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PubMed1973

Transformation and transfection in lysogenic strains of Bacillus subtilis 168.

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.

Bacillus subtilis↗
PubMed1995

Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916.

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of "corncobs", a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.

Adhesins, Bacterial↗
PubMed1983

Regulation of cell proliferation by epidermal growth factor.

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.

Allosteric Regulation↗
PubMed2003

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

Journal Article↗
PubMed1969

Identification of Bacillus subtilis NRRL B-3275 as a strain of Bacillus pumilus.

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.

Amino Acids↗
PubMed1998

Identification and characterization of a novel competence gene, comC, required for DNA binding and uptake in Acinetobacter sp. strain BD413.

A gene (comC) essential for natural transformation was identified in Acinetobacter sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A comC mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.

Acinetobacter↗
PubMed2004

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Agrobacterium tumefaciens↗
PubMedDate not supplied

Genes for breakfast: the have-your-cake-and-eat-it-too of bacterial transformation.

Bacterial transformation, in which cells take up and recombine free strands of DNA, is the simplest process thought to have evolved for genetic exchange, i.e., because of the potential benefits of producing progeny with recombinant genotypes. However, two other functions are equally plausible: acquisition of intact DNA strands to use for recombinational repair of DNA damage, and acquisition of the nutrients contained in DNA molecules. Although the recombinant progeny produced by transformation can be beneficial, the success of genes causing transformation is limited by other factors, especially by the genetic quality of DNA derived from dead cells. Our recent experiments in the naturally transformable bacteria Haemophilus influenzae and Bacillus subtilis suggest that the DNA-repair hypothesis is unlikely to be correct. In H. influenzae, transformation does not detectably increase the cells' ability to survive DNA damage. More importantly, we have found that, although competence (the ability to take up DNA) is induced by nutritional limitation in both H. influenzae and B. subtilis, it is not induced by DNA damage in either. Thus we favor the hypothesis that transformation evolved as a nutrient-uptake system, especially because unrelated DNA is abundant in the environments of many naturally transformable bacteria.

Bacteria↗
PubMed2004

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae.

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.

Bacterial Proteins↗
PubMed1992

Plasmid profiles of Acinetobacter and Enterobacter species of hospital origin: restriction endonuclease analysis of plasmid DNA and transformation of Escherichia coli by R plasmids.

A total of 37 multi-resistant strains (20 Acinetobacter calcoaceticus and 17 Enterobacter cloacae) were isolated from patients of the Intensive Care Units. All the isolates were examined for resistance to a battery of antimicrobial agents by the disk diffusion method. Plasmid profiles and restriction endonuclease analysis of plasmid DNA by EcoR1 revealed the spread of one A. calcoaceticus and two E. cloacae endemic strains. Transformation experiments on Escherichia coli competent cells by three plasmids established the presence of R plasmids in the multi-resistant isolates.

Acinetobacter↗
PubMed2005

[Enrichment of giant panda microsatellite markers using dynal magnet beads].

The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

Animals↗
PubMed2003

ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae.

Natural competence for genetic transformation in Streptococcus pneumoniae is achieved directly by specific proteins that are involved in DNA uptake and chromosomal recombination, and is regulated indirectly by a quorum-sensing system encoded by two loci, comAB and comCDE. The alternative sigma factor, ComX, is thought to be the unique link between quorum sensing and competence-specific genes. To test this hypothesis, we replaced the quorum-sensing inducible promoter (PQ) of the comX gene with either a constitutive promoter (PC) or a raffinose-inducible promoter (PR), so that comX transcription would be independent of quorum sensing. Surprisingly, both competence and expression of late genes, such as ssbB, cglA or celB, were found to depend on CSP in these mutants. An unknown, CSP-dependent regulator was needed when comX was expressed from these ectopic promoters, and it appears to act post-transcriptionally. However, when a multicopy nisin-inducible ComX-overexpressing plasmid was introduced, pneumococcal cells developed competence in the presence of nisin even despite deletion of comE. At 1% of the normal protein peak level, ComX protein stimulated competence without the participation of the pheromone response circuit. Thus, ComX is a unique link to competence-specific genes, but depends on multiple outputs of quorum sensing for maximal expression.

Bacterial Proteins↗