Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. - Related Documents




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44001.0000Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. Previous studies had localised the gene (mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein (Mr 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene (tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.19872828187
632410.9996Genetic and biochemical basis of tetracycline resistance. Properties of several, well characterized, tetracycline resistance determinants were compared. The determinants in Tn1721 and Tn10 (both from Gram-negative bacteria) each contain two genes; one encodes a repressor that regulates both its own transcription and that of a membrane protein that confers resistance by promoting efflux of the drug. Determinants from Gram-positive bacteria also encode efflux proteins, but expression of resistance is probably regulated by translational attenuation. The likely tetracycline binding site (a common dipeptide) in each efflux protein was predicted. The presence of the common binding site is consistent with the ability of an efflux protein originating in Bacillus species to be expressed in Escherichia coli.19863542941
44320.9996Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant. Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.19911954576
38630.9996A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. The neo (neomycin-resistance) gene of transposon Tn5 encodes the enzyme neomycin phosphotransferase II (EC 2.7.1.95), which confers resistance to various aminoglycoside antibiotics, including kanamycin and G418. The gene is widely used as a selectable marker in the transformation of organisms as diverse as bacteria, yeast, plants, and animals. We found a mutation that involves a glutamic to aspartic acid conversion at residue 182 in the protein encoded by the chimeric neomycin phosphotransferase II genes of several commonly used transformation vectors. The mutation substantially reduces phosphotransferase activity but does not appear to affect the stability of the neomycin phosphotransferase II mRNA or protein. Plants and bacteria transformed with the mutant gene are less resistant to antibiotics than those transformed with the normal gene. A simple restriction endonuclease digestion distinguishes between the mutant and the normal gene.19902159150
29240.9996Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Tetracycline-resistance determinants encoding active efflux of the drug are widely distributed in gram-negative bacteria and unique with respect to genetic organization and regulation of expression. Each determinant consists of two genes called tetA and tetR, which are oriented with divergent polarity, and between them is a central regulatory region with overlapping promoters and operators. The amino acid sequences of the encoded proteins are 43-78% identical. The resistance protein TetA is a tetracycline/metal-proton antiporter located in the cytoplasmic membrane, while the regulatory protein TetR is a tetracycline inducible repressor. TetR binds via a helix-turn-helix motif to the two tet operators, resulting in repression of both genes. A detailed model of the repressor-operator complex has been proposed on the basis of biochemical and genetic data. The tet genes are differentially regulated so that repressor synthesis can occur before the resistance protein is expressed. This has been demonstrated for the Tn10-encoded tet genes and may be a common property of all tet determinants, as suggested by the similar locations of operators with respect to promoters. Induction is mediated by a tetracycline-metal complex and requires only nanomolar concentrations of the drug. This is the most sensitive effector-inducible system of transcriptional regulation known to date. The crystal structure of the TetR-tetracycline/metal complex shows the Tet repressor in the induced, non-DNA binding conformation. The structural interpretation of many noninducible TetR mutants has offered insight into the conformational changes associated with the switch between inducing and repressing structures of TetR. Tc is buried in the core of TetR, where it is held in place by multiple contacts to the protein.19947826010
38050.9996Expression of a chloramphenicol-resistance determinant carried on hybrid plasmids in gram-positive and gram-negative bacteria. To analyse the control of chloramphenicol (Cm) resistance conferred by the Staphylococcus aureus plasmid pUB112, a detailed restriction map of this plasmid has been constructed, and the position and orientation of the cat gene have been determined. An MboI restriction fragment carrying the entire cat gene of pUB112 was then cloned in another S. aureus plasmid, the kanamycin (Km) resistance vector pUB110. Depending on the orientation of the incorporated cat fragment, the level of Cm resistance varied dramatically in Bacillus subtilis cells. This effect could not be eliminated by deleting parts of the vector DNA, and only the introduction of a transcription termination signal led to orientation-independent Cm resistance. One such construct was further developed to yield a shuttle vector, replicating both in Escherichia coli and B. subtilis. Using this vector the expression of incorporated genes can be determined in both Gram-positive and Gram-negative bacteria. By in vitro transcription experiments using pUB110 DNA linearized with various restriction endonucleases as template, two pUB110 promoters could be localized and their orientations determined: one promoter controls a gene whose function is unknown, the other regulates the transcription of the KmR gene.19846442250
26360.9995Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria. BACKGROUND: In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. RESULTS: The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. CONCLUSION: We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.200515651989
44270.9995Mercuric reductase in environmental gram-positive bacteria sensitive to mercury. According to existing data, mercury resistance operons (mer operons) are in general thought to be rare in bacteria, other than those from mercury-contaminated sites. We have found that a high proportion of strains in environmental isolates of Gram-positive bacteria express mercuric reductase (MerA protein): the majority of these strains are apparently sensitive to mercury. The expression of MerA was also inducible in all cases. These results imply the presence of phenotypically cryptic mer resistance operons, with both the merA (mercuric reductase) and merR (regulatory) genes still present, but the possible absence of the transport function required to complete the resistance mechanism. This indicates that mer operons or parts thereof are more widely spread in nature than is suggested by the frequency of mercury-resistant bacteria.19921427009
44180.9995Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities. A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.19853994373
44590.9995Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm. We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol.200212390353
6325100.9995Repressed multidrug resistance genes in Streptomyces lividans. Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment. Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump. In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans. The resistance genes of three of these systems were cloned and sequenced. Two of them are accompanied by a repressor gene. These MDR gene sequences are found in most other Streptomyces species investigated. Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes.200312937892
180110.9994Bacterial resistances to inorganic mercury salts and organomercurials. Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).19921311113
385120.9994Introduction of a mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis. A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.200010620668
260130.9994Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species. Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.201121538255
262140.9994Genome scanning in Haemophilus influenzae for identification of essential genes. We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.199910438768
428150.9994Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.19862438417
387160.9994Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli. Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology. The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline. We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose-limited batch and chemostat cultures. If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid-containing bacteria grow at the same rate.19853005111
268170.9994Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis. We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.19979418256
6323180.9994Reduced Susceptibility to Antiseptics Is Conferred by Heterologous Housekeeping Genes. Antimicrobial resistance is common in the microbial inhabitants of the human oral cavity. Antimicrobials are commonly encountered by oral microbes as they are present in our diet, both naturally and anthropogenically, and also used in oral healthcare products and amalgam fillings. We aimed to determine the presence of genes in the oral microbiome conferring reduced susceptibility to common antimicrobials. From an Escherichia coli library, 12,277 clones were screened and ten clones with reduced susceptibility to triclosan were identified. The genes responsible for this phenotype were identified as fabI, originating from a variety of different bacteria. The gene fabI encodes an enoyl-acyl carrier protein reductase (ENR), which is essential for fatty acid synthesis in bacteria. Triclosan binds to ENR, preventing fatty acid synthesis. By introducing the inserts containing fabI, ENR is likely overexpressed in E. coli, reducing the inhibitory effect of triclosan. Another clone was found to have reduced susceptibility to cetyltrimethylammonium bromide and cetylpyridinium chloride. This phenotype was conferred by a UDP-glucose 4-epimerase gene, galE, homologous to one from Veillonella parvula. The product of galE is involved in lipopolysaccharide production. Analysis of the E. coli host cell surface showed that the charge was more positive in the presence of galE, which likely reduces the binding of these positively charged antiseptics to the bacteria. This is the first time galE has been shown to confer resistance against quaternary ammonium compounds and represents a novel, epimerase-based, global cell adaptation, which confers resistance to cationic antimicrobials.201828604259
388190.9994Improved bacterial hosts for regulated expression of genes from lambda pL plasmid vectors. The construction and use of a set of Escherichia coli strains with defective lambda prophages that facilitate expression of genes cloned in lambda pL-plasmid vectors is described. These bacteria allow high and regulated expression of such genes, whereas a kanamycin-resistance marker (KmR) on the prophage allows easy identification and genetic transfer from strain to strain. Optimal conditions for examining gene expression with the pL-vector systems using these strains are discussed.19938406046