Isolation and characterization of a linear plasmid from Streptomyces rimosus

Abstract A linear plasmid was isolated from a strain of Streptomyces rimosus . This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.     

Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis

To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.     

Construction and characterization of recA mutant strains of Corynebacterium glutamicum and Brevibacterium lactofermentum

An internal fragment of the Corynebacterium glutamicum recA gene was amplified by the polymerase chain reaction (PCR) using degenerate primers corresponding to two short sequences that are well conserved homology with RecA sequences from other bacteria including the invariant and functionally conserved amino acids Leu-126, Asp-144, Gly-157, Arg-169 and Asn-193. Highest identity (91%) was shared with the gram-positive Mycobacterium tuberculosis RecA sequence. The amplified fragment was cloned into a conditional suicide vector, pBGS8, and used to generate recA deficient strains of C. glutamicum and Brevibacterium lactofermentum by insertional inactivation. These strains exhibited classical RecA phenotypes including reduced recombinational activity and increased sensitivity to DNA-damaging agents such as UV irradiation, mitomycin C and methyl-methanesulphonate.     

Enhancement of oxytetracycline production in a Streptomyces rimosus strain after treatment with acriflavine

Summary A strain ofStreptomyces rimosus was treated with acriflavine as curing agent; morphological and physiologically altered variants were isolated, and changes in sporulation, aerial and substrate mycelia, pigment formation, oxytetracycline (OTC) resistance and biosynthesis are discussed.     

Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10(-4) to 10(-3) per cell per generation. Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange. Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4BrecA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.     

Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant

Summary The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy. Offprint requests to: K. Esser     

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

The recombination deficient Enterococcus faecalis UV202 strain is a recA mutant

The recA gene of the recombination deficient Enterococcus faecalis strain UV202 was sequenced and found to encode a glycine to aspartic acid mutation at amino acid 265. Both the UV sensitive and recombination deficient phenotypes of the UV202 strain were complemented by expression of the wild-type recA gene cloned under the control of the nisin-inducible promoter of an expression vector.     

鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline.     

Purification and chemical characterization of antimicrobial compounds from a new soil isolate Streptomyces rimosus MTCC 10792

A new isolate of Streptomyces sp. from soil of state Chhattisgarh (India) having broad spectrum antibacterial and antifungal activity was obtained. The active strain was identified as Streptomyces rimosus subsp. rimosus with accession number MTCC 10792 based on physiological, biochemical characteristics and 16S rRNA sequence homology studies. Antimicrobial compound produced by S. rimosus was tested against the drug resistance pathogens by the Bauer and Kirby method. The crude active metabolite was extracted using solvent n-butanol and purified by silica column chromatography and HPLC method. The physicochemical characteristics of the one purified compound viz. color, melting point, solubility, elemental analysis, ESIMS, IR, UV, 1HNMR, ¹³CNMR and chemical reactions have been investigated. Purified antimicrobial compound produced by S. rimosus MTCC 10792 at concentration 25 μg/mL showed antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37R as well as broad activity against all tested bacterial and fungal pathogens.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

Cloning and characterization of the recA gene of Paracoccus denitrificans and construction of a recA-deficient mutant

The recA gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter sphaeroides recA gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy.