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PubMed2001

Molecular characterization of the pneumococcal teichoic acid phosphorylcholine esterase.

A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analysis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain, a structural feature that had not been described for the published choline-binding proteins. The carboxy-terminal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically characterized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistinguishable from the parental strains when whole-mounted cells were observed under the electron microscope and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and M31.

Amino Acid Sequence↗
PubMed1972

Effect of D-cycloserine on transformation in group H streptococcus, strain Challis.

d-Cycloserine (d-CS), a selective inhibitor of bacterial cell wall biosynthesis, inhibited transformation in group H streptococcus, strain Challis, by preventing the development of the competent state. The incubation of strain Challis cells with d-CS resulted in the production of a substance which inhibited the action of competence factor on these cells. d-CS had an enhancing effect on transformation when deoxyribonucleic acid uptake or phenotypic expression was allowed to occur in its presence.

Cell Wall↗
PubMed1989

Lack of correlation between sensitivity to growth inhibition and receptor number for transforming growth factor beta in human squamous carcinoma cell lines.

Four human squamous carcinoma cell lines, SCC-9, SCC-12F, SCC-15G, and SCC-25, were examined for sensitivity to the growth-inhibitory and differentiation-inducing effects of transforming growth factor beta (TGF-beta). None of the four cell lines was induced to differentiate, as measured by staining for keratin and by competence of cells to form cross-linked envelopes, by concentrations of TGF-beta as high as 1000 pM. TGF-beta did not inhibit DNA synthesis or proliferation of SCC-12F cells in monolayer culture. Monolayer growth and DNA synthesis of SCC-15G and SCC-25 cells were markedly inhibited by 10 pM TGF-beta, with maximal inhibition between 10 and 100 pM. Inhibition of SCC-15G cells was apparent as early as 8 h after addition of TGF-beta, but inhibition of SCC-25 cells was not measurable until 2 days. Growth inhibition of SCC-15G cells was completely reversible, whereas inhibition of SCC-25 cells was irreversible. When growth of cells was measured in a defined medium supplemented with 0.5% fetal bovine serum, TGF-beta inhibited both the monolayer and clonal growth of SCC-15G cells. The monolayer growth, but not the clonal growth, of SCC-25 cells was inhibited. Growth of SCC-9 cells in confluent cultures was slightly inhibited by TGF-beta, while growth in subconfluent cultures was unaffected. All four cell lines, when assayed for binding of 125I-labeled TGF-beta, displayed high-affinity (KD = 2-44 pM) binding sites. These sites were present in very low numbers (less than 2000 sites/cell) in SCC-9, SCC-15G, and SCC-25 cells. SCC-12F cells contained 2000-5500 high-affinity sites/cell. Thus, a lack of sensitivity to growth inhibition by TGF-beta does not necessarily correlate with an inability to bind the growth factor to specific cell surface receptors. The possibility remains that altered sensitivity to TGF-beta may be due to loss of or changes in the relative proportions of the various TGF-beta receptor types. TGF-beta is believed to play a primary role in control of normal cellular growth and differentiation, and the study of cells with defective responses to this growth factor should help to identify some of the critical points within the growth cycle at which malignant cells may escape normal regulatory controls. The differential responses of these four human squamous carcinoma cell lines to TGF-beta should provide a useful model for studying such control mechanisms.

Carcinoma, Squamous Cell↗
PubMed1990

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT.

The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.

Bacillus subtilis↗
PubMed1986

Transmission of (LTR, v-src, LTR) without recombination with a helper virus.

We have analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1.

Animals↗
PubMed2001

Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system.

Helicobacter pylori (Hp), a Gram-negative bacterial pathogen and aetiologic agent of gastroduodenal disease in humans, is naturally competent for genetic transformation. Natural competence in bacteria is usually correlated with the presence of type IV pili or type IV pilin-like proteins, which are absent in Hp. Instead, we recently identified the comB operon in Hp, carrying four genes tentatively designated as orf2, comB1, comB2 and comB3. We show here that all ComB proteins and the 37-amino-acid Orf2 peptide display significant primary sequence and structural homology/identity to the basic components of a type IV secretion apparatus. ComB1, ComB2 and ComB3, now renamed ComB8, ComB9 and ComB10, correspond to the Agrobacterium tumefaciens VirB8, VirB9 and VirB10 proteins respectively. The peptide Orf2 carries a lipoprotein motif and a second cysteine residue homologous to VirB7, and was thus designated ComB7. The putative ATPase ComB4, encoded by the open reading frame hp0017 of strain 26695, corresponds to virB4 of the A. tumefaciens type IV secretion system. A Hp comB4 transposon insertion mutant was totally defective in natural transformation. By complementation of a Hp DeltacomB deletion mutant, we demonstrate that each of the proteins from ComB8 to ComB10 is absolutely essential for the development of natural transformation competence. The putative lipoprotein ComB7 is not essential, but apparently stabilizes the apparatus and modulates the transformation efficiency. Thus, pathogenic type I Hp strains contain two functional independent type IV transport systems, one for protein translocation encoded by the cag pathogenicity island and one for uptake of DNA by natural transformation. The latter system indicates a possible novel mechanism for natural DNA transformation in bacteria.

Amino Acid Sequence↗
PubMed2005

Gene expression profiling of cervical tissue during physiological cervical effacement.

OBJECTIVE: The softening and dilation of cervical tissue during parturition requires a rapid reorganization of extracellular matrix and cellular interactions. The purpose of this study was to gain insight into the complex transformational changes in gene expression that lead to cervical effacement. METHODS: Cervical biopsies from effaced cervices of 10 women undergoing spontaneous vaginal delivery and from competent cervices of 10 women undergoing primary cesarean delivery were collected at 37-41 weeks of gestation and subjected to differential complementary DNA (cDNA) microarray analysis. Gene expression results were validated by real-time polymerase chain reaction (PCR). RESULTS: In a cDNA array that enables the analysis of the differential gene expression of more than 600 genes, the messenger (m)RNA expression of 40 genes increased more than 2.5-fold during cervical ripening. The majority of these genes encode cytokines, transcription factors, and cell-matrix-associated proteins. The mRNA expression of 6 genes decreased to less than 0.5-fold. The remaining 556 genes were not significantly altered. Real-time PCR analysis performed for selected, highly up-regulated genes confirmed our cDNA array findings. CONCLUSION: Complete cervical effacement is associated with a characteristic and profound alteration in the gene expression profile of cervical cells. We hypothesize that an understanding of the molecular events that accompany physiological cervical dilation is pivotal to an understanding of pathological conditions such as premature delivery and postterm pregnancy.

Biopsy↗
PubMed1996

Natural transformation of Streptococcus crista.

Over the years Streptococcus gordonii (sanguis) Challis has become the workhorse of genetic manipulations for the sanguis group of oral streptococci. This is because strain Challis was shown in early studies to be highly naturally competent for transformation. However, Challis is not usually the most appropriate strain to use in studies which focus on oral microbial adherence. We report that other members of the newly reorganized sanguis group, particularly within the species S. crista, display reasonable transformation frequencies, with both plasmid and chromosomal DNA, if transformed at the appropriate time during the growth curve. The ability to transform S. crista may be especially important for genetic studies of biological properties that appear to be limited to these specific streptococcal strains.

Genetic Vectors↗
PubMed1997

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci.

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis. For class I pili found in both Neisseria gonorrhoeae and N. meningitidis, PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N.gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.

Amino Acid Sequence↗
PubMed1982

Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture.

A growth-factor-like substance capable of inducing nontransformed mouse AKR-2B, rat NRK, and EGF-receptorless mouse NR6 cells to form progressively growing colonies in soft agar was identified in acid/ethanol extracts of 17-day mouse embryos. This "mouse embryo factor" (MEF) is similar to previously described transforming growth factors in that it is capable of stimulating DNA synthesis and conferring a reversible transformed morphology on nontransformed cells in vitro. Passage of crude embryo extracts over a Bio-Gel P-60 column gave a major peak of soft agar growth-stimulating activity in the 15,000 molecular weight range with a minor peak at about 22,000. This biological activity was sensitive to treatment with either trypsin or dithiothreitol, but was unaffected by heat (56 degrees C for 30 minutes or 100 degrees C for 3 minutes), indicating that the activity is due to a heat-stable polypeptide(s) with disulfide bonds. Separation of these polypeptide(s) by chromatography on carboxymethyl cellulose revealed two peaks of soft agar growth-stimulating activity which did not cochromatograph with a peak of epidermal growth factor receptor-competing activity. The similarities of this mouse embryo-derived growth factor to previously identified transforming growth factors suggest that both fetal development and neoplastic transformation may be affected by similar mechanisms.

Animals↗
PubMed2001

Single thyroid hormone receptor monomers are competent for co-activator-mediated transactivation.

Thyroid hormone receptor (T(3)R) belongs to the superfamily of nuclear receptors containing highly related transcription factors that transform an incoming signal in the form of a lipophilic hormone into an activation of the basal transcriptional machinery. Like many other nuclear receptors, T(3)R acts preferentially as a heterodimer with retinoid X receptor (RXR) but it also has the unique property of binding as a monomer to DNA. This study demonstrates that T(3)R monomers bind preferentially to AGGTCA binding motifs and are able to co-exist with T(3)R-RXR heterodimers in the presence of limiting amounts of RXR. DNA-bound T(3)R monomers efficiently contact all three members of the p160 co-activator family, which in turn boost T(3)R monomer-mediated transactivation. In solution T(3)R monomers take only one agonistic conformation (c2(LPD)), whereas bound to DNA they also stabilize, like T(3)R-RXR heterodimers, a second agonistic conformation (c1(LPD)). Conformation c2(LPD) seems to be of lower ligand sensitivity (10 nM), whereas, both in T(3)R-RXR heterodimers and in DNA-bound T(3)R monomers, c1(LPD) is already activated at a ligand concentration of 1 nM. Taken together, these results suggest that single T(3)R monomers are fully competent for ligand-induced transactivation and that their role in gene regulation by thyroid hormone might have been underestimated.

Animals↗
PubMed2003

Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development.

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50-200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.

Animals↗
PubMed1975

Stability of pathogenic colony types of Neisseria gonorrhoeae in liquid culture by using the parameters of colonial morphology and deoxyribonucleic acid transformation.

This investigation describes the surveillance of the colonial stability of the pathogenic type 1 from the gonococcal strain F62 to the nonvirulent types 3 and 4 in different liquid media. The maintenance of the colony types was monitored by the parameters of colonial morphology and deoxyribonucleic acid-mediated transformation. During growth in a complex medium, Mueller-Hinton broth, only 46.7% of the gonococcal population remained as type 1 after 12 h. The greatest change in the type 1 colony-forming units correlated with the decline in viable count. The conversion process could not be prevented by the continual maintenance of the gonococcus in logarithmic growth. The frequency of transformation from PRO(minus) (proline) to PRO(plus) was proportional to this decrease in type 1 colony-forming units. In contrast to Mueller-Hinton medium, the chemically defined minimal medium Gonococcal Genetic Medium (GGM) was capable of maintaining approximately 90% of the gonococcal population in the type 1 colonial form after 16 h of growth, despite a decrease in the viable count. Although the percentage of type 1 appeared to remain constant in GGM, the apparent transformation frequency increased approximately 24-fold from 0 to 12 h of growth. GGM appears to stimulate or maintain competence, as evidenced by an eightfold increase in transformation when cells are exposed to deoxyribonucleic acid in GGM as compared to Mueller-Hinton.

Culture Media↗
PubMed2000

ComP, a pilin-like protein essential for natural competence in Acinetobacter sp. Strain BD413: regulation, modification, and cellular localization.

We recently identified a pilin-like competence factor, ComP, which is essential for natural transformation of the gram-negative soil bacterium Acinetobacter sp. strain BD413. Here we demonstrate that transcription and synthesis of the pilin-like competence factor ComP are maximal in the late stationary growth phase, whereas competence is induced immediately after inoculation of a stationary-phase culture into fresh medium. Western blot analyses revealed three forms of ComP, one with an apparent molecular mass of 15 kDa, which correlates with the molecular mass deduced from the DNA sequence, one 20-kDa form, which was found to be glycosylated, and one 23-kDa form. The glycosylation of ComP was not required for its function in DNA binding and uptake. The 20-kDa form was present in the cytoplasmic membrane, the periplasm, and the outer membrane, whereas the 23-kDa form was located in the outer membrane and might be due to a further modification. Immunological data suggest that ComP is not a subunit of the pilus structures. Possible functions of ComP in the DNA transformation machinery of Acinetobacter sp. strain BD413 are discussed.

Acinetobacter calcoaceticus↗
PubMed1994

Three highly homologous membrane-bound lipoproteins participate in oligopeptide transport by the Ami system of the gram-positive Streptococcus pneumoniae.

Oligopeptides are an important source of nutrients, but can serve also as signals for intercellular communication. Oligopeptide-binding proteins seem likely to play a role both in oligopeptide transport and in communication processes. One such protein, AmiA, has been identified in Streptococcus pneumoniae. amiA is the first gene of an operon, ami, which encodes a multicomponent oligopeptide transporter belonging to the family of ABC transporters (or traffic ATPases). This transporter was the first system of this type described in Gram-positive bacteria. To investigate the role and the subcellular location of the putative oligopeptide-binding protein in a bacterium devoid of periplasm, AmiA null mutants were first constructed. None was affected for oligopeptide uptake by the Ami system. Since this apparent dispensability of AmiA could result from a functional redundancy, we looked for chromosomal genes encoding homologues of AmiA. Two homologous genes were identified by DNA-DNA hybridization at low stringency with an amiA probe. Both genes (aliA and aliB) were cloned and shown to encode putative lipoproteins highly homologous to AmiA (close to 60% amino acid identity). Examination of all combinations of amiA, aliA and aliB mutations indicated that these proteins have overlapping specificities toward oligopeptides. The triple mutant is as deficient for oligopeptide transport as mutants in the amiCDE or F genes, which demonstrates that an oligopeptide-binding component is absolutely required for transport by the Ami system. Metabolic labelling with [3H]palmitic acid and cell fractionation were used to demonstrate that the three proteins are indeed membrane-bound lipoproteins in S. pneumoniae. This supports our previous hypothesis that substrate-binding lipoproteins are functionally equivalent to the periplasmic substrate-binding component of ABC transporters of Gram-negative bacteria. Finally, the observation that competence for genetic transformation was drastically reduced in a particular AliB mutant suggests that oligopeptide sensing is important for triggering competence.

Amino Acid Sequence↗