many antibiotics have become less effective as microorganisms develop resistance. Besides antibiotic resistance, another bacterial strategy that contributes to the capacity to withstand antimicrobials is biofilm formation. Because of these bacterial survival strategies, the desired response cannot be achieved with conventional treatment. Considering the limited discovery of new compounds, the most logical approach is to reconstruct existing antimicrobial molecules with nano-drug delivery systems. With this scientific approach, the aim of the study is to develop a novel nano-antibiotic hydrogel formulation containing silver nanoparticles, chitosan, and amoxicillin. Endodontic disease was used as a model of biofilm-mediated infection, and the antibacterial activity of nano-antibiotic hydrogel was evaluated with the E. faecalis standard bacterial strain. By adopting the Box-Behnken design for the optimisation of formulation variables, an innovative pharmaceutical formulation with antimicrobial and antibiofilm activity was successfully obtained. Further characterisation studies, including nanoparticle characterisation, in vitro cytotoxicity, and ex vivo activity studies, were carried out on dental samples using the optimised formulation. All results were compared with antimicrobial agents routinely used in endodontic treatment. The findings mainly conclude that the optimised nano-antibiotic hydrogel may be an alternative antimicrobial formulation since it is non-cytotoxic and exhibits high antibiofilm activity.

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Escherichia coli cells evade inducible parE toxin expression by reducing plasmid copy number.

This research has increased our understanding of how bacteria respond to the pressure from plasmid-borne toxic genes, such as those found in toxin-antitoxin systems. Surprisingly, we found that bacteria survived toxic ParE protein expression by reducing the number of these plasmids in the cells. This discovery reveals another way in which bacteria can balance toxin expression with antibiotic selection to attenuate the effects of deleterious genes. This insight is not only valuable for understanding bacterial survival strategies but may also influence the development of better tools in biotechnology, where plasmids are often used to study the functional roles of genes.

Combating Multidrug Resistance: The Potential of Antimicrobial Peptides and Biofilm Challenges

The escalating crisis of antibiotic resistance represents a formidable challenge to global public health, necessitating urgent and innovative solutions. This review delves into the multifaceted nature of antibiotic resistance, emphasizing the pivotal role of biofilms and the genetic mechanisms underpinning resistance in both Gram-positive and Gram-negative bacteria. A significant focus is placed on Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), and its mechanisms of resistance, including the SCCmec element and the agr quorum sensing system. The review also explores the alarming rise of resistance in Gram-negative pathogens, such as E. coli and K. pneumoniae, highlighting the perilous spread of extended-spectrum β- lactamases (ESBLs) and carbapenemases. Amidst this dire landscape, antimicrobial peptides (AMPs), particularly melittin from bee venom (BV), emerge as promising agents capable of breaching microbial defenses, including those of dormant cells within biofilms, thus offering a potential strategy to circumvent traditional resistance mechanisms. The review underscores the necessity of understanding bacterial survival strategies, such as biofilm formation and genetic adaptation, to develop effective countermeasures against antibiotic-resistant infections.

Use of a combined antibacterial synergy approach and the ANNOgesic tool to identify novel targets within the gene networks of multidrug-resistant Klebsiella pneumoniae.

Since the 1980s, the development of new drug classes for the treatment of multidrug-resistant Klebsiella pneumoniae has become limited, highlighting the urgent need for novel antibiotics. To address this challenge, this study aimed to explore the synergistic interactions between chemical compounds and representative antibiotics, such as carbapenem and colistin. The primary objective of this study was not only to mitigate the adverse impact of multidrug-resistant K. pneumoniae on public health but also to establish a sustainable balance among humans, animals, and the environment. Phenotypical measurements were conducted using the broth microdilution technique to determine the drug sensitivity of bacterial strains. Additionally, a genotypical approach was employed, involving traditional RNA sequencing analysis to identify differentially expressed genes and the computational ANNOgesic tool to detect noncoding RNAs. This study revealed the existence of various pathways and regulatory RNA elements that form a functional network. These pathways, characterized by the expression of specific genes, contribute to the combined treatment effect and bacterial survival strategies. The connections between pathways are facilitated by regulatory RNA elements that respond to environmental changes. These findings suggest an adaptive response of bacteria to harsh environmental conditions.IMPORTANCENoncoding RNAs were identified as key players in post-transcriptional regulation. Moreover, this study predicted the presence of novel small regulatory RNAs that interact with target genes, as well as the involvement of riboswitches and RNA thermometers in conjunction with associated genes. These findings will contribute to the discovery of potential antimicrobial therapeutic candidates. Overall, this study offers valuable insights into the synergistic effects of chemical compounds and antibiotics, highlighting the role of regulatory RNA elements in bacterial response, and survival strategies. The identification of novel noncoding RNAs and their interactions with target genes, riboswitches, and RNA thermometers holds promise for the development of antimicrobial therapies.

Unveiling potential virulence determinants in Vibrio isolates from Anadara tuberculosa through whole genome analyses.

The genus Vibrio includes pathogenic bacteria able to cause disease in humans and aquatic organisms, leading to disease outbreaks and significant economic losses in the fishery industry. Despite much work on Vibrio in several marine organisms, no specific studies have been conducted on Anadara tuberculosa. This is a commercially important bivalve species, known as "piangua hembra," along Colombia's Pacific coast. Therefore, this study aimed to identify and characterize the genomes of Vibrio isolates obtained from A. tuberculosa. Bacterial isolates were obtained from 14 A. tuberculosa specimens collected from two locations along the Colombian Pacific coast, of which 17 strains were identified as Vibrio: V. parahaemolyticus (n = 12), V. alginolyticus (n = 3), V. fluvialis (n = 1), and V. natriegens (n = 1). Whole genome sequence of these isolates was done using Oxford Nanopore Technologies (ONT). The analysis revealed the presence of genes conferring resistance to β-lactams, tetracyclines, chloramphenicol, and macrolides, indicating potential resistance to these antimicrobial agents. Genes associated with virulence were also found, suggesting the potential pathogenicity of these Vibrio isolates, as well as genes for Type III Secretion Systems (T3SS) and Type VI Secretion Systems (T6SS), which play crucial roles in delivering virulence factors and in interbacterial competition. This study represents the first genomic analysis of bacteria within A. tuberculosa, shedding light on Vibrio genetic factors and contributing to a comprehensive understanding of the pathogenic potential of these Vibrio isolates.IMPORTANCEThis study presents the first comprehensive report on the whole genome analysis of Vibrio isolates obtained from Anadara tuberculosa, a bivalve species of great significance for social and economic matters on the Pacific coast of Colombia. Research findings have significant implications for the field, as they provide crucial information on the genetic factors and possible pathogenicity of Vibrio isolates associated with A. tuberculosa. The identification of antimicrobial resistance genes and virulence factors within these isolates emphasizes the potential risks they pose to both human and animal health. Furthermore, the presence of genes associated with Type III and Type VI Secretion Systems suggests their critical role in virulence and interbacterial competition. Understanding the genetic factors that contribute to Vibrio bacterial virulence and survival strategies within their ecological niche is of utmost importance for the effective prevention and management of diseases in aquaculture practices.

Regulation of major bacterial survival strategies by transcripts sequestration in a membraneless organelle

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA invivo and invitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.

An Insight into the Microbiology, Epidemiology, and Host Cell Biology of Legionella Pneumophila: A Review of Literature

Legionnaires’ disease (LD) is a type of severe pneumonia that mainly caused by bacteria of the genus Legionella. LD bacteria reside in the water systems of facilities where lack of water exchange or flow plays a crucial role in enhancing bacterial growth. The under-recognition of the dangers of Legionella along with easing of Coronavirus disease 2019 (COVID-19) lockdown restrictions and global reopening, pose a potential increased risk of developing LD. Various Legionella species can lead to legionellosis infections, including LD and Pontiac fever. Legionellosis cases is generally found in natural or artificial aquatic environments such as cooling towers, hot water tanks, or air conditioning. The bacteria elude the host’s immune responses by various strategies, including releasing effector proteins. Thus, this review provides insight into the microbiology, epidemiology, and host cell biology of L. pneumophila, as well as an emphasis on the bacterial novel survival strategies of L. pneumophila. Also, suggests taking intensive actions towards closed buildings as a potential source of bacterial infection.

Listeria innocua Biofilm Assay Using NanoLuc Luciferase.

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution. Graphic abstract: Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

Bacterial survival strategies in sludge alkaline fermentation for volatile fatty acids production: Study on the physiological properties, temporal evolution and spatial distribution of bacterial community

This study investigated the dynamics of ATP synthase activity, phospholipid fatty acid (PLFA) profile, and temporal evolution and spatial distribution of bacterial community to analyze bacterial survival strategies in sludge alkaline fermentation (SAF) for volatile fatty acids (VFAs) production. The results revealed a significant increase in ATP synthase activity at pH 9 and 10 (p < 0.05), which could contribute to proton entry into cells and benefit bacterial survival. PLFA analysis indicated that the unsaturated fatty acids content increased with the increase of pH. Firmicutes were the dominant microorganisms in the running stage of the pH 10 reactor (35.81–62.34%) and might have been the key microbes that influenced VFAs production. Further analysis of the spatial distribution of microbial community suggested that Firmicutes mainly lived inside flocs during SAF. These findings provide an understanding for bacterial survival strategies in SAF, which could help to develop methods to further improve VFAs yield.

Metal-Pseudomonas veronii 2E Interactions as Strategies for Innovative Process Developments in Environmental Biotechnology.

The increase of industrial discharges is the first cause of the contamination of water bodies. The bacterial survival strategies contribute to the equilibrium restoration of ecosystems being useful tools for the development of innovative environmental biotechnologies. The aim of this work was to study the Cu(II) and Cd(II) biosensing, removal and recovery, mediated by whole cells, exopolymeric substances (EPS) and biosurfactants of the indigenous and non-pathogenic Pseudomonas veronii 2E to be applied in the development of wastewater biotreatments. An electrochemical biosensor was developed using P. veronii 2E biosorption mechanism mediated by the cell surface associated to bound exopolymeric substances. A Carbon Paste Electrode modified with P. veronii 2E (CPEM) was built using mineral oil, pre-washed graphite power and 24 h-dried cells. For Cd(II) quantification the CPEM was immersed in Cd(II) (1–25 μM), detected by Square Wave Voltammetry. A similar procedure was used for 1–50 μM Cu(II). Regarding Cd(II), removal mediated by immobilized EPS was tested in a 50 ml bioreactor with 0.13 mM Cd(II), pH 7.5. A 54% metal retention by EPS was achieved after 7 h of continuous operation, while a 40% was removed by a control resin. In addition, surfactants produced by P. veronii 2E were studied for recovery of Cd(II) adsorbed on diatomite, obtaining a 36% desorption efficiency at pH 6.5. Cu(II) adsorption from a 1 mM solution was tested using P. veronii 2E purified soluble EPS in 50 mL- batch reactors (pH = 5.5, 32°C). An 80% of the initial Cu(II) was retained using 1.04 g immobilized EPS. Focusing on metal recovery, Cu nanoparticles (NPs) biosynthesis by P. veronii 2E was carried out in Cu(II)-PYG Broth at 25°C for 5 days. Extracellular CuNPs were characterized by UV-Vis spectral analysis while both extracellular and intracellular NPs were analyzed by SEM and TEM techniques. Responses of P. veronii 2E and its products as biosurfactants, bound and soluble EPS allowed Cu(II) and Cd(II) removal, recovery and biosensing resulting in a multiple and versatile tool for sustainable wastewater biotreatments.

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System.

A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor (acid, nutrition, and salt) were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.

Deciphering Streptococcal Biofilms.

Streptococci are a diverse group of bacteria, which are mostly commensals but also cause a considerable proportion of life-threatening infections. They colonize many different host niches such as the oral cavity, the respiratory, gastrointestinal, and urogenital tract. While these host compartments impose different environmental conditions, many streptococci form biofilms on mucosal membranes facilitating their prolonged survival. In response to environmental conditions or stimuli, bacteria experience profound physiologic and metabolic changes during biofilm formation. While investigating bacterial cells under planktonic and biofilm conditions, various genes have been identified that are important for the initial step of biofilm formation. Expression patterns of these genes during the transition from planktonic to biofilm growth suggest a highly regulated and complex process. Biofilms as a bacterial survival strategy allow evasion of host immunity and protection against antibiotic therapy. However, the exact mechanisms by which biofilm-associated bacteria cause disease are poorly understood. Therefore, advanced molecular techniques are employed to identify gene(s) or protein(s) as targets for the development of antibiofilm therapeutic approaches. We review our current understanding of biofilm formation in different streptococci and how biofilm production may alter virulence-associated characteristics of these species. In addition, we have summarized the role of surface proteins especially pili proteins in biofilm formation. This review will provide an overview of strategies which may be exploited for developing novel approaches against biofilm-related streptococcal infections.

Dynamics of Acetyl Homeostasis in Bacillus subtilis

Bacillus subtilis biofilms are effective models for studying prokaryotic multicellular systems. These biofilms, multicellular bacterial communities encased in a protective polysaccharide and protein matrix, are associated with several bacterial and chronic infections but can also serve beneficial purposes in agriculture and wastewater treatment. Recent investigation into post‐translational protein modification in bacteria has elucidated important regulatory mechanisms for growth and biofilm formation. Protein acetylation has been observed to regulate bacterial multicellularity, and it is closely linked to cellular metabolism by using acetyl metabolites as donors of acetyl groups. The goal of this study is to examine the role of protein lysine acetylation as a regulatory mechanism for metabolism B. subtilis. We hypothesize that bacteria use acetylation as a buffering mechanism for acetyl metabolites to balance levels and prevent metabolite toxicity by increasing acetylation during times of high acetyl metabolite levels and deacetylating proteins to restore metabolites during times of low levels. Wild type bacteria as well as knockout mutants affecting protein acetylation and the carbon overflow pathway are grown in biofilm‐inducing minimal media, and cell and supernatant samples are collected at various ODs. Acetyl‐CoA, acetyl‐phosphate, and acetate levels over the course of growth are measured using fluorometric and colorimetric metabolite test level kits. Dynamics of global lysine acetylation levels are also measured by an anti‐acetyl lysine Western blot. Work so far has focused primarily on optimizing and troubleshooting metabolite assay kits by modifying growth and sample preparation conditions. Acetate dynamics have been measured for the wild type bacteria, showing a continuous secretion of acetate into the supernatant throughout exponential growth. This result is expected, as rapid growth should coincide with continuous utilization of the carbon overflow pathway. Initial Western blots show increased acetylation in the ΔackAΔacuCΔsrtN triple knockout mutant, which is expected due to a buildup of acetyl phosphate and inhibition of deacetylase activity. Future research will also focus on analyzing mutated genes in Δpta suppressor mutants to uncover genes related to acetyl‐CoA‐mediated cell death. Findings from this study will promote the understanding of the regulatory mechanisms behind biofilm formation and potentially uncover novel information about bacterial survival strategies and programmed cell death.Support or Funding InformationThis project was funded by grants from the National Science Foundation, the Northeastern University Honors Program, and the Northeastern University Office of Undergraduate Research and Fellowships.

The role of bacterial cell envelope structures in acid stress resistance in E. coli.

Acid resistance (AR) is an indispensable mechanism for the survival of neutralophilic bacteria, such as Escherichia coli (E. coli) strains that survive in the gastrointestinal tract. E. coli acid tolerance has been extensively studied during past decades, with most studies focused on gene regulation and mechanisms. However, the role of cell membrane structure in the context of acid stress resistance has not been discussed in depth. Here, we provide a comprehensive review of the roles and mechanisms of the E. coli cell envelope from different membrane components, such as membrane proteins, fatty acids, chaperones, and proton-consuming systems, and particularly focus on the innovative effects revealed by recent studies. We hope that the information guides us to understand the bacterial survival strategies under acid stress and to further explore the AR regulatory mechanisms to prevent or treat E. coli and other related Gram-negative bacteria infection, or to enhance the AR of engineering E. coli.

The application of phage reactivation capacity to sens bacterial viability and activity after photocatalytic treatment

ABSTRACT We purpose in this study to develop a reliable and low-cost method for the detection of Viable but nonculturable (VBNC) bacteria. Indeed, after water disinfection, injured-VBNC bacteria can be underestimated using conventional assessment methods, causing false-negative results and, posing a significant and potential health risk. The VBNC bacterial survival strategy can hide the real microbial quality of treated water. To overcome this bacterial assessment limitation, we were used a specific and lytic phage to monitor the presence of active bacteria; Pseudomonas aeruginosa after photocatalytic treatment. Within 2 h of phage-target bacteria contact, the reduction of phage amplification rate (At ) can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of new infectious phages release despite their lose of cultivability in the usual media. The determination of phage reactivation coefficient (Rt ) after 2 and 8 h of phage-target cell contact time reveals the ability of phages to reactive their infectivity and their amplification in positive correlation with their host cells viability and activity. The increase in phage reactivation coefficient (Rt ) after an extension of the latent period was directly related to the positive interaction between infectious phages and potential active bacteria. The use of this method can improve the water disinfection process and avoid public health-hazardous especially related to the resuscitation of active-nonculturable bacteria mainly for pathogenic bacteria.

Antibiotics: Combatting Tolerance To Stop Resistance.

Antibiotic resistance poses an alarming and ever-increasing threat to modern health care. Although the current antibiotic crisis is widely acknowledged, actions taken so far have proved insufficient to slow down the rampant spread of resistant pathogens. Problematically, routine screening methods and strategies to restrict therapy failure almost exclusively focus on genetic resistance, while evidence for dangers posed by other bacterial survival strategies is mounting. Antibiotic tolerance, occurring either population-wide or in a subpopulation of cells, allows bacteria to transiently overcome antibiotic treatment and is overlooked in clinical practice. In addition to prolonging treatment and causing relapsing infections, recent studies have revealed that tolerance also accelerates the emergence of resistance. These critical findings emphasize the need for strategies to combat tolerance, not only to improve treatment of recurrent infections but also to effectively address the problem of antibiotic resistance at the root.

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status.

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

Roles of Cholesteryl-α-Glucoside Transferase and Cholesteryl Glucosides in Maintenance of Helicobacter pylori Morphology, Cell Wall Integrity, and Resistance to Antibiotics.

Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori, and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori, their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains (Hp26695Δ421 and Hp76Δ421) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics.IMPORTANCEHelicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics.

Bacterial Survival Strategies in an Alkaline Tailing Site and the Physiological Mechanisms of Dominant Phylotypes As Revealed by Metagenomic Analyses.

Microorganisms inhabiting mine tailings require specific metabolic strategies to survive, which may hold potential for pollution clean up. Effective in situ bioremediation will rely on an in-depth understanding of the function of the bacterial communities, especially the abundant and metabolically active phylotypes. In this study, the bacterial communities collected from an alkaline tailing site were profiled by 16S rRNA gene amplicon sequencing as well as shotgun metagenomic analysis. Our results indicated that potentials for carbon and nitrogen fixation as well as metal resistance and transformation were widespread among the bacterial community members, especially in highly enriched phylotypes, such as members of Thiobacillus and Meiothermus. Important functional microbial guilds including carbon and nitrogen fixers may contribute to phytoremediation by providing nutrients for hyperaccumulator plants. In addition, metal-metabolizing bacteria may influence metal speciation and solubility. This discovery provides an understanding for microbial survival strategies in the tailings and lays the foundation for future potential manipulation of the tailing microbiome for in situ bioremediation.