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619500.9737Differential gene expression analysis shows that cephalosporin resistance is intrinsic to Clostridioides difficile strain 630. Clostridioides difficile infection (CDI) is the most common nosocomial infection in the US. CDI has become a growing concern due to C. difficile's resistance to several antibiotics, including cephalosporins. Furthermore, patients administered cephalosporins are at higher risk of contracting CDI. Cephalosporins are β-lactam antibiotics, which prevent bacterial cell wall synthesis by inhibiting penicillin-binding proteins (PBPs). β-lactam-resistant bacteria evade these antibiotics by producing β-lactamases or by harboring low-affinity PBPs. A genomic analysis of C. difficile strain 630 identified 31 putative β-lactam resistance genes. Upon cefoxitin exposure, few C. difficile strain 630 putative antibiotic-resistant genes were overexpressed. Most notably, the β-lactamase blaCDD gene was upregulated approximately 600-fold, as previously reported. Deletion of the blaCDD locus did not change in cephalosporin susceptibility. Deletion of the second most upregulated gene, the PBP vanY, was also ineffective at decreasing cephalosporin resistance. Cefoxitin exposure of the C. difficile strain 630ΔblaCDD mutant did not increase upregulation of other putative antibiotic resistance genes compared to wildtype C. difficile strain 630. Transcriptomic analyses of wildtype C. difficile strain 630 exposed to cephradine, cefoxitin, ceftazidime, or cefepime revealed the shared upregulation of a putative heterodimeric ABC transporter encoded by loci CD630_04590 (ABC transporter ATP-binding protein) and CD630_04600 (ABC transporter permease). These genes are genomically located directly downstream of blaCDD (CD630_04580). The deletion mutant CD630_04600 remained resistant to a number of antibiotics. Thus, even though blaCDD, CD630_04590, and CD630_04600 are all upregulated when exposed to cephalosporins, they do not seem to be involved in antibiotic resistance in C. difficile strain 630.202539672901
74810.9734Contact-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
62120.9729Activation of ChvG-ChvI regulon by cell wall stress confers resistance to β-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens. A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens. Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a stress response, resembling those of sigma factors in other bacteria. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with β-lactam antibiotics, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria that is independent of the ChvG-ChvI repressor ExoR.202236480495
902730.9726Scorpion 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
999340.9723Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases. Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σ(V) is required for lysozyme resistance, and σ(V) is activated in response to lysozyme. Once activated, σ(V), encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σ(V) regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficileIMPORTANCEClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.202032868404
67450.9721Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes. Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR(2)S(2), and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR(2) were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR(2)S(2) interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.202336892288
11760.9720Acyl depsipeptide (ADEP) resistance in Streptomyces. ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.201121636652
872670.9719CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02. Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.202337802457
973480.9718Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria. Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.202337264114
11690.9716The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor. The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpP(ADEP)) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpP(ADEP) in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpP(ADEP)) as an ADEP resistance gene. ClpP(ADEP) constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.201931399403
8729100.9715Protein S-Acyl Transferase GhPAT27 Was Associated with Verticillium wilt Resistance in Cotton. Protein palmitoylation is an ability of the frame of the cell marker protein is one of the most notable reversible changes after translation. However, studies on protein palmitoylation in cotton have not yet been performed. In our current research, the PAT gene family was systematically identified and bioinformatically analyzed in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, and 211 PAT genes were authenticated and classified into six subfamilies. Sixty-nine PAT genes were identified in upland cotton, mainly at the ends of its the 26 chromosomes of upland cotton. The majority of these genes are located in the nucleus of the plant. Gene structure analysis revealed that each member encodes a protein that which contains at least one DHHC structural domain. Cis-acting element analysis indicated that GhPATs genes are mainly involved in hormone production, light response and stress response. Gene expression pattern analysis indicated that most GhPATs genes were differentially expressed upon induction by pathogenic bacteria, drought, salt, hot and cold stresses, and some GhPATs could be induced by multiple abiotic stresses simultaneously. GhPATs genes showed different expression patterns in tissue-specific assays and were found to be preferentially expressed in roots, followed by expression in stems and leaves. Virus-induced gene silencing (VIGS) experiments showed that cotton was significantly less resistant to Verticillium dahliae when GhPAT27 was silenced. We conclude that the GhPAT27 gene, which mediates S-palmitoylation acetylation, may be involved in the regulation of upland cotton resistance to Verticillium wilt (VW). Overall, this work has provided a fundamental framework for understanding the latent capabilities of GhPATs and a solid foundation for molecular breeding and plant pathogen resistance in cotton.202236297782
661110.9715A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase. The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.201627601569
623120.9714The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin. The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.202031964794
307130.9713Escape from Lethal Bacterial Competition through Coupled Activation of Antibiotic Resistance and a Mobilized Subpopulation. Bacteria have diverse mechanisms for competition that include biosynthesis of extracellular enzymes and antibiotic metabolites, as well as changes in community physiology, such as biofilm formation or motility. Considered collectively, networks of competitive functions for any organism determine success or failure in competition. How bacteria integrate different mechanisms to optimize competitive fitness is not well studied. Here we study a model competitive interaction between two soil bacteria: Bacillus subtilis and Streptomyces sp. Mg1 (S. Mg1). On an agar surface, colonies of B. subtilis suffer cellular lysis and progressive degradation caused by S. Mg1 cultured at a distance. We identify the lytic and degradative activity (LDA) as linearmycins, which are produced by S. Mg1 and are sufficient to cause lysis of B. subtilis. We obtained B. subtilis mutants spontaneously resistant to LDA (LDAR) that have visibly distinctive morphology and spread across the agar surface. Every LDAR mutant identified had a missense mutation in yfiJK, which encodes a previously uncharacterized two-component signaling system. We confirmed that gain-of-function alleles in yfiJK cause a combination of LDAR, changes in colony morphology, and motility. Downstream of yfiJK are the yfiLMN genes, which encode an ATP-binding cassette transporter. We show that yfiLMN genes are necessary for LDA resistance. The developmental phenotypes of LDAR mutants are genetically separable from LDA resistance, suggesting that the two competitive functions are distinct, but regulated by a single two-component system. Our findings suggest that a subpopulation of B. subtilis activate an array of defensive responses to counter lytic stress imposed by competition. Coordinated regulation of development and antibiotic resistance is a streamlined mechanism to promote competitive fitness of bacteria.201526647299
9037140.9712Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BACKGROUND: Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. RESULTS: To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. CONCLUSION: Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.200919761586
9014150.9712Role of acid responsive genes in the susceptibility of Escherichia coli to ciclopirox. Antibiotic resistance poses a huge threat to the effective treatment of bacterial infections. To circumvent the limitations in developing new antibiotics, researchers are attempting to repurpose pre-developed drugs that are known to be safe. Ciclopirox, an off-patent antifungal agent, inhibits the growth of Gram-negative bacteria, and genes involved in galactose metabolism and lipopolysaccharide (LPS) biosynthesis are plausible antibacterial targets for ciclopirox, since their expression levels partially increase susceptibility at restrictive concentrations. In the present study, to identify new target genes involved in the susceptibility of Escherichia coli to ciclopirox, genome-wide mRNA profiling was performed following ciclopirox addition at sublethal concentrations, and glutamate-dependent acid resistance (GDAR) genes were differentially regulated. Additional susceptibility testing, growth analyses and viability assays of GDAR regulatory genes revealed that down-regulation of evgS or hns strongly enhanced susceptibility to ciclopirox. Further microscopy and phenotypic analyses revealed that down-regulation of these genes increased cell size and decreased motility. Our findings could help to maximise the efficacy of ciclopirox against hard-to-treat Gram-negative pathogens.201829654752
9088160.9712Cocrystallizing and Codelivering Complementary Drugs to Multidrugresistant Tuberculosis Bacteria in Perfecting Multidrug Therapy. Bacteria cells exhibit multidrug resistance in one of two ways: by raising the genetic expression of multidrug efflux pumps or by accumulating several drug-resistant components in many genes. Multidrug-resistive tuberculosis bacteria are treated by multidrug therapy, where a few certain antibacterial drugs are administered together to kill a bacterium jointly. A major drawback of conventional multidrug therapy is that the administration never ensures the reaching of different drug molecules to a particular bacterium cell at the same time, which promotes growing drug resistivity step-wise. As a result, it enhances the treatment time. With additional tabletability and plasticity, the formation of a cocrystal of multidrug can ensure administrating the multidrug chemically together to a target bacterium cell. With properly maintaining the basic philosophy of multidrug therapy here, the synergistic effects of drug molecules can ensure killing the bacteria, even before getting the option to raise the drug resistance against them. This can minimize the treatment span, expenditure and drug resistance. A potential threat of epidemic from tuberculosis has appeared after the Covid-19 outbreak. An unwanted loop of finding molecules with the potential to kill tuberculosis, getting their corresponding drug approvals, and abandoning the drug after facing drug resistance can be suppressed here. This perspective aims to develop the universal drug regimen by postulating the principles of drug molecule selection, cocrystallization, and subsequent harmonisation within a short period to address multidrug-resistant bacteria.202337150990
750170.9711Mutations 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
147180.9711Regulation of Multidrug Efflux Pumps by TetR Family Transcriptional Repressor Negatively Affects Secondary Metabolism in Streptomyces coelicolor A3(2). Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.202336790176
9997190.9711RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.201021209831