# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 6194 | 0 | 1.0000 | Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome. In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes. A 4.4-kb fragment encoding the S. typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC. This gene organization is conserved between E. coli and S. typhimurium. We determined that the S. typhimurium sdiA gene was able to weakly complement the E. coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele. To better understand the function of sdiA in S. typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S. typhimurium virulence plasmid. The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes. | 1998 | 9495757 |
| 445 | 1 | 0.9993 | 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 |
| 8890 | 2 | 0.9993 | Eavesdropping by bacteria: the role of SdiA in Escherichia coli and Salmonella enterica serovar Typhimurium quorum sensing. Many gram-negative bacteria utilize N-acyl-L-homoserine lactones (AHLs) to bind to transcriptional regulators leading to activation or repression of target genes. Escherichia coli and Salmonella enterica do not synthesize AHLs but do contain the AHL receptor, SdiA. Studies reveal that SdiA can bind AHLs produced by other bacterial species and thereby allow E. coli and S. enterica to regulate gene transcription. The Salmonella sdiA gene regulates the rck gene, which mediates Salmonella adhesion and invasion of epithelial cells and the resistance of the organism to complement. In E. coli, there is some evidence that SdiA may regulate genes associated with acid resistance, virulence, motility, biofilm formation, and autoinducer-2 transport and processing. However, there is a lack of information concerning the role of SdiA in regulating growth and survival of E. coli and Salmonella in food environments, and therefore studies in this area are needed. | 2011 | 21034261 |
| 380 | 3 | 0.9993 | Expression 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. | 1984 | 6442250 |
| 6324 | 4 | 0.9992 | 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 |
| 439 | 5 | 0.9992 | 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 |
| 6217 | 6 | 0.9992 | Identification of the sigmaB regulon of Bacillus cereus and conservation of sigmaB-regulated genes in low-GC-content gram-positive bacteria. The alternative sigma factor sigma(B) has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of sigma(B)-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being sigma(B) dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor sigma(B) and a crucial positive regulator of sigma(B) activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified sigma(B)-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the sigma(B) regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where sigma(B) of the B. cereus group was placed close to the ancestral form of sigma(B) in gram-positive bacteria. The data described in this study and previous studies in which the complete sigma(B) regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of sigma(B)-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their sigma(B) dependency in all four bacteria, suggesting that the sigma(B) regulon of the different gram-positive bacteria has evolved to perform niche-specific functions. | 2007 | 17416654 |
| 385 | 7 | 0.9991 | Introduction 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. | 2000 | 10620668 |
| 440 | 8 | 0.9991 | 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 |
| 641 | 9 | 0.9991 | Bile salts induce resistance to polymyxin in enterohemorrhagic Escherichia coli O157:H7. Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin. | 2011 | 21725004 |
| 180 | 10 | 0.9991 | 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 | 11 | 0.9991 | 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 |
| 6322 | 12 | 0.9991 | A soxRS-constitutive mutation contributing to antibiotic resistance in a clinical isolate of Salmonella enterica (Serovar typhimurium). The soxRS regulon is activated by redox-cycling drugs such as paraquat and by nitric oxide. The >15 genes of this system provide resistance to both oxidants and multiple antibiotics. An association between clinical quinolone resistance and elevated expression of the soxRS regulon has been observed in Escherichia coli, but this association has not been explored for other enteropathogenic bacteria. Here we describe a soxRS-constitutive mutation in a clinical strain of Salmonella enterica (serovar Typhimurium) that arose with the development of resistance to quinolones during treatment. The elevated quinolone resistance in this strain derived from a point mutation in the soxR gene and could be suppressed in trans by multicopy wild-type soxRS. Multiple-antibiotic resistance was also transferred to a laboratory strain of S. enterica by introducing the cloned mutant soxR gene from the clinical strain. The results show that constitutive expression of soxRS can contribute to antibiotic resistance in clinically relevant S. enterica. | 2001 | 11120941 |
| 292 | 13 | 0.9991 | Mechanisms 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. | 1994 | 7826010 |
| 644 | 14 | 0.9991 | The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds. BACKGROUND: The marR gene of Escherichia coli encodes a repressor of the marRAB operon, a regulatory locus controlling multiple antibiotic resistance in this organism. Inactivation of marR results in increased expression of marA, which acts at several target genes in the cell leading to reduced antibiotic accumulation. Exposure of E. coli to sodium salicylate (SAL) induces marRAB operon transcription and antibiotic resistance. The mechanism by which SAL antagonizes MarR repressor activity is unclear. MATERIALS AND METHODS: Recombinant plasmid libraries were introduced into a reporter strain designed to identify cloned genes encoding MarR repressor activity. Computer analysis of sequence databases was also used to search for proteins related to MarR. RESULTS: A second E. coli gene, MprA, that exhibits MarR repressor activity was identified. Subsequent database searching revealed a family of 10 proteins from a variety of bacteria that share significant amino acid sequence similarity to MarR and MprA. At least four of these proteins are transcriptional repressors whose activity is antagonized by SAL or by phenolic agents structurally related to SAL. CONCLUSIONS: The MarR family is identified as a group of regulatory factors whose activity is modulated in response to environmental signals in the form of phenolic compounds. Many of these agents are plant derived. Some of the MarR homologs appear more likely to control systems expressed in animal hosts, suggesting that phenolic sensing by bacteria is important in a variety of environments and in the regulation of numerous processes. | 1995 | 8521301 |
| 8455 | 15 | 0.9990 | 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 |
| 443 | 16 | 0.9990 | 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 |
| 289 | 17 | 0.9990 | A genetic system that reports transient activation of genes in Bacillus. Site-specific recombination is a powerful tool for precise excision of DNA fragments. We used this characteristic to construct a genetic system to report the transient activation of a promoter by promoting the stable acquisition of an antibiotic resistance marker by the bacterium. The system is composed of two compatible plasmid derivatives from Gram-positive bacteria. One of the plasmids allows the insertion of promoters upstream from tnpI, which encodes the site-specific recombinase of Tn4430. The second plasmid carries two selectable resistance genes: one is flanked by two site-specific recombination sequences and is lost following recombination; in contrast, the other resistance gene becomes functional after the site-specific recombination event. By inserting conditionally controlled promoters (the xylose-inducible xylA promoter or the plcA promoter whose expression is dependent on the growth medium) upstream of tnpI, we demonstrated that our genetic system responds to signals inducing transcription by conferring a new resistance phenotype to the host bacteria. Thus, this system can be used to identify genes which are transiently or conditionally expressed. | 1997 | 9427554 |
| 377 | 18 | 0.9990 | Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other gram-negative bacteria. We report the construction of two broad host range promoter-probe plasmid vectors for rapid analysis of promoters in Gram-negative bacteria. The new vectors, pME4507 and pME4510, carry carbenicillin and gentamycin resistance genes, respectively, and are small sized (4 kb) with a flexible multiple cloning site to facilitate directional cloning of putative promoter elements. The vectors allow rapid plate-based screening for promoter activities, using beta-galactosidase as the reporter enzyme. In the absence of an inserted promoter fragment, they display very low background activity, making them a useful tool for analysis of low expression level promoters. | 1998 | 9851050 |
| 378 | 19 | 0.9990 | Construction and use of a self-cloning promoter probe vector for gram-negative bacteria. Transposon Tn5 has been used extensively for the genetic analysis of Gram- bacteria. We describe here the construction and use of a Tn5 derivative which contains the ColE1 origin of DNA replication, thereby allowing the cloning of DNA adjacent to the Tn without the need for construction of genomic libraries. The Tn is derived from Tn5-B21 [Simon et al., Gene 80 (1989) 161-169] and contains a promoter-probe lacZ gene and genes encoding resistance to tetracycline and beta-lactams. It is housed within a mobilisable suicide plasmid which can be transferred to a wide range of Gram- bacteria. The Tn was tested using pyoverdine siderophore-synthesis genes (pvd) from Pseudomonas aeruginosa. The simple cloning procedure allowed 15.9 kb of pvd-associated DNA to be cloned; in addition, the lacZ reporter gene allowed the transcription of pvd genes to be studied. The bacteria were resistant to carbenicillin only if the Tn (and hence the beta-lactamase-encoding gene) was downstream from an active promoter. | 1993 | 8386128 |