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At least 91 records · Page 5Linked to original sources
PubMed1995

Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene.

Upon the cessation of exponential growth, Bacillus subtilis enters a transition phase leading to either sporulation or a non-sporulating stationary phase. During this transition period, cells secrete degradative enzymes, become competent for DNA transformation, are motile and acquire resistance to oxidative killing. We now report that mrgA, originally identified as a gene repressed by metal ions, encodes a member of the Dps/PexB family of general stress proteins. Like Escherichia coli Dps(PexB), MrgA forms highly stable, multimeric protein-DNA complexes which accumulate in stationary-phase cells and protect against oxidative killing. MrgA is part of an inducible oxidative stress response in B. subtilis: mrgA is induced by hydrogen peroxide, and a strain lacking MrgA displays increased sensitivity to oxidative killing. In addition, a hydrogen peroxide-resistant mutant, which constitutively overproduces catalase and alkyl hydroperoxide reductase, also overproduces MrgA. These results indicate a complex interplay between metal ions and the expression of the B. subtilis oxidative stress response.

Amino Acid Sequence↗
PubMed1995

Characterization of the pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae.

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Amino Acid Sequence↗
PubMed2005

[Screening and cloning of hepatitis C virus non-structural protein 4A interacting protein gene in hepatocytes].

OBJECTIVE: To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A). METHODS: Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods. RESULTS: Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase. CONCLUSIONS: Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins↗
PubMed1973

Interaction between deoxyribonucliec acid and distamycin A studied by transformation in Bacillus subtilis.

Distamycin A inhibits deoxyribonucleic acid (DNA)-mediated transformation and transfection in Bacillus subtilis at doses with little or no antibacterial effect. The inhibition of transformation parallels the inhibition of DNA uptake; further-more, during the transformation process, donor DNA molecules become distamycin and deoxyribonuclease resistant at the same time. This demonstrates that the drug acts by inhibiting DNA uptake. Although the drug is known to bind DNA, the inhibition is not related to affinity of the drug for DNA. This is shown by the lack of dependence of the extent of inhibition on DNA concentration, whereas the degree of inhibition depends upon cell concentration. Supporting this view is the fact that transformation by single-stranded DNA was also inhibited, even though the drug does not bind to denatured DNA. Distamycin A probably interferes with transformation by competing with DNA for some unknown bacterial component involved in transport of DNA into the cell.

Amidines↗
PubMed1991

In vitro mutation of Haemophilus influenzae transforming deoxyribonucleic acid by ultraviolet radiation at -70 degrees C.

Previous studies have shown the non-mutability of Haemophilus influenzae either by UV irradiation of the cells or by irradiating the transforming DNA and transformation of competent cells. In the present work, we present evidence of transforming DNA mutation in vitro by UV irradiation at -70 degrees C, which upon transformation of competent cells showed a rise in the mutation frequencies of novobiocin resistance of the order of several hundredfold. Also we performed experiments using the UV-irradiated DNA either sonicated or DNase-treated, which allowed us to propose that such rise in mutation frequency is probably due to the integration of DNA carrying premutagenic photoproducts to the recipient cells' genome. We think that the key point was the low temperature at which the DNA was irradiated in order to obtain the mutagenic effects, since it is likely that at -70 degrees C, the main photoproducts are not the cyclobutane dimers, but are the spore photoproducts, which are probably responsible for the damage that leads to mutagenic effects.

Cold Temperature↗
PubMed2005

[Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes].

OBJECTIVE: To investigate biological functions of non-structural protein 4B (NS4B) of hepatitis C virus (HCV), yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B. METHODS: HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT7 and transformed into yeast cells AH109 (type alpha). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics. RESULTS: Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit III, one retinol binding protein 4, one reticulon 3-A (RTN3) and one fibrinogen gamma polypeptide (FGG). CONCLUSION: Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.

Blotting, Western↗
PubMed1970

Quantitive autoradiographic study of competence and deoxyribonucleic acid incorporation in Bacillus subtilis.

The relationships among deoxyribonucleic acid (DNA) uptake, transformation, and autoradiographic labeling were investigated. It is shown that: (i) autoradiography is a good method for measuring the total fraction of competent cells able to incorporate transforming DNA; in our best experiment that fraction was 11.5%. (ii) Computation of the fraction of competent cells in a culture of Bacillus subtilis, by comparing the frequencies of single and double transformants for two unlinked markers, gives values which are somewhat different from those obtained by auto-radiography. (iii) On the average, a competent cell of B. subtilis irreversibly takes up from 0.9 x 10(-10) to 1.4 x 10(-10) mug of DNA; this amount represents about 1/55 to 1/35 of the bacterial chromosome weighing 5 x 10(-9) mug, and corresponds to three to six DNA molecules having a molecular weight of 1.6 x 10(7).

Autoradiography↗
PubMed1989

Genetic transformation in some cariogenic Streptococcus milleri.

Only one of six Streptococcus milleri strains, known to produce dental caries in gnotobiotic rats, was found to be spontaneously transformable. At optimal competence (after 60 min incubation in transformation medium) DNA uptake by this strain, CR 287, was rapid; maximum DNA was taken up by cells within 15 min of its addition to give a transformation frequency of 1 x 10(-2) transformants per colony-forming unit (cfu). With post-optimally competent cells, the rate of DNA uptake was markedly decreased, although a transformation frequency similar to that of optimally competent cells was obtained. For example, after 240 min incubation in transformation medium the cells required approximately 90 min to reach maximum DNA uptake and gave a transformation frequency of 6 x 10(-3) transformants per cfu. Cultures retained the ability to give their maximum transformation frequency for at least 4 h, but only if a DNA-cell contact time of 2 h was used. Strain CR 287 and S. milleri strain NCTC 10707, a transformable non-cariogenic strain, produced competence factor (CF). The CF of CR 287, but not that of NCTC 10707, induced high competence in the non-spontaneously transformable cariogenic S. milleri strain NCTC 11169. Like S. sanguis, S. milleri must therefore produce CFs of different constitutional types. The three transformable strains could be transformed by plasmid shuttle cloning vector pVA838 DNA.

Animals↗
PubMed1986

Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer.

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

[Comparative study of plasmid transformation in competent cells and protoplasts of Bacillus subtilis].

The effect of different factors which inhibit transformation with chromosomal DNA on plasmid transformation of competent cells and protoplasts was examined. The treatment with EDTA, dinitrophenol, CdCl2 and different temperature shifts affects significantly less plasmid transformation of protoplasts than plasmid and chromosomal transformation of competent cells. These data point out the different mechanisms of DNA uptake by competent cells and protoplasts.

Bacillus subtilis↗
PubMedDate not supplied

Method and parameters for genetic transformation of Streptococcus sanguis Challis.

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth containing 10% foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 micrograms/ml. At 10 micrograms/ml, more than 2.2% of the colony-forming units were transformed to streptomycin resistance, while transforming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The transformation reaction was inhibited at 0 and 4 degrees C, whereas it occurred efficiently both at 25 and 37 degrees C.

DNA, Bacterial↗
PubMed1989

DNA-binding proteins in cells and membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb fractions BI and BII of Neisseria gonorrhoeae contain both linear and circular DNAs. Because little is known about the interactions between DNA and blebs, studies were initiated to identify specific proteins that bind DNA in elaborated membrane blebs. Western immunoblots of whole-cell and bleb proteins from transformation-competent and DNA-uptake-deficient (dud) mutants were probed with single- or double-stranded gonococcal DNA, pBR322, or synthetic DNA oligomers containing intact or altered gonococcal transformation uptake sequences. The specificity and sensitivity of a nonradioactive DNA-binding protein assay was evaluated, and the assay was used to visualize DNA-protein complexes on the blots. The complexes were then characterized by molecular mass, DNA-binding specificity, and expression in bleb fractions. The assay effectively detected blotted DNA-binding proteins. At least 17 gonococcal DNA-binding proteins were identified; unique subsets occurred in BI and BII. Certain DNA-binding proteins had varied affinities for single- and double-stranded DNA, and the intact transformation uptake sequence competitively displaced the altered sequence from a BI protein at 11 kilodaltons (kDa). A dud mutant, strain FA660, lacked DNA-binding activity at the 11-kDa protein in BI. The segregation of DNA-binding proteins within BI and BII correlates with their distinct protein profiles and suggests that these vesicles may play different roles. Although the DNA-binding proteins expressed in BII may influence the nuclease-resistant export of plasmids within BII vesicles, the BI 11-kDa protein may bind transforming DNA.

Base Sequence↗
PubMed1980

Mechanism of homospecific DNA uptake in Haemophilus influenzae transformation.

DNA uptake by competent H. influenzae cells requires the presence of a specific base sequence (uptake site) on the entering DNA duplex. This sequence is probably recognized by a receptor on the cell surface. We have examined the kinetics and stoichiometry of DNA uptake by competent cells and have shown that the results are consistent with a simple model involving: 1) reversible binding of the DNA uptake site to a cell surface receptor, 2) an irreversible step resulting in a commitment toward DNA uptake, and 3) transport of the DNA duplex into the cell. We have also shown that a competent H. influenzae cell can absorb only 4 to 8 molecules of DNA, regardless of their length. To explain this counting mechanism, we suggest that each cell has only 4 to 8 receptors and that each receptor can be used to transport only one molecule of DNA.

Base Sequence↗
PubMed1991

Genetic competence in Bacillus subtilis.

Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several histidine kinase and response regulator members of the bacterial two-component signal transduction machinery, as well as a number of known transcriptionally active proteins. Results of genetic studies are consistent with the notion that the regulatory proteins interact in a hierarchical way to make up a regulatory pathway, and it is possible to propose a provisional scheme for the organization of this pathway. It is remarkable that almost all of the regulatory gene products appear to play roles in the control of various forms of postexponential expression in addition to competence, e.g., sporulation, degradative-enzyme production, motility, and antibiotic production. This has led to the notion of a signal transduction network which transduces environmental information to determine the levels and timing of expression of the ultimate products characteristic of each of these systems.

Amino Acid Sequence↗
PubMed1982

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.

Calcium Chloride↗