# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 439 | 0 | 1.0000 | Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance. Acinetobacter baumannii 19606 harbors pMAC, a 9540-bp plasmid that contains 11 predicted open-reading frames (ORFs). Cloning and transformation experiments using Acinetobacter calcoaceticus BD413 mapped replication functions within a region containing four 21-bp direct repeats (ori) and ORF 1, which codes for a predicted replication protein. Subcloning and tri-parental mating experiments mapped mobilization functions to the product of ORF 11 and an adjacent predicted oriT. Three ORFs code for proteins that share similarity to hypothetical proteins encoded by plasmid genes found in other bacteria, while the predicted products of three others do not match any known sequence. The product of ORF 8 is similar to Ohr, a hydroperoxide reductase responsible for organic peroxide detoxification and resistance in bacteria. This ORF is immediately upstream of a coding region whose product is related to the MarR family of transcriptional regulators. Disk diffusion assays showed that A. baumannii 19606 is resistant to the organic peroxide-generating compounds cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (t-BHP), although to levels lower than those detected in Pseudomonas aeruginosa PAO1. Cloning and introduction of the ohr and marR ORFs into Escherichia coli was associated with an increase in resistance to CHP and t-BHP. This appears to be the first case in which the genetic determinants involved in organic peroxide resistance are located in an extrachromosomal element, a situation that can facilitate the horizontal transfer of genetic elements coding for a function that protects bacterial cells from oxidative damage. | 2006 | 16530832 |
| 443 | 1 | 0.9995 | Deletion 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. | 1991 | 1954576 |
| 442 | 2 | 0.9995 | Mercuric 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. | 1992 | 1427009 |
| 180 | 3 | 0.9995 | Bacterial 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). | 1992 | 1311113 |
| 426 | 4 | 0.9995 | Plasmid-determined resistance to serum bactericidal activity: a major outer membrane protein, the traT gene product, is responsible for plasmid-specified serum resistance in Escherichia coli. Resistance to the bactericidal activity of serum appears to be an important virulence property of invasive bacteria. The conjugative multiple-antibiotic-resistance plasmid R6-5 was found to confer upon Escherichia coli host bacteria increased resistance against rabbit serum. Gene-cloning techniques were used to localize the serum resistance determinant of R6-5 to a segment of the plasmid that encodes conjugal transfer functions, and a pACYC184 hybrid plasmid, designated pKT107, that contains this segment was constructed. The generation and analysis of deletion and insertion mutant derivatives of the pKT107 plasmid that no longer specify serum resistance permitted precise localization of the serum-resistance cistron on the R6-5 map and demonstrated that this locus is coincident with that of traT, one of the two surface exclusion genes of R6-5. Examination of the proteins synthesized in E. coli minicells of pKT107 and its serum-sensitive mutant derivative plasmids confirmed that the serum-resistance gene product of R6-5 is the traT protein and showed that this protein is a major structural component (about 21,000 copies per cell) of the bacterial outer membrane. | 1980 | 6995306 |
| 262 | 5 | 0.9994 | Genome 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. | 1999 | 10438768 |
| 263 | 6 | 0.9994 | Selection 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. | 2005 | 15651989 |
| 6324 | 7 | 0.9994 | Genetic 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. | 1986 | 3542941 |
| 445 | 8 | 0.9994 | Selection 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. | 2002 | 12390353 |
| 188 | 9 | 0.9994 | Resistance to ag(i) cations in bacteria: environments, genes and proteins. Bacterial resistance to Ag(I) has been reported periodically with isolates from many environments where toxic levels of silver might be expected to occur, but initial reports were limited to the occurrence of resistant bacteria. The availability of silver-resistance conferring DNA sequences now allow genetic and mechanistic studies that had basically been missing. The genes determining Ag(I) resistance were sequenced from a plasmid found in a burn ward isolate. The 14.2 kb determinant contains seven recognized genes, arranged in three mRNA transcriptional units. The silE gene determines an extracellular (periplasmic space) metal-binding protein of 123 amino acids, including ten histidine residues implicated in Ag(I) binding. SilE is homologous to PcoE, of copper resistance. The next two genes, silR and silS, determine a two protein, histidine-kinase membrane sensor and aspartyl phosphate transcriptional responder, similar to other two component systems such as CzcR and CzcS (for cadmium, zinc and cobalt resistance) and PcoR and PcoS (for copper resistance). The remaining four genes, silCBAP, are co-transcribed and appear to determine Ag(+) efflux, with SilCBA homologous to CzcCBA, a three component cation/proton antiporter, and SilP a novel P-type ATPase with a amino-terminal histidine-rich cation-specificity region. The effects of increasing Ag(+) concentrations and growth medium halides (Cl-, Br- and I-) have been characterized, with lower Cl- concentrations facilitating resistance and higher concentrations toxicity. The properties of this unique Ag(I)-binding SilE protein are being characterized. Sequences similar to the silver-resistance DNA are being characterized by Southern blot DNA/DNA hybridization, PCR in vitro DNA synthesis and DNA sequencing. More than 25 additional closely related sequences have been identified in bacteria from diverse sources. Initial DNA sequencing results shows approximately 5-20% differences in DNA sequences. | 1999 | 18475907 |
| 184 | 10 | 0.9994 | Plasmid chromate resistance and chromate reduction. Compounds of hexavalent chromium (chromates and dichromates) are highly toxic. Plasmid genetic determinants for chromate resistance have been described in several bacterial genera, most notably in Pseudomonas. Resistance to chromate is associated with decreased chromate transport by the resistant cells. The genes for a hydrophobic polypeptide, ChrA, were identified in chromate resistance plasmids of Pseudomonas aeruginosa and Alcaligenes eutrophus. ChrA is postulated to be responsible for the outward membrane translocation of chromate anions. Widespread bacterial reduction of hexavalent chromate to the less toxic trivalent chromic ions is also known. Chromate reduction determinants have not, however, been found on bacterial plasmids or transposons. In different bacteria, chromate reduction is either an aerobic or an anaerobic process (but not both) and is carried out either by soluble proteins or by cell membranes. Chromate reduction may also be a mechanism of resistance to chromate, but this has not been unequivocally shown. | 1992 | 1741461 |
| 8455 | 11 | 0.9994 | RT-PCR: characterization of long multi-gene operons and multiple transcript gene clusters in bacteria. Reverse transcription (RT)-PCR is a valuable tool widely used for analysis of gene expression. In bacteria, RT-PCR is helpful beyond standard protocols of northern blot RNA/DNA hybridization (to identify transcripts) and primer extension (to locate their start points), as these methods have been difficult with transcripts that are low in abundance or unstable, similar to long multi-gene operons. In this report, RT-PCR is adapted to analyze transcripts that form long multi-gene operons--where they start and where they stop. The transcripts can also be semiquantitated to follow the expression of genes under different growth conditions. Examples using RT-PCR are presented with two different multi-gene systems for metal cation resistance to silver and mercury ions. The silver resistance system [9 open reading frames (ORFs); 12.5 kb] is shown by RT-PCR to synthesize three nonoverlapping messenger RNAs that are transcribed divergently. In the mercury resistance system (8 ORFs; 6.3 kb), all the genes are transcribed in the same orientation, and two promoter sites produce overlapping transcripts. For RT-PCR, reverse transcriptase enzyme is used to synthesize first-strand cDNA that is used as a template for PCR amplification of single-gene products, from the beginning, middle or end of long multi-gene, multi-transcript gene clusters. | 1999 | 10572645 |
| 444 | 12 | 0.9994 | The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases. Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria. | 2004 | 16329852 |
| 428 | 13 | 0.9994 | Identification 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. | 1986 | 2438417 |
| 440 | 14 | 0.9994 | Nucleotide 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. | 1987 | 2828187 |
| 179 | 15 | 0.9994 | The genetics and biochemistry of mercury resistance. The ability of bacteria to detoxify mercurial compounds by reduction and volatilization is conferred by mer genes, which are usually plasmid located. The narrow spectrum (Hg2+ detoxifying) Tn501 and R100 determinants have been subjected to molecular genetic and DNA sequence analysis. Biochemical studies on the flavoprotein mercuric reductase have elucidated the mechanism of reduction of Hg2+ to Hg0. The mer genes have been mapped and sequenced and their protein products studied in minicells. Based on the deduced amino acid sequences, these proteins have been assigned a role in a mechanistic scheme for mercury flux in resistant bacteria. The mer genes are inducible, with regulatory control being exerted at the transcriptional level both positively and negatively. Attention is now focusing on broad-spectrum resistance involving detoxification of organomercurials by an additional enzyme, organomercurial lyase. Lyase genes have recently been cloned and sequencing studies are in progress. | 1987 | 2827958 |
| 8456 | 16 | 0.9994 | Identification of genes required by Bacillus thuringiensis for survival in soil by transposon-directed insertion site sequencing. Transposon-directed insertion site sequencing was used to identify genes required by Bacillus thuringiensis to survive in non-axenic plant/soil microcosms. A total of 516 genetic loci fulfilled the criteria as conferring survival characteristics. Of these, 127 (24.6 %) were associated with uptake and transport systems; 227 loci (44.0 %) coded for enzymatic properties; 49 (9.5 %) were gene regulation or sensory loci; 40 (7.8 %) were structural proteins found in the cell envelope or had enzymatic activities related to it and 24 (4.7 %) were involved in the production of antibiotics or resistance to them. Eighty-three (16.1 %) encoded hypothetical proteins or those of unknown function. The ability to form spores was a key survival characteristic in the microcosms: bacteria, inoculated in either spore or vegetative form, were able to multiply and colonise the soil, whereas a sporulation-deficient mutant was not. The presence of grass seedlings was critical to colonisation. Bacteria labelled with green fluorescent protein were observed to adhere to plant roots. The sporulation-specific promoter of spo0A, the key regulator of sporulation, was strongly activated in the rhizosphere. In contrast, the vegetative-specific promoters of spo0A and PlcR, a pleiotropic regulator of genes with diverse activities, were only very weakly activated. | 2014 | 24310935 |
| 187 | 17 | 0.9994 | Functional coexistence of twin arsenic resistance systems in Pseudomonas putida KT2440. The genome of the soil bacterium Pseudomonas putida KT2440 bears two virtually identical arsRBCH operons putatively encoding resistance to inorganic arsenic species. Single and double chromosomal deletions in each of these ars clusters of this bacterium were tested for arsenic sensitivity and found that the contribution of each operon to the resistance to the metalloid was not additive, as either cluster sufficed to endow cells with high-level resistance. However, otherwise identical traits linked to each of the ars sites diverged when temperature was decreased. Growth of the various mutants at 15°C (instead of the standard 30°C for P. putida) uncovered that ars2 affords a much higher resistance to As (III) than the ars1 counterpart. Reverse transcription polymerase chain reaction of arsB1 and arsB2 genes as well as lacZ fusions to the Pars1 and Pars2 promoters traced the difference to variations in transcription of the corresponding gene sets at each temperature. Functional redundancy may thus be selected as a stable condition - rather than just as transient state - if it affords one key activity to be expressed under a wider range of physicochemical settings. This seems to provide a straightforward solution to regulatory problems in environmental bacteria that thrive under changing scenarios. | 2015 | 24673935 |
| 494 | 18 | 0.9993 | The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons. The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis. From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA. The level of DNA sequence similarity between these genes and those of the mer determinant of Tn21 was between 56 center dot 4 and 62 center dot 4%. A neighbour-joining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date. Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid. The cloned determinant has been shown to express mercury resistance constitutively. Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant. This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the -35 and -10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region. Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions. Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1 center dot 37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment. This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments. | 1996 | 8932707 |
| 386 | 19 | 0.9993 | A 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. | 1990 | 2159150 |