Identification of a Staphylococcus aureus transposon (Tn4291) that carries the methicillin resistance gene(s).

We isolated a transposon (Tn4291) that carries the resistance gene(s) for methicillin in a secondary insertion site on the penicillinase plasmid pI524. Transposition of Tn4291 into pI524 occurred during the transduction of the tetracycline resistance plasmid pSN1 from a methicillin-resistant donor into a recipient that carried the mec allele in the primary site on the chromosome. Insertion of Tn4291 caused extensive rearrangement of pI524 and resulted in the formation of a 27.9-kilobase-pair plasmid (pIT103) which coded for resistance to methicillin and cadmium, but not penicillin. Although resistance to methicillin and cadmium were always linked, Tn4291 was stably maintained only in the presence of a chromosomal mec allele, while in its absence the plasmid was unstable and transposition to the primary site occurred. Subsequently, a 20.1-kilobase-pair plasmid, pIT203, was formed which retained cadmium resistance and regained the ability to express beta-lactamase activity.     

Duplication of the streptokinase gene in the chromosome of Streptococcus equisimilis H46A

The erythromycin resistance plasmid pSM752 carrying the cloned streptokinase gene, skc, was introduced by protoplast transformation into Streptococcus equisimilis H46A from which skc was originally cloned. Cells transiently supporting the replication of pSM752 gave rise to an erythromycin-resistant clone designated H46SM which was plasmid free and produced streptokinase at levels approximately twice as high as the wild type. Southern hybridization of total cell DNA with an skc-containing probe provided evidence for the duplication of the skc gene in the H46SM chromosome. The results, which have some bearing on industrial streptokinase production, can be best explained by a single cross-over event between the chromosome and the plasmid in the region of shared homology leading to the integration of pSM752 in a Campbell-like manner.     

Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei

A gene (nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNAT1. The nat gene was localized on a 1-kb SalI-MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of NcR in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.     

Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome

Cloning of an autonomously replicating sequence (ars) from the origin region of Bacillus subtilis was previously unsuccessful because of the strong incompatibility exerted by sequences located within the oriC region. Using an ars searching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of the oriC region, we have succeeded in isolating ars fragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA-box regions (non-translatable regions containing multiple repeats of DnaA-box) separated by the dnaA gene. Neither one of the DnaA-box regions by itself showed ars activity. When constructed as oriC plasmids, the dnaA coding region could be removed without affecting ars activity. The minimum distance between the two DnaA-box regions obtained so far is 274 bp. The copy number of the oriC plasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into chromosome.     

Cloning of the gene of Saccharomyces cerevisiae yeasts that determines resistance to the toxic action of cadmium ions

A gene conferring resistance to cadmium in Saccharomyces cerevisiae was isolated from a yeast gene library created on the basis of the pL3 vector. The phenotype of resistance is only expressed in the yeast cells with cloned DNA inserted into a multicopy plasmid. Integration of the plasmid into chromosome or introduction of the centromeric region into the plasmid decreases the level of cadmium resistance. The cloned Sau3A I fragment of the yeast chromosome is 3.5 kbp in size. Restriction analysis and subcloning experiments showed the gene to be located within 1.6 kbp of the XhoI-Sau3A I fragment of DNA. Instability was observed in the vicinity of the XhoI-Sau3A I fragment of the yeast DNA in Escherichia coli.     

Molecular cloning and DNA sequencing of the radC gene of Escherichia coli K-12.

The radC102 mutation that sensitizes E. coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome. E. coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR. The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector. A library of EcoRI/KpnI DNA fragments of E. coli strain BH20 was inserted into pUC19. One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10. The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment. The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced. The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500. The order of the genes in this region of the E. coli chromosome is: fpg--rpmBG--radC--pyrE.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Molecular cloning of a gene affecting the autolysin level and flagellation in Bacillus subtilis

A 2.8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HB101 and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRG1), of 10.2 kb, complemented flaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the HindIII and XbaI sites (1.0 kb apart) in pBRG1. The integration of a plasmid having chloramphenicol resistance closely linked to the flaD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the flaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.     

Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions.

The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by beta-galactosidase activity. The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome. Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.     

The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair,the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family

Plasmid pB4 is a conjugative antibiotic resistance plasmid, originally isolated from a microbial community growing in activated sludge, by means of an exogenous isolation method with Pseudomonas sp. B13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 bp long and contains at least 81 complete coding regions. A suite of coding regions predicted to be involved in plasmid replication, plasmid maintenance, and conjugative transfer revealed significant similarity to the IncP-1beta backbone of R751. Four resistance gene regions comprising mobile genetic elements are inserted in the IncP-1beta backbone of pB4. The modular 'gene load' of pB4 includes (1) the novel transposon Tn 5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn 5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene bla(NPS-1) flanked by highly conserved sequences characteristic of integrons, and (4) a tripartite antibiotic resistance determinant comprising an efflux protein of the resistance-nodulation-division (RND) family, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF). The components of the RND-MFP-OMF efflux system showed the highest similarity to the products of the mexCD-oprJ determinant from the Pseudomonas aeruginosa chromosome. Functional analysis of the cloned resistance region from pB4 in Pseudomonas sp. B13 indicated that the RND-MFP-OMF efflux system conferred high-level resistance to erythromycin and roxithromycin resistance on the host strain. This is the first example of an RND-MFP-OMF-type antibiotic resistance determinant to be found in a plasmid genome. The global genetic organization of pB4 implies that its gene load might be disseminated between bacteria in different habitats by the combined action of the conjugation apparatus and the mobility of its component elements.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA

The region of temperate bacteriophage T12 responsible for integration into the chromosome of Streptococcus pyogenes has been identified. The integrase gene ( int ) and the phage attachment site ( attP ) are found immediately upstream of the gene for speA , the latter of which is known to be responsible for the production of erythrogenic toxin A (also known as pyrogenic exotoxin A). The integrase gene has a coding capacity for a protein of 41 457 Da, and the C-terminus of the deduced protein is similar to other conserved C-terminal regions typical of phage integrases. Upstream of int is a second open reading frame, which is capable of encoding an acidic protein of 72 amino acids (8744 Da); the position of this region in relation to int suggests it to be the phage excisionase gene ( xis ). The arms flanking the integrated prophage ( attL and attR ) were identified, allowing determination of the sequences of the phage ( attP ) and bacterial ( attB ) attachment sites. A fragment containing the integrase gene and attP was cloned into a streptococcal suicide vector; when introduced into S. pyogenes by electrotransformation, this plasmid stably integrated into the bacterial chromosome at attB . The insertion site for the phage into the S. pyogenes chromosome was found to be in the anticodon loop of a putative type II gene for a serine tRNA. attP and attB share a region of identity that is 96 bp in length; this region of identity corresponds to the 3' end of the tRNA gene such that the coding sequence remains intact after integration of the prophage. The symmetry of the core region of att may set this region apart from previously described phage attachment sites (Campbell, 1992), and may play a role in the biology of this medically important bacteriophage.     

Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies.

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment which contains a small open reading frame that could encode an 85-amino-acid peptide. When an omega-Tet insertion was made into the putative pmrD open reading frame (pmrD2::omega-Tet), the resulting plasmid no longer conferred polymyxin resistance, whereas an omega-Tet insertion into vector sequences had no effect. Maxicell analysis confirmed that a protein of the expected size is made in vivo. The PmrD protein shows no significant homology to any known protein, but it does show limited homology across the active site of the p15 acid protease from Rous sarcoma virus, indicating that the protein may have proteolytic activity. However, changing the aspartic acid residue at the putative active site to alanine reduced but did not eliminate polymyxin resistance. When pmrD2::omega-Tet replaced the chromosomal copy of pmrD, the resulting strain showed wild-type sensitivity to polymyxin and could be complemented to resistance by a plasmid that carried pmrD. The pmrA505 allele confers resistance to polymyxin when present in single copy on the chromosome or when present on a plasmid in pmrA+ pmrD+ cells. In combination with the pmrD(2)::-Tet mutation, the effect o the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present in the chromosome or on a plasmid. Conversely, a strain carrying an insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmrD. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene product and PmrD.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Molecular Genetics and Genomics - The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the...     

Molecular cloning of a chromosomal DNA region encompassing the dihydrofolate reductase gene ofStreptococcus pneumoniae

Natural competence ofStreptococcus pneumoniae was used to locate and enrich DNA restriction fragments, biologically active for transformation of thymidine-deficient to thymidine-proficient cells. Mutations in the dihydrofolate reductase gene are accompanied by resistance to the drug trimethoprim (Tp). A 6.5-kb region of the pneumococcal chromosome encompassing the dihydrofolate reductase gene has been cloned in plasmid pLS1.Escherichia coli mutants, resistant to Tp, became fully sensitive to the drug when they harbored the recombinant plasmid. The pneumococcaldfrA mutation has been mapped within a 500-bp DNA region.     

Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760

A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.