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819300.9880Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation. Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.202134311575
819010.9874Identification of Quorum-Sensing Inhibitors Disrupting Signaling between Rgg and Short Hydrophobic Peptides in Streptococci. Bacteria coordinate a variety of social behaviors, important for both environmental and pathogenic bacteria, through a process of intercellular chemical signaling known as quorum sensing (QS). As microbial resistance to antibiotics grows more common, a critical need has emerged to develop novel anti-infective therapies, such as an ability to attenuate bacterial pathogens by means of QS interference. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development in response to the short hydrophobic peptides SHP2 and SHP3. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that could specifically inhibit Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt complexes of Rgg and FITC (fluorescein isothiocyanate)-conjugated SHP using a fluorescence polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with submicromolar affinities, to specifically abolish transcription of Rgg2/3-controlled genes, and to prevent biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit, cyclosporine A, as well as its nonimmunosuppressive analog, valspodar, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The Rgg-FITC-peptide-based screen provides a platform to identify inhibitors specific for each Rgg type. Discovery of Rgg inhibitors constitutes a step toward the goal of manipulating bacterial behavior for purposes of improving health. IMPORTANCE: The global emergence of antibiotic-resistant bacterial infections necessitates discovery not only of new antimicrobials but also of novel drug targets. Since antibiotics restrict microbial growth, strong selective pressures to develop resistance emerge quickly in bacteria. A new strategy to fight microbial infections has been proposed, namely, development of therapies that decrease pathogenicity of invading organisms while not directly inhibiting their growth, thus decreasing selective pressure to establish resistance. One possible means to this goal is to interfere with chemical communication networks used by bacteria to coordinate group behaviors, which can include the synchronized expression of genes that lead to disease. In this study, we identified chemical compounds that disrupt communication pathways regulated by Rgg proteins in species of Streptococcus. Treatment of cultures of S. pyogenes with the inhibitors diminished the development of biofilms, demonstrating an ability to control bacterial behavior with chemicals that do not inhibit growth.201525968646
823920.9871Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis. Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.201121628502
909430.9871Pathogen-Specific Polymeric Antimicrobials with Significant Membrane Disruption and Enhanced Photodynamic Damage To Inhibit Highly Opportunistic Bacteria. Highly pathogenic Gram-negative bacteria and their drug resistance are a severe public health threat with high mortality. Gram-negative bacteria are hard to kill due to the complex cell envelopes with low permeability and extra defense mechanisms. It is challenging to treat them with current strategies, mainly including antibiotics, peptides, polymers, and some hybrid materials, which still face the issue of drug resistance, limited antibacterial selectivity, and severe side effects. Together with precise bacteria targeting, synergistic therapeutic modalities, including physical membrane damage and photodynamic eradication, are promising to combat Gram-negative bacteria. Herein, pathogen-specific polymeric antimicrobials were formulated from amphiphilic block copolymers, poly(butyl methacrylate)- b-poly(2-(dimethylamino) ethyl methacrylate- co-eosin)- b-ubiquicidin, PBMA- b-P(DMAEMA- co-EoS)-UBI, in which pathogen-targeting peptide ubiquicidin (UBI) was tethered in the hydrophilic chain terminal, and Eosin-Y was copolymerized in the hydrophilic block. The micelles could selectively adhere to bacteria instead of mammalian cells, inserting into the bacteria membrane to induce physical membrane damage and out-diffusion of intracellular milieu. Furthermore, significant in situ generation of reactive oxygen species was observed upon light irradiation, achieving further photodynamic eradication. Broad-spectrum bacterial inhibition was demonstrated for the polymeric antimicrobials, especially highly opportunistic Gram-negative bacteria, such as Pseudomona aeruginosa ( P. aeruginosa) based on the synergy of physical destruction and photodynamic therapy, without detectable resistance. In vivo P. aeruginosa-infected knife injury model and burn model both proved good potency of bacteria eradication and promoted wound healing, which was comparable with commercial antibiotics, yet no risk of drug resistance. It is promising to hurdle the infection and resistance suffered from highly opportunistic bacteria.201930632740
902740.9871Scorpion Venom Antimicrobial Peptides Induce Siderophore Biosynthesis and Oxidative Stress Responses in Escherichia coli. The increasing development of microbial resistance to classical antimicrobial agents has led to the search for novel antimicrobials. Antimicrobial peptides (AMPs) derived from scorpion and snake venoms offer an attractive source for the development of novel therapeutics. Smp24 (24 amino acids [aa]) and Smp43 (43 aa) are broad-spectrum AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio mauruspalmatus and subsequently characterized. Using a DNA microarray approach, we examined the transcriptomic responses of Escherichia coli to subinhibitory concentrations of Smp24 and Smp43 peptides following 5 h of incubation. Seventy-two genes were downregulated by Smp24, and 79 genes were downregulated by Smp43. Of these genes, 14 genes were downregulated in common and were associated with bacterial respiration. Fifty-two genes were specifically upregulated by Smp24. These genes were predominantly related to cation transport, particularly iron transport. Three diverse genes were independently upregulated by Smp43. Strains with knockouts of differentially regulated genes were screened to assess the effect on susceptibility to Smp peptides. Ten mutants in the knockout library had increased levels of resistance to Smp24. These genes were predominantly associated with cation transport and binding. Two mutants increased resistance to Smp43. There was no cross-resistance in mutants resistant to Smp24 or Smp43. Five mutants showed increased susceptibility to Smp24, and seven mutants showed increased susceptibility to Smp43. Of these mutants, formate dehydrogenase knockout (fdnG) resulted in increased susceptibility to both peptides. While the electrostatic association between pore-forming AMPs and bacterial membranes followed by integration of the peptide into the membrane is the initial starting point, it is clear that there are numerous subsequent additional intracellular mechanisms that contribute to their overall antimicrobial effect.IMPORTANCE The development of life-threatening resistance of pathogenic bacteria to the antibiotics typically in use in hospitals and the community today has led to an urgent need to discover novel antimicrobial agents with different mechanisms of action. As an ancient host defense mechanism of the innate immune system, antimicrobial peptides (AMPs) are attractive candidates to fill that role. Scorpion venoms have proven to be a rich source of AMPs. Smp24 and Smp43 are new AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, and these peptides can kill a wide range of bacterial pathogens. By better understanding how these AMPs affect bacterial cells, we can modify their structure to make better drugs in the future.202133980680
75050.9871Mutations in Genes with a Role in Cell Envelope Biosynthesis Render Gram-Negative Bacteria Highly Susceptible to the Anti-Infective Small Molecule D66. Anti-infectives include molecules that target microbes in the context of infection but lack antimicrobial activity under conventional growth conditions. We previously described D66, a small molecule that kills the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) within cultured macrophages and murine tissues, with low host toxicity. While D66 fails to inhibit bacterial growth in standard media, the compound is bacteriostatic and disrupts the cell membrane voltage gradient without lysis under growth conditions that permeabilize the outer membrane or reduce efflux pump activity. To gain insights into specific bacterial targets of D66, we pursued two genetic approaches. Selection for resistance to D66 revealed spontaneous point mutations that mapped within the gmhB gene, which encodes a protein involved in the biosynthesis of the lipopolysaccharide core molecule. E. coli and S. Typhimurium gmhB mutants exhibited increased resistance to antibiotics, indicating a more robust barrier to entry. Conversely, S. Typhimurium transposon insertions in genes involved in outer membrane permeability or efflux pump activity reduced fitness in the presence of D66. Together, these observations underscore the significance of the bacterial cell envelope in safeguarding Gram-negative bacteria from small molecules.202540732029
74860.9870Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways. Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.201526305955
908570.9869Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink. While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.201626802120
829780.9868Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase. Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE: Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.201727821607
61390.98674-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen. Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Although studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here, we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS-dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise sensitive organism in vitro and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.202133955352
8596100.9867Stringent response-mediated ferroptosis-like death resistance underlies Novosphingobium persistence during ciprofloxacin stress. Antibiotics, as emerging hazardous materials in the environment, pose significant risks to ecosystems and contribute to the spread of antibiotic-resistant bacteria. Although extensive knowledge has been accumulated on antibiotic-resistance mechanisms in individual bacteria, less is understood about how the bacterial communities respond to antibiotic exposure under natural environmental conditions, where nutrient supplies are often limited and fluctuating. Here, we report that Novosphingobium dominated in a wetland bacterial community under 1 µg/mL ciprofloxacin (CIP) exposure and persisted during DL-serine hydroxamate-induced starvation, where the stringent response alarmer (p)ppGpp was detected. Metagenome sequencing revealed that genes associated with siderophore transport, cytochrome c, and glutathione S-transferase were significantly enriched in Novosphingobium, linking its dominance under CIP stress to iron homeostasis and oxidative stress responses. Further study on the survival mechanism of Novosphingobium pentaromativorans US6-1 under 8 µg/mL CIP stress demonstrated that stringent response regulated the growth rate and maintained cell viability by suppressing the TCA cycle and oxidative phosphorylation, deterring the entry of CIP and siderophore into cells, reducing intracellular ferrous iron and malondialdehyde, and balancing cellular redox status, thereby protecting cells from ferroptosis-like death. This study is the first to report Novosphingobium's dominance and persistence in a bacterial community during CIP stress in natural environmental conditions and to propose the stringent response-mediated ferroptosis-like death resistance as one of its key survival mechanisms.IMPORTANCEAntibiotics in the environment are increasingly recognized as a new class of pollutants that accelerate the evolutionary selection of antibiotic-resistant bacteria. However, little is known about how this selection occurs under natural conditions, including how specific bacteria taxa and mechanisms respond to particular antibiotics. This study reveals for the first time the selection effect of CIP on Novosphingobium under nutrient-limited conditions, during which stringent response and iron homeostasis play important roles. An innovative linkage between stringent response and ferroptosis-like death resistance is proposed in N. pentaromativorans US6-1, which serves as the CIP resistance mechanism for Novosphingobium. These findings may help inform strategies to combat antimicrobial resistance in the natural environment.202540952106
8192110.9867Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation. Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.202134017857
8675120.9867Adaptive Strategies in a Poly-Extreme Environment: Differentiation of Vegetative Cells in Serratia ureilytica and Resistance to Extreme Conditions. Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.201930804904
8293130.9866Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms. Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate. IMPORTANCE: Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain significant quantities of eDNA in their biofilms. In some bacteria, eDNA is derived from dead cells, but that does not appear to be the case for all eDNA-containing organisms, including NTM. In this study, we found that eDNA export in NTM is conditionally dependent on the molecules to which the bacteria are exposed and that bicarbonate positively influences eDNA export. We also identified genes and proteins important for eDNA export, which begins to piece together a description of a mechanism for eDNA. Better understanding of eDNA export can give new targets for the development of antivirulence drugs, which are attractive because resistance to classical antibiotics is currently a significant problem.201627923918
508140.9866Insights into the chaotropic tolerance of the desert cyanobacterium Chroococcidiopsis sp. 029 (Chroococcidiopsales, Cyanobacteria). The mechanism of perchlorate resistance of the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated by assessing whether the pathways associated with its desiccation tolerance might play a role against the destabilizing effects of this chaotropic agent. During 3 weeks of growth in the presence of 2.4 mM perchlorate, an upregulation of trehalose and sucrose biosynthetic pathways was detected. This suggested that in response to the water stress triggered by perchlorate salts, these two compatible solutes play a role in the stabilization of macromolecules and membranes as they do in response to dehydration. During the perchlorate exposure, the production of oxidizing species was observed by using an oxidant-sensing fluorochrome and determining the expression of the antioxidant defense genes, namely superoxide dismutases and catalases, while the presence of oxidative DNA damage was highlighted by the over-expression of genes of the base excision repair. The involvement of desiccation-tolerance mechanisms in the perchlorate resistance of this desert cyanobacterium is interesting since, so far, chaotropic-tolerant bacteria have been identified among halophiles. Hence, it is anticipated that desert microorganisms might possess an unrevealed capability of adapting to perchlorate concentrations exceeding those naturally occurring in dry environments. Furthermore, in the endeavor of supporting future human outposts on Mars, the identified mechanisms might contribute to enhance the perchlorate resistance of microorganisms relevant for biologically driven utilization of the perchlorate-rich soil of the red planet.202438156502
8325150.9866The Effects of Airflow on the Mechanosensitive Channels of Escherichia coli MG1655 and the Impact of Survival Mechanisms Triggered. Understanding how bacteria respond to ventilated environments is a crucial concept, especially when considering accurate airflow modeling and detection limits. To properly design facilities for aseptic conditions, we must minimize the parameters for pathogenic bacteria to thrive. Identifying how pathogenic bacteria continue to survive, particularly due to their multi-drug resistance characteristics, is necessary for designing sterile environments and minimizing pathogen exposure. A conserved characteristic among bacterial organisms is their ability to maintain intracellular homeostasis for survival and growth in hostile environments. Mechanosensitive (MS) channels are one of the characteristics that guide this phenomenon. Interestingly, during extreme stress, bacteria will forgo favorable homeostasis to execute fast-acting survival strategies. Physiological sensors, such as MS channels, that trigger this survival mechanism are not clearly understood, leaving a gap in how bacteria translate physical stress to an intracellular response. In this paper, we study the role of mechanosensitive ion channels that are potentially triggered by aerosolization. We hypothesize that change in antimicrobial uptake is affected by aerosolization stress. Bacteria regulate their defense mechanisms against antimicrobials, which leads to varying susceptibility. Based on this information we hypothesize that aerosolization stress affects the antimicrobial resistance defense mechanisms of Escherichia coli (E. coli). We analyzed the culturability of knockout E. coli strains with different numbers of mechanosensitive channels and compared antibiotic susceptibility under stressed and unstressed airflow conditions. As a result of this study, we can identify how the defensive mechanisms of resistant bacteria are triggered for their survival in built environments. By changing ventilation airflow velocity and observing the change in antibiotic responses, we show how pathogenic bacteria respond to ventilated environments via mechanosensitive ion channels.202337764080
8261160.9865Studies on Bd0934 and Bd3507, Two Secreted Nucleases from Bdellovibrio bacteriovorus, Reveal Sequential Release of Nucleases during the Predatory Cycle. Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a "living antibiotic." During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorusIMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.202032601070
729170.9865Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa. Extracellular DNA (eDNA) is in the environment, bodily fluids, in the matrix of biofilms, and accumulates at infection sites. eDNA can function as a nutrient source, a universal biofilm matrix component, and an innate immune effector in eDNA traps. In biofilms, eDNA is required for attachment, aggregation, and stabilization of microcolonies. We have recently shown that eDNA can sequester divalent metal cations, which has interesting implications on antibiotic resistance. eDNA binds metal cations and thus activates the Mg(2+)-responsive PhoPQ and PmrAB two-component systems. In Pseudomonas aeruginosa and many other Gram-negative bacteria, the PhoPQ/PmrAB systems control various genes required for virulence and resisting killing by antimicrobial peptides (APs), including the pmr genes (PA3552-PA3559) that are responsible for the addition of aminoarabinose to lipid A. The PA4773-PA4775 genes are a second DNA-induced cluster and are required for the production of spermidine on the outer surface, which protects the outer membrane from AP treatment. Both modifications mask the negative surface charges and limit membrane damage by APs. DNA-enriched biofilms or planktonic cultures have increased antibiotic resistance phenotypes to APs and aminoglycosides. These dual antibiotic resistance and immune evasion strategies may be expressed in DNA-rich environments and contribute to long-term survival.201323419933
8772180.9865The role of drought response genes and plant growth promoting bacteria on plant growth promotion under sustainable agriculture: A review. Drought is a major stressor that poses significant challenges for agricultural practices. It becomes difficult to meet the global demand for food crops and fodder. Plant physiology, physico-chemistry and morphology changes in plants like decreased photosynthesis and transpiration rate, overproduction of reactive oxygen species, repressed shoot and root shoot growth and modified stress signalling pathways by drought, lead to detrimental impacts on plant development and output. Coping with drought stress requires a variety of adaptations and mitigation techniques. Crop yields could be effectively increased by employing plant growth-promoting rhizobacteria (PGPR), which operate through many mechanisms. These vital microbes colonise the rhizosphere of crops and promote drought resistance by producing exopolysaccharides (EPS), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and phytohormones including volatile compounds. The upregulation or downregulation of stress-responsive genes causes changes in root architecture due to acquiring drought resistance. Further, PGPR induces osmolyte and antioxidant accumulation. Another key feature of microbial communities associated with crops includes induced systemic tolerance and the production of free radical-scavenging enzymes. This review is focused on detailing the role of PGPR in assisting plants to adapt to drought stress.202439002396
9148190.9864Biofilms as Battlefield Armor for Bacteria against Antibiotics: Challenges and Combating Strategies. Bacterial biofilms are formed by communities, which are encased in a matrix of extracellular polymeric substances (EPS). Notably, bacteria in biofilms display a set of 'emergent properties' that vary considerably from free-living bacterial cells. Biofilms help bacteria to survive under multiple stressful conditions such as providing immunity against antibiotics. Apart from the provision of multi-layered defense for enabling poor antibiotic absorption and adaptive persistor cells, biofilms utilize their extracellular components, e.g., extracellular DNA (eDNA), chemical-like catalase, various genes and their regulators to combat antibiotics. The response of biofilms depends on the type of antibiotic that comes into contact with biofilms. For example, excessive production of eDNA exerts resistance against cell wall and DNA targeting antibiotics and the release of antagonist chemicals neutralizes cell membrane inhibitors, whereas the induction of protein and folic acid antibiotics inside cells is lowered by mutating genes and their regulators. Here, we review the current state of knowledge of biofilm-based resistance to various antibiotic classes in bacteria and genes responsible for biofilm development, and the key role of quorum sensing in developing biofilms and antibiotic resistance is also discussed. In this review, we also highlight new and modified techniques such as CRISPR/Cas, nanotechnology and bacteriophage therapy. These technologies might be useful to eliminate pathogens residing in biofilms by combating biofilm-induced antibiotic resistance and making this world free of antibiotic resistance.202337894253