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PubMed2001

The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403.

Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group.

Amino Acids↗
PubMed2006

Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.

Natural transformation is a widespread mechanism for genetic exchange in bacteria. Aminoglycoside and fluoroquinolone antibiotics, as well as mitomycin C, a DNA-damaging agent, induced transformation in Streptococcus pneumoniae. This induction required an intact competence regulatory cascade. Furthermore, mitomycin C induction of recA was strictly dependent on the development of competence. In response to antibiotic stress, S. pneumoniae, which lacks an SOS-like system, exhibited genetic transformation. The design of antibiotherapy should take into consideration this potential of a major human pathogen to increase its rate of genetic exchange in response to antibiotics.

Aminoglycosides↗
PubMed1969

Killing of Haemophilus influenzae cells by integrated ultraviolet-induced lesions from transforming deoxyribonucleic acid.

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.

DNA, Bacterial↗
PubMed1984

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.

Aminopterin↗
PubMedDate not supplied

Early stages of HIV replication: how to hijack cellular functions for a successful infection.

From the cell surface to the nucleus, the human immunodeficiency virus type 1 (HIV-1) will face multiple obstacles, crossing the plasma and nuclear membranes, but also finding its path within the cytoplasm in which elements from the cytoskeleton, organelles, and high a protein concentration, limit intracellular movements. At the same time, HIV-1 has to counteract cellular defenses--known as restriction factors--interfering with early steps of the virus cycle. Although the general outcomes of these early stages have been identified since several decades, the stepwise interactions taking place between cellular and viral components during this early journey, which will transform the incoming viral-RNA genome into a double-strand DNA competent for integration, remain largely unknown. In that sense, the uncoating process and the molecular basis of intracellular trafficking of preintegration complexes (PICs) are still poorly defined. Additionally, other key stages, which have been the focus of many reports, still require some clarifications, as is the case for the precise determinants of nuclear import of PICs. Finally, whereas the molecular mechanisms of integration, the last event of the early phase of retroviral life cycle, are now well understood, the choice of the integration site remains mysterious. Fully elucidating the early steps of HIV-1 replication is therefore crucial, not only for developing new antiretroviral drugs, but also for improving the design of lentiviral vectors for gene therapy. Since the mechanisms of HIV-1 entry and innate cell defenses were recently the topic of excellent reviews, we will focus here on uncoating and intracellular trafficking of HIV-1.

Cell Membrane↗
PubMed1989

Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens.

Simian virus 40 T antigen (TAg) exhibits nonspecific and origin-specific DNA binding (ori binding) and ATPase and helicase activities, all of which are related to its roles in viral DNA replication. We have characterized some of the properties of four replication-defective but transformation-competent mutant TAgs, C6-2, T22, C11, and C8A. C6-2 and T22 TAgs were each previously determined to lack ori-binding properties, while C11 TAg was reported to lack ATPase activity. The C8A TAg did not exhibit defects in either ori-binding or ATPase functions. We have analyzed additional aspects of these mutant TAgs pertaining to their helicase, DNA-binding, and immunological properties. With the exception of the C11 TAg, all the other TAgs exhibited helicase activity. The lack of helicase activity by C11 TAg was consistent with its previously shown inability to hydrolyze ATP or to replicate viral DNA. These results therefore show that ori-binding and helicase activities are separate functions of TAg. Wild-type and mutant TAgs bound with similar efficiency to either native or denatured calf thymus DNA-cellulose, indicating no marked differences in their nonspecific DNA-binding properties. We also tested the binding of wild-type and mutant TAgs to a monoclonal antibody, PAb 100, that was previously shown to recognize an extremely small class of TAg that may represent a unique conformational form of the protein. Interestingly, while less than 10% of the wild-type, C6-2, C11, and T22 mutant TAgs were recognized by PAb 100, more than 60% of the C8A mutant TAg was bound by this antibody. Therefore, although no defect in biochemical function was observed with the C8A TAg, its deficiency in viral DNA replication may be related to an unusual conformation, as detected by its dramatically increased recognition by PAb 100. These results show that the helicase activity of TAg is not required for its transformation function.

Antibodies, Monoclonal↗
PubMed1983

[Recombination in the plasmid transformation of Bacillus subtilis: the role of superhelical DNA].

The inhibitor of DNA gyrase, novobiocine affects recombination between homologous plasmids in the course of plasmid transformation. Novobiocine decreased significantly the yield of recombinants when added to competent cells during the first 10 minutes. Later, after 30 to 40 min, addition of the antibiotic had no effect. Probably, recombination is completed, in general, in 10 to 20 min after DNA uptake. Plasmid recombination was depressed totally after preincubation of competent cells with the drug. Evidently, inhibition of recombination was due to the loss of superhelicity in resident plasmid DNA.

Bacillus subtilis↗
PubMed1989

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Alleles↗
PubMed1992

A periplasmic protein disulfide oxidoreductase is required for transformation of Haemophilus influenzae Rd.

The mutated gene in JG16, a Haemophilus influenzae strain deficient in competence-induced DNA binding and uptake, was cloned and the wild-type allele was sequenced. The gene was shown by Northern analysis to be constitutively expressed on a 1.7-kilobase transcript. The gene product was identified as a 20.6-kDa protein targeted to the periplasm. The protein contains the sequence Cys-Pro-His-Cys (CPHC) and is highly similar to two other periplasmic CPHC motif-containing proteins: DsbA, an Escherichia coli protein (45% identity, 87% homology) and TcpG, a Vibrio cholerae protein (32% identity, 74% homology). Both DsbA and TcpG promote disulfide bond formation in periplasmic proteins, are required for pilus biogenesis, and, like thioredoxin, are capable of reducing insulin in vitro. The Haemophilus protein was shown to complement an E. coli mutation in DsbA and was named Por (periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did not occur. These findings suggest that Por is required for the correct assembly and/or folding of one or more disulfide-containing cell envelope protein involved either in competence development or in the DNA-binding and -uptake machinery.

Amino Acid Sequence↗
PubMed1992

One-step transformation of yeast in stationary phase.

A fast yeast-transformation technique has been developed by adding thio compounds to alkali-ion based protocols and incubating at 45 degrees C. This procedure is especially recommended for cells from stationary phase at a density up to 2.5 x 10(8) cells/ml. It involves only one step for the preparation and transformation of competent cells within 30 min. The yield was more than 10(4) transformants/microgram plasmid DNA. This protocol is easy to scale up for many DNA samples and is also applicable for yeast cells from different types of storages.

DNA, Fungal↗
PubMed1985

HSV- and chemical carcinogen-induced amplification of SV40 DNA sequences in transformed cells is cell-line-dependent.

Eleven simian virus 40-transformed cell lines from 5 different species were tested for their ability to amplify integrated simian virus 40 DNA upon infection with herpes simplex virus type I or treatment with various chemical carcinogens. Four cell lines were positive only for virus-induced gene amplification and two lines were positive for both carcinogen- and virus-induced gene amplification. Individual cell lines were assayed for the presence of an intact SV40 origin of replication, the expression of a functional SV40 T-antigen, and permissivity to herpes simplex virus replication. These parameters were found to be positive in all 6 amplification-competent cell lines. The ability of herpes simplex virus to amplify SV40 DNA sequences in transformed cells is greater than that of chemical carcinogens and can be suppressed by specific inhibitors of the herpes virus-encoded DNA polymerase.

Animals↗
PubMed2004

Characterization of three new competence-regulated operons in Haemophilus influenzae.

Haemophilus influenzae is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized. To date, several operons involved in transformation in this organism have been described. These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media. The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in H. influenzae. In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis. These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes. The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in Pseudomonas aeruginosa. An insertion in HI0939 abolished both DNA binding and uptake. The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in Klebsiella oxytoca.

Base Sequence↗
PubMed1980

DNA-mediated transformation in mutants of phage group 2 Staphylococcus aureus.

A large pool of antibiotic resistant and auxotrophic mutants was isolated from the Staphylococcus aureus phage group 2 strains UT0002-19 and UT0017 by (1) antibiotic gradient plates, (2) trimethoprim selection, and (3) nitrosoguanidine mutagenesis, which sometimes was coupled by enrichment with either penicillin or methicillin. Strain UT0002-19 has a chromosomal determinant for exfoliative toxin (ET), which causes "scalded skin syndrome" in man. A few mutants were isolated from the phage group 1 strain UT0080, which also produces ET. Two transformation regimens, called the broth and plate methods, were devised for the phage group 2 strains. They employed 80 alpha as helper phage, and recipient cells were incubated with transforming DNA in the presence of Ca2+. Strain UT0080 was transformed using phage 55 as helper. Maximum competence of the phage group 2 strains occurred during early logarithmic growth in trypticase soy broth, but cells grown overnight on heart infusion agar were also competent. Transformation frequencies of all markers ranged from 10(-6) to 10(-8). For phage 80 alpha, a multiplicity of infection of 4 was optimal in transforming a mutant of strain UT0002-19. Transformation of gly, lin, met, ole, rif, and ser markers in S. aureus is reported for the first time. Ery and ole markers in all three strains exhibited cross-resistance. Mapping studies, similar to those performed by DNA-mediated transformation in the phage group 3 strain 8325, can now be commenced for phage group 2 strains of S. aureus in order to elucidate the molecular genetics of this medically important bacterium.

Anti-Bacterial Agents↗
PubMed2004

Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli.

Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.

DNA, Bacterial↗
PubMed2000

[Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium fredii RT19].

A 23 kb DNA fragment related to salt tolerance was obtained from the gene library of S. fredii strain RT19. In this study, BamH I was selected to digest 23 kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4 mol/L NaCl. Thus, a 4.4 kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4 kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained.

Amino Acid Sequence↗
PubMed2000

Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease.

A genetic-based search for surface proteins of Streptococcus pneumoniae involved in adhesion identified a putative zinc metalloprotease (ZmpB). ZmpB shared high amino acid sequence similarities with IgA1 proteases of Gram-positive bacteria, but ZmpB had neither IgA1 nor IgA2 protease activity. Analysis of a family of surface-expressed proteins, the choline-binding proteins (Cbp's), in a zmpB-deficient mutant demonstrated a global loss of surface expression of CbpA, CbpE, CbpF and CbpJ. CbpA was detected within the cytoplasm. The zmpB-deficient mutant also failed to lyse with penicillin, a sign of lack of function of the Cbp LytA. Immunodetection studies revealed that the autolysin (LytA), normally located on the cell wall, was trapped in the cytoplasm colocalized with DNA and the transformation protein CinA. Trafficking of CinA and RecA to the cell membrane during genetic competence was also not observed in the zmpB-deficient mutant. These results suggest a protease dependent regulatory mechanism governing the translocation of CinA and the Cbp's LytA and CbpA of S. pneumoniae.

Alleles↗
PubMed1982

Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.

Cytochrome b Group↗