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1.  Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum. 
The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.
PMCID: PMC184227  PMID: 2543291
2.  Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria 
Microbial Resistance to antibiotics is on the rise, in part because of inappropriate use of antibiotics in human medicine but also because of practices in the agricultural industry. Intensive animal production involves giving livestock animals large quantities of antibiotics to promote growth and prevent infection. These uses promote the selection of antibiotic resistance in bacterial populations. The resistant bacteria from agricultural environments may be transmitted to humans, in whom they cause disease that cannot be treated by conventional antibiotics. The author reviews trends in antibiotic use in animal husbandry and agriculture in general. The development of resistance is described, along with the genetic mechanisms that create resistance and facilitate its spread among bacterial species. Particular aspects of resistance in bacterial species common to both the human population and the agrifood industry are emphasized. Control measures that might reverse the current trends are highlighted.
PMCID: PMC1229782  PMID: 9835883
3.  Antibiotic resistance shaping multi-level population biology of bacteria 
Antibiotics have natural functions, mostly involving cell-to-cell signaling networks. The anthropogenic production of antibiotics, and its release in the microbiosphere results in a disturbance of these networks, antibiotic resistance tending to preserve its integrity. The cost of such adaptation is the emergence and dissemination of antibiotic resistance genes, and of all genetic and cellular vehicles in which these genes are located. Selection of the combinations of the different evolutionary units (genes, integrons, transposons, plasmids, cells, communities and microbiomes, hosts) is highly asymmetrical. Each unit of selection is a self-interested entity, exploiting the higher hierarchical unit for its own benefit, but in doing so the higher hierarchical unit might acquire critical traits for its spread because of the exploitation of the lower hierarchical unit. This interactive trade-off shapes the population biology of antibiotic resistance, a composed-complex array of the independent “population biologies.” Antibiotics modify the abundance and the interactive field of each of these units. Antibiotics increase the number and evolvability of “clinical” antibiotic resistance genes, but probably also many other genes with different primary functions but with a resistance phenotype present in the environmental resistome. Antibiotics influence the abundance, modularity, and spread of integrons, transposons, and plasmids, mostly acting on structures present before the antibiotic era. Antibiotics enrich particular bacterial lineages and clones and contribute to local clonalization processes. Antibiotics amplify particular genetic exchange communities sharing antibiotic resistance genes and platforms within microbiomes. In particular human or animal hosts, the microbiomic composition might facilitate the interactions between evolutionary units involved in antibiotic resistance. The understanding of antibiotic resistance implies expanding our knowledge on multi-level population biology of bacteria.
PMCID: PMC3589745  PMID: 23508522
antibiotics; resistance; population biology; multi-level selection; evolution; evolvability; resistome; microbiome
4.  Antibiotics From Microbes: Converging To Kill 
Current opinion in microbiology  2009;12(5):520-527.
As genetically encoded small molecules, antibiotics are phenotypes that have resulted from mutation and natural selection. Advances in genetics, biochemistry, and bioinformatics have connected hundreds of antibiotics to the gene clusters that encode them, allowing these molecules to be analyzed using the tools of evolutionary biology. This review surveys examples of convergent evolution from microbially produced antibiotics, including the convergence of distinct gene clusters on similar phenotypes and the merger of distinct gene clusters into a single functional unit. Examining antibiotics through an evolutionary lens highlights the versatility of biosynthetic pathways, reveals lessons for combating antibiotic resistance, and provides an entry point for studying the natural roles of these natural products.
PMCID: PMC3176294  PMID: 19695947
5.  Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment 
The ISME Journal  2011;5(8):1253-1261.
The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 1019 bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.
PMCID: PMC3146270  PMID: 21368907
integron; pollution; sewage; agriculture; horizontal gene transfer; antibiotic resistance
6.  Regulation of the Streptomyces coelicolor Calcium-Dependent Antibiotic by absA, Encoding a Cluster-Linked Two-Component System 
Journal of Bacteriology  2002;184(3):794-805.
The Streptomyces coelicolor absA two-component system was initially identified through analysis of mutations in the sensor kinase absA1 that caused inhibition of all four antibiotics synthesized by this strain. Previous genetic analysis had suggested that the phosphorylated form of AbsA2 acted as a negative regulator of antibiotic biosynthesis in S. coelicolor (T. B. Anderson, P. Brian, and W. C. Champness, Mol. Microbiol. 39:553–566, 2001). Genomic sequence data subsequently provided by the Sanger Centre (Cambridge, United Kingdom) revealed that absA was located within the gene cluster for production of one of the four antibiotics, calcium-dependent antibiotic (CDA). In this paper we have identified numerous transcriptional start sites within the CDA cluster and have shown that the original antibiotic-negative mutants used to identify absA exhibit a stronger negative regulation of promoters upstream of the proposed CDA biosynthetic genes than of promoters in the clusters responsible for production of actinorhodin and undecylprodigiosin. The same antibiotic-negative mutants also showed an increase in transcription from a promoter divergent to that of absA, upstream of a putative ABC transporter, in addition to an increase in transcription of absA itself. Interestingly, the negative regulation of the biosynthetic transcripts did not appear to be mediated by transcriptional regulation of cdaR (a gene encoding a homolog of the pathway-specific regulators of the act and red clusters) or by any other recognizable transcriptional regulator associated with the cluster. The role of absA in regulating the expression of the diverse antibiotic biosynthesis clusters in the genome is discussed in light of its location in the cda cluster.
PMCID: PMC139508  PMID: 11790750
7.  Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements 
PLoS ONE  2011;6(2):e17038.
The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.
PMCID: PMC3040208  PMID: 21359229
8.  Pharmacodynamics, Population Dynamics, and the Evolution of Persistence in Staphylococcus aureus 
PLoS Genetics  2013;9(1):e1003123.
When growing populations of bacteria are confronted with bactericidal antibiotics, the vast majority of cells are killed, but subpopulations of genetically susceptible but phenotypically resistant bacteria survive. In accord with the prevailing view, these “persisters” are non- or slowly dividing cells randomly generated from the dominant population. Antibiotics enrich populations for pre-existing persisters but play no role in their generation. The results of recent studies with Escherichia coli suggest that at least one antibiotic, ciprofloxacin, can contribute to the generation of persisters. To more generally elucidate the role of antibiotics in the generation of and selection for persisters and the nature of persistence in general, we use mathematical models and experiments with Staphylococcus aureus (Newman) and the antibiotics ciprofloxacin, gentamicin, vancomycin, and oxacillin. Our results indicate that the level of persistence varies among these drugs and their concentrations, and there is considerable variation in this level among independent cultures and mixtures of independent cultures. A model that assumes that the rate of production of persisters is low and persisters grow slowly in the presence of antibiotics can account for these observations. As predicted by this model, pre-treatment with sub-MIC concentrations of antibiotics substantially increases the level of persistence to drugs other than those with which the population is pre-treated. Collectively, the results of this jointly theoretical and experimental study along with other observations support the hypothesis that persistence is the product of many different kinds of errors in cell replication that result in transient periods of non-replication and/or slowed metabolism by individual cells in growing populations. This Persistence as Stuff Happens (PaSH) hypothesis can account for the ubiquity of this phenomenon. Like mutation, persistence is inevitable rather than an evolved character. What evolved and have been identified are genes and processes that affect the frequency of persisters.
Author Summary
Because of its importance to therapy, a great deal of effort has been devoted to understanding the mechanisms responsible for and the genetic basis of persistence in inherently susceptible but phenotypically antibiotic-resistant subpopulations of bacteria. Much of this research is based on the premise that persisters are produced at random from the susceptible population and the antibiotics used to detect them play no role in their generation. The results of this jointly theoretical and experimental study are inconsistent with this hypothesis. These results, along with observations reported by other investigators, including the failure to find bacteria that do not produce persisters but an abundance of genes modifying their frequency, support the hypothesis that there are many mechanisms responsible for persistence. Based on these collective theoretical and experimental results, along with evolutionary considerations, we postulate that persistence is analogous to mutation. It is an inevitable product of errors and glitches in cell replication and metabolism rather than an evolved character.
PMCID: PMC3536638  PMID: 23300474
9.  Evolutionary consequences of antibiotic use for the resistome, mobilome and microbial pangenome 
The widespread use and abuse of antibiotic therapy has evolutionary and ecological consequences, some of which are only just beginning to be examined. One well known consequence is the fixation of mutations and lateral gene transfer (LGT) events that confer antibiotic resistance. Sequential selection events, driven by different classes of antibiotics, have resulted in the assembly of diverse resistance determinants and mobile DNAs into novel genetic elements of ever-growing complexity and flexibility. These novel plasmids, integrons, and genomic islands have now become fixed at high frequency in diverse cell lineages by human antibiotic use. Consequently they can be regarded as xenogenetic pollutants, analogous to xenobiotic compounds, but with the critical distinction that they replicate rather than degrade when released to pollute natural environments. Antibiotics themselves must also be regarded as pollutants, since human production overwhelms natural synthesis, and a major proportion of ingested antibiotic is excreted unchanged into waste streams. Such antibiotic pollutants have non-target effects, raising the general rates of mutation, recombination, and LGT in all the microbiome, and simultaneously providing the selective force to fix such changes. This has the consequence of recruiting more genes into the resistome and mobilome, and of increasing the overlap between these two components of microbial genomes. Thus the human use and environmental release of antibiotics is having second order effects on the microbial world, because these small molecules act as drivers of bacterial evolution. Continued pollution with both xenogenetic elements and the selective agents that fix such elements in populations has potentially adverse consequences for human welfare.
PMCID: PMC3560386  PMID: 23386843
metagenomics; evolvability; pollution; pangenome; resistome; parvome; mobilome
10.  The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella 
Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the environment.
PMCID: PMC3920066  PMID: 24575089
Salmonella; bacteriophage; antibiotic; carbadox; prophage; gene transfer; transduction
11.  Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). 
Journal of Bacteriology  1996;178(24):7276-7284.
A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.
PMCID: PMC178644  PMID: 8955413
12.  A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog 
Journal of Bacteriology  1999;181(19):6142-6151.
Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner. S. coelicolor mutants globally deficient in antibiotic production (Abs− phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that the absB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in the rnc open reading frame. Constructed disruptions of rnc in both S. coelicolor 1501 and Streptomyces lividans 1326 caused an Abs− phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rnc mutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.
PMCID: PMC103644  PMID: 10498729
13.  Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics 
Many of the antibiotics used today are made by a group of bacteria called Streptomyces. Streptomycetes evolved about 450 million years ago as branched filamentous organisms adapted to the utilization of plant remains. They reproduce by sending up specialized aerial branches, which form spores. Aerial growth is parasitic on the primary colony, which is digested and reused for aerial growth. The reproductive phase is coordinated with the secretion of antibiotics, which may protect the colony against invading bacteria during aerial growth. A clue to the integration of antibiotic production and aerial growth is provided by bldA mutants, which are defective in both processes. These mutants lack the ability to translate a particularly rare codon, UUA, in the genetic code. The UUA codon (TTA in DNA) is present in several regulatory genes that control sets of antibiotic production genes, and in one, bldH that controls aerial mycelium formation. The regulatory genes for antibiotic production are all involved in self-reinforcing regulatory systems that potentially amplify the regulatory significance of small changes in the efficiency of translation of UUA codons. One of the regulatory targets of bldH is an extracellular protease inhibitor protein that is likely to delay the digestion of the primary biomass until the colony is ready for aerial growth. The use of the UUA codon to orchestrate different aspects of extracellular biology appeared very early in Streptomyces evolution.
PMCID: PMC1609407  PMID: 16627293
Streptomyces coelicolor; bldA; codon usage; protease inhibitor; evolution of Streptomyces; tRNA
14.  Metagenomic Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in a Tannery Wastewater Treatment Plant 
PLoS ONE  2013;8(10):e76079.
Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.
PMCID: PMC3787945  PMID: 24098424
15.  Identification of Three New Genes Involved in Morphogenesis and Antibiotic Production in Streptomyces coelicolor 
Journal of Bacteriology  2003;185(20):6147-6157.
We report the isolation and partial characterization of three new mutants of Streptomyces coelicolor that are defective in morphogenesis and antibiotic production. The genes identified by the mutations were located and cloned by using a combination of Tn5 in vitro mutagenesis, cotransformation, and genetic complementation. Mutant SE69 produces lower amounts of antibiotics than the wild type produces, produces spores only after prolonged incubation on rich media, and identifies a gene whose predicted protein product is similar to the GntR family of transcriptional regulators; also, production of aerial mycelia on both rich and poor media is significantly delayed in this mutant. Mutant SE293 is defective in morphogenesis, overproduces antibiotics on rich media, fails to grow on minimal media, and identifies a gene whose predicted protein product is similar to the TetR family of transcriptional regulators. Preliminary evidence suggests that the SE293 gene product may control a molybdopterin binding protein located immediately adjacent to it. Mutant SJ175 sporulates sooner and more abundantly than the wild type and overproduces antibiotics on rich media, and it identifies a gene whose predicted protein product contains regions of predominantly hydrophobic residues similar to those of integral membrane proteins.
PMCID: PMC225029  PMID: 14526027
16.  Genetic analysis of microcin H47 antibiotic system. 
Journal of Bacteriology  1993;175(17):5420-5427.
The microcin H47 genetic determinants span a DNA region of ca. 10 kb and represent the first description of an enterobacterial antibiotic system located in the chromosome of the producing strain. Transcriptional and translational fusions to lacZ showed a complex transcriptional organization of the microcin H47 system. Complementation tests identified six genes that are necessary for the production of the antibiotic; the products of two of them are involved in the export of microcin to the extracellular medium. The immunity determinant was located in an 0.8-kb DNA fragment. There is a putative "silent region" of ca. 3 kb inside the system that could not be clearly related to any antibiotic function. Protein products were identified and assigned to three production genes and also to a gene from the silent region.
PMCID: PMC206597  PMID: 8366029
17.  Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in Streptomyces coelicolor▿ †  
Journal of Bacteriology  2010;192(19):4973-4982.
Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNA-encoding gene (α-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was α-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, α-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III.
PMCID: PMC2944504  PMID: 20675485
18.  Characterization of Genes Involved in Biosynthesis of a Novel Antibiotic from Burkholderia cepacia BC11 and Their Role in Biological Control of Rhizoctonia solani 
Applied and Environmental Microbiology  1998;64(10):3939-3947.
Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.
PMCID: PMC106582  PMID: 9758823
19.  Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli. 
Journal of Bacteriology  1993;175(10):2888-2894.
A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids. The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E. coli proteins. One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype. Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus. We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants. However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA. These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.
PMCID: PMC204606  PMID: 8491710
20.  Genetic Diversity among Enterococcus faecalis 
PLoS ONE  2007;2(7):e582.
Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.
PMCID: PMC1899230  PMID: 17611618
21.  Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics. 
Journal of Bacteriology  1989;171(3):1435-1444.
A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.
PMCID: PMC209764  PMID: 2522090
22.  Mechanism of Action of the Arylomycin Antibiotics and Effects of Signal Peptidase I Inhibition 
Antimicrobial Agents and Chemotherapy  2012;56(10):5054-5060.
Clinically approved antibiotics inhibit only a small number of conserved pathways that are essential for bacterial viability, and the physiological effects of inhibiting these pathways have been studied in great detail. Likewise, characterizing the effects of candidate antibiotics that function via novel mechanisms of action is critical for their development, which is of increasing importance due to the ever-growing problem of resistance. The arylomycins are a novel class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), which is an essential enzyme that functions as part of the general secretory pathway and is not the target of any clinically deployed antibiotic. Correspondingly, little is known about the effects of SPase inhibition or how bacteria may respond to mitigate the associated secretion stress. Using genetically sensitized Escherichia coli and Staphylococcus aureus as model organisms, we examine the activity of arylomycin as a function of its concentration, bacterial cell density, target expression levels, and bacterial growth phase. The results reveal that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. Interestingly, this has profoundly different effects on E. coli and S. aureus. Finally, we examine the activity of arylomycin in combination with distinct classes of antibiotics and demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.
PMCID: PMC3457390  PMID: 22802255
23.  A Glutamate Mutase Is Involved in the Biosynthesis of the Lipopeptide Antibiotic Friulimicin in Actinoplanes friuliensis 
Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family Enterobacteriaceae. The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.
PMCID: PMC151761  PMID: 12543643
24.  Two-component systems in Streptomyces: key regulators of antibiotic complex pathways 
Streptomyces, the main antibiotic-producing bacteria, responds to changing environmental conditions through a complex sensing mechanism and two-component systems (TCSs) play a crucial role in this extraordinary “sensing” device.
Moreover, TCSs are involved in the biosynthetic control of a wide range of secondary metabolites, among them commercial antibiotics. Increased knowledge about TCSs can be a powerful asset in the manipulation of bacteria through genetic engineering with a view to obtaining higher efficiencies in secondary metabolite production. In this review we summarise the available information about Streptomyces TCSs, focusing specifically on their connections to antibiotic production.
PMCID: PMC3881020  PMID: 24354561
Streptomyces; Two-component systems; Antibiotic production; Secondary metabolism
25.  Genetic analysis of erythromycin production in Streptomyces erythreus. 
Journal of Bacteriology  1985;164(1):425-433.
Streptomyces erythreus produces the 14-membered macrolide antibiotic erythromycin A. The properties of erythromycin A nonproducing mutants and their genetic linkage to chromosomal markers were used to establish the rudiments of genetic organization of antibiotic production. Thirty-three Ery- mutants, produced by mutagenesis of S. erythreus NRRL 2338 and affecting the formation of the macrolactone and deoxysugar intermediates of erythromycin A biosynthesis, were classified into four phenotypically different groups based on their cosynthesis behavior, the type of biosynthetic intermediate accumulated, and their ability to biotransform known biochemical intermediates of erythromycin A. Demonstration of the occurrence of natural genetic recombination during conjugal mating in S. erythreus enabled comparison of the genetic linkage relationships of three different ery mutations with seven other markers on a simple chromosome map. This established a chromosomal location for the ery mutations, which appear to be located in at least two positions within one interval of the map.
PMCID: PMC214262  PMID: 4044528

Results 1-25 (208721)