Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.
Ontology highlight
ABSTRACT:
SUBMITTER: Cain BD
PROVIDER: S-EPMC204795 | biostudies-other | 1993 Jun
REPOSITORIES: biostudies-other
Publications
Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.
Cain B D BD Norton P J PJ Eubanks W W Nick H S HS Allen C M CM
Journal of bacteriology 19930601 12
An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification. One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance. The bacA gene mapped to approximately 67 m ...[more]
Similar Datasets
Project description:A small open reading frame from the Escherichia coli chromosome, bcrC(EC), encodes a homologue to the BcrC subunit of the bacitracin permease from Bacillus licheniformis. We show that disruption of the chromosomal bcrC(EC) gene causes bacitracin sensitivity and, conversely, that BcrC(EC) confers bacitracin resistance when expressed from a multicopy plasmid.
| S-EPMC103648 | biostudies-literature
Project description:Although the TEM-1 ?-lactamase (BlaTEM-1) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to ?-lactam/?-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only blaTEM-1 One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher ?-lactamase activity and BlaTEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in blaTEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in blaTEM-1 gene dosage, allowing BlaTEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the blaTEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the blaTEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.IMPORTANCE Understanding how bacteria acquire resistance to antibiotics is essential for treating infected patients effectively, as well as preventing the spread of resistant organisms. In this study, a clinical isolate of E. coli was identified that dedicated more than 15% of its genome toward tandem amplification of a ~10-kb resistance module, allowing it to escape antibiotic-mediated killing. Our research is significant in that it provides one possible explanation for clinical isolates that exhibit discordant behavior when tested for antibiotic resistance by different phenotypic methods. Our research also shows that GRM amplification is difficult to detect by short-read WGS technologies. Analysis of raw long-read sequence data was required to confirm GRM amplification as a mechanism of antibiotic resistance.
| S-EPMC5915731 | biostudies-literature
Project description:Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (?16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.
| S-EPMC3029738 | biostudies-literature
Project description:The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F(2) populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.
| S-EPMC2824275 | biostudies-literature
Project description:When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells-with their large populations and short generation times-frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.
| S-EPMC6116729 | biostudies-literature
Project description:UnlabelledTo survive environmental challenges, biological systems rely on proteins that were selected by evolution to function in particular cellular and conditional settings. With the advent of protein design and synthetic biology, it is now possible to construct novel proteins that are not biased by eons of selection in natural hosts. The availability of these sequences prompts us to ask whether natural biological organisms can use naïve-non-biological-proteins to enhance fitness in stressful environments. To address this question, we transformed a library of DNA sequences encoding ?1.5 × 10(6) binary patterned de novo proteins into E. coli, and selected for sequences that enable growth in concentrations of copper that would otherwise be toxic. Several novel sequences were discovered, and one of them, called Construct K (ConK), was studied in detail. Cells expressing ConK accumulate approximately 50% less copper than control cells. The function of ConK does not involve an oxidase, nor does it require two of the best characterized copper efflux systems. However, the ability of ConK to rescue cells from toxic concentrations of copper does require an active proton motive force. Further selections for growth in higher concentrations of copper led to the laboratory evolution of variants of ConK with enhanced levels of activity in vivo. These studies demonstrate that novel proteins, unbiased by evolutionary history in the natural world, can enhance the fitness of biological systems.SynopsisLiving systems evolve to adapt to potentially lethal environmental changes. This normally involves repurposing existing genetic information (i.e. sequences that were selected by billions of years of evolution). Here we show that a completely de novo protein, not derived from nature, can enable E. coli cells to grow in otherwise toxic concentrations of copper, demonstrating that living systems also have the capacity to incorporate and protopurpose entirely novel genetic information.
| S-EPMC4918413 | biostudies-literature
Project description:After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of trimethoprim resistance worldwide, a better understanding of drug-resistance mechanisms is required. In this study, we screened the single-gene knockout library of Escherichia coli, and identified mgrB and other several genes involved in trimethoprim resistance. Subsequent comparative transcriptional analysis between ΔmgrB and the wild-type strain showed that expression levels of phoP, phoQ, and folA were significantly upregulated in ΔmgrB. Further deleting phoP or phoQ could partially restore trimethoprim sensitivity to ΔmgrB, and co-overexpression of phoP/Q caused TMP resistance, suggesting the involvement of PhoP/Q in trimethoprim resistance. Correspondingly, MgrB and PhoP were shown to be able to modulated folA expression in vivo. After that, efforts were made to test if PhoP could directly modulate the expression of folA. Though phosphorylated PhoP could bind to the promotor region of folA in vitro, the former only provided a weak protection on the latter as shown by the DNA footprinting assay. In addition, deleting the deduced PhoP box in ΔmgrB could only slightly reverse the TMP resistance phenotype, suggesting that it is less likely for PhoP to directly modulate the transcription of folA. Taken together, our data suggested that, in E. coli, MgrB affects susceptibility to trimethoprim by modulating the expression of folA with the involvement of PhoP/Q. This work broadens our understanding of the regulation of folate metabolism and the mechanisms of TMP resistance in bacteria.
| S-EPMC8355897 | biostudies-literature
Project description:A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5? and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5?. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5?. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5?, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5?, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5?.
| S-EPMC4671525 | biostudies-literature
Project description:Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bloodstream and other extraintestinal infections in human and animals. The greatest challenge encountered by ExPEC during an infection is posed by the host defense mechanisms, including lysozyme. ExPEC have developed diverse strategies to overcome this challenge. The aim of this study was to characterize the molecular mechanism of ExPEC resistance to lysozyme. For this, 15,000 transposon mutants of a lysozyme-resistant ExPEC strain NMEC38 were screened; 20 genes were identified as involved in ExPEC resistance to lysozyme-of which five were located in the gene cluster between galF and gnd, and were further confirmed to be involved in O-specific polysaccharide biosynthesis. The O-specific polysaccharide was able to inhibit the hydrolytic activity of lysozyme; it was also required by the complete lipopolysaccharide (LPS)-mediated protection of ExPEC against the bactericidal activity of lysozyme. The O-specific polysaccharide was further shown to be able to directly interact with lysozyme. Furthermore, LPS from ExPEC strains of different O serotypes was also able to inhibit the hydrolytic activity of lysozyme. Because of their cell surface localization and wide distribution in Gram-negative bacteria, O-specific polysaccharides appear to play a long-overlooked role in protecting bacteria against exogenous lysozyme.
| S-EPMC5955474 | biostudies-literature
Project description:Extensive use of colistin in food animals is deemed a major driving force for the emergence and transmission of mcr-1 However, a non-colistin usage factor(s) contributing to mobile colistin resistance may also exist in animal production systems. Given that polymyxin, a bacterium-derived peptide antibiotic, has been successfully used as a surrogate to study bacterial resistance to antimicrobial peptides (AMPs), acquisition of MCR-1 may confer cross-resistance to the unrelated AMPs implicated in practical applications. To test this, we first constructed Escherichia coli recombinant strains differing only in the presence or absence of functional MCR-1. Among diverse tested AMPs, MCR-1 was observed to confer cross-resistance to bacitracin, an in-feed antibiotic widely used in animal industry. The significantly (2-fold) increased bacitracin MIC was confirmed by using different bacitracin products, broth media, and laboratory host strains for susceptibility tests. Subsequently, an original mcr-1 gene-bearing plasmid, pSLy21, was conjugatively transferred to eight clinical E. coli recipient strains isolated from diarrheic pigs, which also led to significantly increased MICs of both colistin (4-fold to 8-fold) and bacitracin (2-fold). Growth curve examination further demonstrated that MCR-1 provides a growth advantage to various E. coli strains in the presence of bacitracin. Given that bacitracin, a feed additive displaying low absorption in the intestine, can be used in food animals with no withdrawal required, imprudent use of bacitracin in food animals may serve as a risk factor to enhance the ecological fitness of MCR-1-positive E. coli strains, consequently facilitating the persistence and transmission of plasmid-mediated colistin resistance in agricultural ecosystem.IMPORTANCE Polymyxins (e.g., colistin) are the drugs of last resort to treat multidrug-resistant infections in humans. To control mobile colistin resistance, there is a worldwide trend to limit colistin use in animal production. However, simply limiting colistin use in animal production may still not effectively mitigate colistin resistance due to an overlooked non-colistin usage factor(s). Using controlled systems, in this study, we observed that MCR-1 confers cross-resistance to bacitracin, a popular in-feed antibiotic used in food animals. Thus, imprudent and extensive usage of bacitracin in food animals may serve as a non-colistin usage risk factor for the transmissible colistin resistance. Further comprehensive in vitro and in vivo studies are highly warranted to generate science-based information for risk assessment and risk management of colistin resistance, consequently facilitating the development of proactive and effective strategies to mitigate colistin resistance in animal production system and protect public health.
| S-EPMC6126145 | biostudies-literature