Intercellular nanotubes mediate bacterial communication. - Related Documents




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933201.0000Intercellular nanotubes mediate bacterial communication. Bacteria are known to communicate primarily via secreted extracellular factors. Here we identify a previously uncharacterized type of bacterial communication mediated by nanotubes that bridge neighboring cells. Using Bacillus subtilis as a model organism, we visualized transfer of cytoplasmic fluorescent molecules between adjacent cells. Additionally, by coculturing strains harboring different antibiotic resistance genes, we demonstrated that molecular exchange enables cells to transiently acquire nonhereditary resistance. Furthermore, nonconjugative plasmids could be transferred from one cell to another, thereby conferring hereditary features to recipient cells. Electron microscopy revealed the existence of variously sized tubular extensions bridging neighboring cells, serving as a route for exchange of intracellular molecules. These nanotubes also formed in an interspecies manner, between B. subtilis and Staphylococcus aureus, and even between B. subtilis and the evolutionary distant bacterium Escherichia coli. We propose that nanotubes represent a major form of bacterial communication in nature, providing a network for exchange of cellular molecules within and between species.201121335240
928310.9996Vibrio cholerae: Measuring Natural Transformation Frequency. Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation.201425367272
933320.9996Exclusion systems preserve host cell homeostasis and fitness, ensuring successful dissemination of conjugative plasmids and associated resistance genes. Plasmid conjugation is a major driver of antibiotic resistance dissemination in bacteria. In addition to genes required for transfer and maintenance, conjugative plasmids encode exclusion systems that prevent host cells from acquiring identical or redundant plasmids. Despite their ubiquity, the biological impact of these systems remains poorly understood. Here, we investigate the importance of the exclusion mechanism for plasmid dynamics and bacterial physiology at the single-cell level. Using real-time microscopy, we directly visualize how the absence of exclusion results in plasmid unregulated self-transfer, causing continuous and repeated plasmid exchange among host cells. This runaway conjugation severely compromises cell integrity, viability, and fitness, a largely undescribed phenomenon termed lethal zygosis. We demonstrate that lethal zygosis is associated with membrane stress, activation of the SOS response, and potential reactivation of SOS-inducible prophages, as well as chromosome replication and segregation defects. This study highlights how exclusion systems maintain host cell homeostasis by limiting plasmid transfer. Paradoxically, this restriction is critical to the successful dissemination of conjugative plasmids by conferring a selective advantage, which explains their evolutionary conservation and underscores their role in the spread of antibiotic resistance among pathogenic bacteria.202540966505
928230.9996Could DNA uptake be a side effect of bacterial adhesion and twitching motility? DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence-a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.201323381940
933140.9996Pneumococcal extracellular vesicles mediate horizontal gene transfer via the transformation machinery. Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria and, in doing so, may promote the spread of drug resistance genes in the population.IMPORTANCEThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here, we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface and that this DNA can be imported by the transformation machinery, facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.202439503503
935750.9996The bifunctional enzymes of antibiotic resistance. The evolutionary union of two genes--each encoding proteins of complementary enzymatic activity--into a single gene so as to allow the coordinated expression of these activities as a fusion polypeptide, is an increasingly recognized biological occurrence. The result of this genetic union is the bifunctional enzyme. This fusion of separate catalytic activities into a single protein, whose gene is regulated by a single promoter, is seen especially where the coordinated expression of the separate activities is highly desirable. Increasingly, a circumstance driving the evolution of the bifunctional enzyme in bacteria is the resistance response of bacteria to antibiotic chemotherapy. We summarize the knowledge on bifunctional antibiotic-resistance enzymes, as possible harbingers of clinically significant resistance mechanisms of the future.200919615931
822360.9996Biogenic ammonia modifies antibiotic resistance at a distance in physically separated bacteria. Bacteria release low-molecular-weight by-products called secondary metabolites, which contribute to bacterial ecology and biology. Whereas volatile compounds constitute a large class of potential infochemicals, their role in bacteria-bacteria interactions remains vastly unexplored. Here we report that exposure to gaseous ammonia released from stationary-phase bacterial cultures modifies the antibiotic resistance spectrum of all tested Gram-negative and Gram-positive bacteria. Using Escherichia coli K12 as a model organism, and increased resistance to tetracycline as the phenotypic read-out, we demonstrate that exposure to ammonia generated by the catabolism of l-aspartate increases the level of intracellular polyamines, in turn leading to modifications in membrane permeability to different antibiotics as well as increased resistance to oxidative stress. We show that the inability to import ammonia via the Amt gas channel or to synthesize polyamines prevent modification in the resistance profile of aerially exposed bacteria. We therefore provide here the first detailed molecular characterization of widespread, long-range chemical interference between physically separated bacteria.201121651627
935570.9996Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer. Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the genetic level and provide the foundation for future mechanistic studies.202438664513
930680.9996Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways. During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts' defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and-importantly-transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.202134946067
29390.9996Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram-negative bacteria. The resistance protein TetA, a membrane-spanning H+-[tc.M]+ antiporter, must be sensitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs' concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly specific tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selectively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mammals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR-based eukaryotic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure--function studies in bacteria and specific requirements of particular applications in eukaryotes have made it a versatile and highly adaptable regulatory system.200312869186
9303100.9995A Conserved Class II Type Thioester Domain-Containing Adhesin Is Required for Efficient Conjugation in Bacillus subtilis. Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.202133727345
9338110.9995Polyamines in bacteria: pleiotropic effects yet specific mechanisms. Extensive data in a wide range of organisms point to the importance of polyamine homeostasis for growth. The two most common polyamines found in bacteria are putrescine and spermidine. The investigation of polyamine function in bacteria has revealed that they are involved in a number of functions other than growth, which include incorporation into the cell wall and biosynthesis of siderophores. They are also important in acid resistance and can act as a free radical ion scavenger. More recently it has been suggested that polyamines play a potential role in signaling cellular differentiation in Proteus mirabilis. Polyamines have also been shown to be essential in biofilm formation in Yersinia pestis. The pleiotropic nature of polyamines has made their investigation difficult, particularly in discerning any specific effect from more global growth effects. Here we describe key developments in the investigation of the function of polyamines in bacteria that have revealed new roles for polyamines distinct from growth. We describe the bacterial genes necessary for biosynthesis and transport, with a focus on Y. pestis. Finally we review a novel role for polyamines in the regulation of biofilm development in Y. pestis and provide evidence that the investigation of polyamines in Y. pestis may provide a model for understanding the mechanism through which polyamines regulate biofilm formation.200717966408
9330120.9995Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery. Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population.202338168155
9248130.9995Towards an integrated model of bacterial conjugation. Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.201525154632
9426140.9995Determination of Effects and Mechanisms of Action of Bacterial Amyloids on Antibiotic Resistance. Bacterial functional amyloids, apart from their many other functions, can influence the resistance of bacteria to antibiotics and other antibacterial agents. Mechanisms of modulation of susceptibility of bacterial cells to antimicrobials can be either indirect or direct. The former mechanisms are exemplified by the contribution of functional amyloids to biofilm formation, which may effectively prevent the penetration of various compounds into bacterial cells. The direct mechanisms include the effects of bacterial proteins revealing amyloid-like structures, like the C-terminal region of the Escherichia coli Hfq protein, on the expression of genes involved in antibiotic resistance. Therefore, in this paper, we describe methods by which effects and mechanisms of action of bacterial amyloids on antibiotic resistance can be studied. Assessment of formation of biofilms, determination of the efficiency of antibiotic resistance in solid and liquid media, and determination of the effects on gene expression at levels of mRNA abundance and stability and protein abundance are described.202235951301
9334150.9995Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Antibiotic resistance, virulence, and other plasmids in bacteria use toxin-antitoxin gene pairs to ensure their persistence during host replication. The toxin-antitoxin system eliminates plasmid-free cells that emerge as a result of segregation or replication defects and contributes to intra- and interspecies plasmid dissemination. Chromosomal homologs of toxin-antitoxin genes are widely distributed in pathogenic and other bacteria and induce reversible cell cycle arrest or programmed cell death in response to starvation or other adverse conditions. The dissection of the interaction of the toxins with intracellular targets and the elucidation of the tertiary structures of toxin-antitoxin complexes have provided exciting insights into toxin-antitoxin behavior.200312970556
9291160.9995Highlights of Streptomyces genetics. Sixty years ago, the actinomycetes, which include members of the genus Streptomyces, with their bacterial cellular dimensions but a mycelial growth habit like fungi, were generally regarded as a possible intermediate group, and virtually nothing was known about their genetics. We now know that they are bacteria, but with many original features. Their genome is linear with a unique mode of replication, not circular like those of nearly all other bacteria. They transfer their chromosome from donor to recipient by a conjugation mechanism, but this is radically different from the E. coli paradigm. They have twice as many genes as a typical rod-shaped bacterium like Escherichia coli or Bacillus subtilis, and the genome typically carries 20 or more gene clusters encoding the biosynthesis of antibiotics and other specialised metabolites, only a small proportion of which are expressed under typical laboratory screening conditions. This means that there is a vast number of potentially valuable compounds to be discovered when these 'sleeping' genes are activated. Streptomyces genetics has revolutionised natural product chemistry by facilitating the analysis of novel biosynthetic steps and has led to the ability to engineer novel biosynthetic pathways and hence 'unnatural natural products', with potential to generate lead compounds for use in the struggle to combat the rise of antimicrobial resistance.201931189905
3795170.9995Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells. Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.200211914355
8895180.9995Loss of DNA mismatch repair genes leads to acquisition of antibiotic resistance independent of secondary mutations. Antibiotic resistant bacteria have been a rising clinical concern for decades. Beyond acquisition of alleles conferring resistance, bacteria under stress (e.g., from changing environmental conditions or mutations) can have higher intrinsic resistance to antibiotics than unstressed cells. This concern is expanded for gram-negative bacteria which have a protective outer membrane serving as an additional barrier against harmful molecules such as antibiotics. Here, we report a pathway which increases antibiotic resistance (i.e., minimum inhibitory concentration) in response to inactivation of the DNA Mismatch Repair pathway (MMR). This pathway led to increased intrinsic resistance and was independent of secondary mutations. Specifically, deletion of the DNA mismatch repair genes mutL or mutS caused resistance to various antibiotics spanning different classes, molecular sizes, and mechanisms of action in several different E. coli K-12 MG1655 strains, and in Salmonella enterica serovar Typhimurium LT2. This pathway was independent of the SOS response (severe DNA damage response). However, the patterns of resistance correlated with previously reported increases in MMR mutants in rates of homoeologous recombination, homologous recombination between non-identical DNA strands. Mutations expected to lower rates of recombination in MMR mutants also decreased the resistance to most antibiotics. Finally, we found lysis occurs in MMR mutants and may contribute to resistance to other antibiotics. Our results have demonstrated a novel mechanism that increases antibiotic resistance in direct response to loss of MMR genes, and we propose this resistance involves increased rates of homoeologous recombination and cell lysis. The increased antibiotic resistance of MMR mutants provides a path for these cells to survive in antibiotics long enough to develop more specific resistance mutations and so may contribute to the development of new clinical resistance alleles.202540667202
9132190.9995Antibiotic resistance: a survival strategy. Antibiotics are natural, semi-synthetic, or synthetic molecules that target the cell wall of bacteria, DNA replication, RNA transcription, or mRNA translation, the cellular machinery responsible for the synthesis of precursor molecules. Bacteria have evolved and adopted numerous strategies to counteract the action of antibiotics. Antibiotic resistance is intrinsic and an inherent characteristic of the microorganism. Intrinsic resistance is due to cell wall impermeability, efflux, biofilm formation, and the expression of genes mediating inactivating enzymes. Antibiotic resistance can also arise by the acquisition of extracellular DNA and is expressed phenotypically as efflux, modification or acquisition of target sites, and enzymatic inactivation of the antibiotic. Not only have bacteria acquired the mechanisms necessary to withstand the effects of antibiotics, they have also acquired elaborate mechanisms to mobilize and disseminate these successful strategies: plasmids, transposons, insertion sequences, and cassettes. Antibiotic resistance is a major worldwide clinical problem of public health concern because of the reduced efficacy caused by the various mechanisms of resistance. Global strategies are emerging to help address this critical problem.200516134477