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PubMed1999

Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation.

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.

Bacterial Proteins↗
PubMed1984

Cytoplasmic transformation of Saccharomyces cerevisiae using purified mtDNA.

Transformation of cytoplasmic mutants of Saccharomyces cerevisiae proved to be successful using plasmid or intact mitochondria, but not when mtDNA was used as the transforming principle. The sphaeroplasts of two cytoplasmic mutants, respiratory deficient and erythromycin sensitive cells, were transformed using purified mtDNA from respiratory competent or erythromycin resistant donor strains.

Cytoplasm↗
PubMed1982

Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities.

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

Bacillus subtilis↗
PubMed1987

Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA.

Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

Bacillus thuringiensis↗
PubMed1988

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells.

UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.

DNA Repair↗
PubMed1993

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

Natural competence of Streptococcus 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 pneumococcal dfrA mutation has been mapped within a 500-bp DNA region.

Chromosome Mapping↗
PubMed2002

Opportunistic colonization of Ralstonia solanacearum-infected plants by Acinetobacter sp. and its natural competence development.

The behavior of the soil bacterium Acinetobacter sp. BD413 was monitored in Ralstonia solanacearum-infected and non-infected tomato plants after direct injection into the stem or natural infection by roots. In healthy plants, Acinetobacter sp. BD413 failed to colonize plant tissue. In plants infected simultaneously by the pathogen R. solanacearum,the Acinetobacter population increased linearly to about 3.1 x 10(7) cells per gram plant material and was maintained at a high level until the death of the plant. Moreover, Acinetobacter sp. BD413 was found to develop a competent state when multiplying in planta, indicating it could possibly be transformed by bacterial or plant DNA.

Acinetobacter↗
PubMed2005

The Mre11/Rad50/Xrs2 complex and non-homologous end-joining of incompatible ends in S. cerevisiae.

In Saccharomyces cerevisiae, the Mre11/Rad50/Xrs2 (MRX) complex plays important roles in both homologous and non-homologous pathways of DNA repair. In this study, we investigated the role of the MRX complex and its enzymatic functions in non-homologous repair of DNA ends containing incompatible end structures. Using a plasmid transformation assay, we found that mre11 and rad50 null strains are extremely deficient in joining of incompatible DNA ends. Expression of the nuclease-deficient Mre11 mutant H125N fully complemented the mre11 strain for joining of mismatched ends in the absence of homology, while a mutant of Rad50 deficient in ATP-dependent activities exhibited levels of end-joining similar to a rad50 deletion strain. Although the majority of non-homologous end-joining (NHEJ) products isolated did not contain microhomologies, introduction of an 8bp microhomology at mismatched ends resulted in microhomology-mediated joining in all of the products recovered, demonstrating that a microhomology exerts a dominant effect on processing events that occur during NHEJ. Nuclease-deficient Mre11p was less efficient in promoting microhomology-mediated end-joining in comparison to its ability to stimulate non-microhomology-mediated events, suggesting that Mre11p influences, but is not essential for, microhomology-mediated repair. When the linearized DNA was transformed in the presence of an intact homologous plasmid to facilitate gap repair, there was no decrease in NHEJ products obtained, suggesting that NHEJ and homologous repair do not compete for DNA ends in vivo. These results suggest that the MRX complex is essential for joining of incompatible ends by NHEJ, and the ATP-dependent activities of Rad50 are critical for this process.

Adenosine Triphosphate↗
PubMed1999

Expression of beta-defensin genes by human salivary glands.

This study investigated expression of genes encoding human beta-defensins 1 and 2 by human salivary glands. Tissues from surgical biopsies were collected fresh onto ice and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent and human beta-defensin messenger RNA detected by reverse transcriptase polymerase chain reaction amplification. DNA sequencing of amplified fragments, after ligation into pGEM-T Easy vector and transformation of competent Escherichia coli, confirmed identities of cloned fragments. Human beta-defensin 1 messenger RNA was detected in all 25 samples that generated amplifiable cDNA, as assessed using abl-specific primers. Three of 13 submandibular gland samples (two normal, one chronically inflamed), and 2 of 2 minor salivary gland samples (one normal, one chronically inflamed) expressed human beta-defensin 2 messenger RNA. All six parotid gland samples studied were negative for human beta-defensin 2 messenger RNA. Thus, human beta-defensin 1 gene expression occurred in all human major and minor salivary glands studied, whereas human beta-defensin 2 expression occurred only in a small number of gland samples.

Defensins↗
PubMed2006

Genomic distribution and functions of uptake signal sequences in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is naturally competent for transformation, with a transformation system similar to that of Haemophilus influenzae that preferentially takes up DNA bearing uptake signal sequences (USS) with the same 9-base USS core. This study examined the function of the extended 29-base USS, which comprises a highly conserved 1st region (containing the 9-base core) and 2nd and 3rd semi-conserved AT-rich regions, in transformation of A. actinomycetemcomitans. Transformation frequency was not affected by either location (in middle or at 5' end) or quantity (one or two) of USS in donor DNA. Relative transformation efficiencies (in comparison to the positive control) were 28-67 % for linear DNA with single-base mutations in the USS 1st region, and 47 % and 73 %, respectively, for linear DNA with USS that contained either a non-consensus 2nd or a non-consensus 3rd region. Plasmids with a stand-alone 1st or a stand-alone 2nd-3rd region exhibited 21 % and 6 % relative transformation efficiencies, respectively. It was also noted that A. actinomycetemcomitans and H. influenzae were similar in the frequencies and distribution patterns of USS in their genomes. In conclusion, all three regions of the extended 29-base USS are required for optimum transformation in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1966

Genetic Recombination of Transforming Deoxyribonucleic Acid Molecules with the Recipient Genome and Among Themselves in Protoplasts of Bacillus subtilis.

Hirokawa, Hideo (Southwest Center for Advanced Studies, Dallas, Tex.), and Yonosuke Ikeda. Genetic recombination of transforming deoxyribonucleic acid molecules with the recipient genome and among themselves in protoplasts of Bacillus subtilis. J. Bacteriol. 92:455-463. 1966.-Re-extraction of transforming deoxyribonucleic acid (DNA) from protoplasts of Bacillus subtilis is much more efficient than from intact competent cells. This facilitated the detection of physical recombination between donor and recipient DNA molecules, as indicated by a high cotransfer index of ind(+) and his(+) markers which were originally located in exogenous and endogenous DNA molecules, respectively. This recombinant DNA was extracted after 30 min of incubation of ind his(+) protoplasts with ind(+)his DNA, previously extracted from a corresponding mutant strain of B. subtilis. The intracellular formation of recombinant molecules (ind(+)his(+)) bearing markers from two different exogenous DNA species was also detected 15 min after exposure of ind his recipient protoplasts to a mixture of ind(+)his and ind his(+) donor DNA molecules. The unity of the recombinant molecule was ascertained by dilution experiments and by its being resistant to ribonuclease and trypsin treatment (but being sensitive to deoxyribonuclease). The formation of recombinant molecules showed an inverse kinetics to that of the intracellularly induced loss of linkage between the corresponding markers in the wild-type DNA, thus suggesting a breakage and reunion process which is also favored by the absence of DNA synthesis in the protoplasts and the effect of some specific inhibitors.

Journal Article↗
PubMed2004

Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential.

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

Animals↗
PubMed2006

[Cloning and expression of HLA-A*0201-BSP].

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.

Biotin↗
PubMed1996

A novel peptidoglycan-linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae.

A novel peptidoglycan-linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species-related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild-type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild-type strain. comL is a single-copy gene and maps downstream of the previously described comA gene. It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5' end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA'). In E. coli, cloned ComL can be labelled with [3H]-palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseria meningitidis as well as in several nonpathogenic Neisseria species.

Amino Acid Sequence↗
PubMed1986

Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not further cut during uptake, the endonucleolytic activity associated with entry of plasmid DNA appeared to act preferentially on circular DNA. Although linear plasmid DNA was taken up into a DNase-resistant state as efficiently as circular DNA, linear plasmid DNA transformed much less efficiently than circular plasmid DNA. These data suggest that during entry transforming plasmid DNA often is processed to double-stranded linear molecules; transformants may arise when some molecules are repaired to form circles. Occasional molecules which enter as intact circles may also lead to transformants.

DNA, Bacterial↗
PubMed1997

DNA binding and transcriptional enhancement by purified TCDD.Ah receptor complex.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional enhancer which mediates the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related chemicals and which may have a role in the normal development of some tissues. We have previously reported the purification of the transformed TCDD.receptor complex from rat liver cytosol based on binding to its dioxin-responsive enhancer sequence (DRE) and that it comprises the AhR ligand-binding monomer and its dimerization partner, ARNT. The present studies were designed to compare the DRE-binding characteristics of the purified receptor with the cruder preparations that are commonly used and ultimately to determine whether the purified receptor complex itself (in the absence of additional cytosolic or nuclear factors) is capable of enhancing transcription in an in vitro system. The purified AhR retained in vitro DRE binding activity in the presence of carrier protein and dithiothreitol, and its affinity for the DRE oligonucleotide was equivalent to that of the other receptor preparations (crude and partially purified cytosolic and crude nuclear). When the ligand.receptor complex was bound to a DRE oligonucleotide containing BrdU and then UV-irradiated, two proteins in each of the receptor preparations were found to crosslink to BrdU-DRE, and we concluded that they are the AhR monomer and ARNT protein. All receptor preparations also gave a similar footprint of interaction with G-residues within the DRE consensus sequence, as assessed by methylation interference. Furthermore, purified and partially purified receptors were able to stimulate transcription from a DRE-containing template in a cell-free system in the presence of HeLa cell nuclear extract. Transcriptional enhancement was receptor dose-dependent, TCDD-dependent, and specific for the DRE sequence upstream of the promotor in our template construct. These data document for the first time that a purified TCDD.Ah receptor complex retains both specific DNA binding and transcriptional activities. This observation constitutes an important step toward understanding the mechanism of gene regulation by TCDD since it implies that the transformed receptor.ligand complex itself is competent as a transcriptional enhancer without a requirement for other factors.

Animals↗