Biomedical subjects
Publications and source records attributed to D B Clewell.
Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.
pAD1 is a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis that encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. The response is regulated by a cluster of genes that includes a positive regulatory determinant, traE1, able to activate key structural genes involved in the conjugative process. A negative regulatory determinant, traA, affects the expression of traE1 and is sensitive to the pheromone signal. Between the two determinants is a gene, iad, which encodes a small peptide, iAD1, a competitive inhibitor of cAD1. The determinants (traE1-iad-traA) are oriented such that iad and traE1 are transcribed in the same direction, opposite that of traA. Transcription of iad and traA starts between these determinants and moves outward in each case. A recent report from our laboratory, dealing with transcriptional fusions in the traE1-iad region (L. T. Pontius and D. B. Clewell, J. Bacteriol. 174:3152-3160, 1992), indicated that traE1 expression may be dependent on transcriptional read-through of a terminator(s) between iad and traE1. The present report provides direct analyses of relevant RNA species before and during induction and shows that indeed transcriptional read-through from iad is important in the initial expression of traE1. However, the data show that once traE1 is activated, it can then be expressed independently, probably because of TraE1 activating its own promoter. This view is also supported by genetic complementation studies. In addition, DNA binding studies with TraA showed that the protein binds to the promoter of iad. Binding of TraA to the region between iad and traE1 could not be detected; however, the involvement of TraA in influencing transcription termination in this region is still not ruled out, since additional factors could be involved. A model for the regulation of the pheromone response is presented.
A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.
Phase variation of Streptococcus gordonii between high (Spp+) and low (Spp-) levels of glucosyltransferase (GTF) activity resulted in the greater adhesion of Spp- strains to saliva-coated hydroxyapatite (S-HA) in a washed-cell adhesion test. Specific GTF mutants did not show this response. Although washed Spp+ cells produced 5-fold or more glucan from sucrose than Spp- cells did under the conditions of the adhesion test, sucrose elevated the adhesion of both phenotypes to hydroxyapatite (HA) equally, but had no effect on adhesion to S-HA. This effect was not sucrose-specific, however, because equimolar amounts of other carbohydrates and NaCl elevated adhesion of both Spp types to levels similar to those seen with sucrose. Adhesion did not correlate with relative changes in cell hydrophobicity. These results suggest that, in addition to changes in GTF activity, other changes relevant to adhesion may occur during Spp phase variation.
The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 of Enterococcus faecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.
The Enterococcus faecalis plasmid pAD1 undergoes conjugative transfer in response to cAD1, a peptide sex pheromone emitted by potential bacterial recipients. Regulation of pAD1 transfer involves a number of plasmid-encoded determinants:iad, which determines a peptide-competitive inhibitor iAD1; signal sensing and transducing elements; and negative and positive regulators. The key positive regulator(s) of the pheromone response is believed to be encoded within a segment designated the E region of the plasmid. In this study, we analyzed the nucleotide sequence and transcription within the E region. An open reading frame designated traE1 was identified; its inferred protein consists of 118 amino acids. Insertional mutagenesis of traE1 resulted in a complete loss in plasmid transfer capability. Analysis of Tn917-lac insertions giving rise to transcriptional lacZ fusions showed that traE1 is transcribed only under cAD1 inducing conditions. Analysis of additional lacZ fusions within the region provided some insight into the roles of potential regulatory signals within and around the nucleotide sequences reported here. A regulatory role appearing to involve read-through of certain key transcription termination sequences seemed evident.
Streptococcus gordonii Challis was previously shown to give rise to phase variants expressing high (Spp+) or low (Spp-) levels of extracellular glucosyltransferase (GTF) activity. Here, shotgun cloning of an S. gordonii Spp+ chromosomal digest resulted in a chimeric plasmid (pAM5010) able to complement the Spp- phenotype. In addition, introduction of pAM5010 into an Spp+ strain resulted in a 10-fold increase in GTF expression. Deletion analysis of pAM5010 identified a 1.2-kb DNA segment which exhibited the same functional properties as pAM5010. Nucleotide sequence analysis of this region revealed a gene approximately 1 kb in size. The gene was designated rgg. Disruption of the chromosomal rgg gene open reading frame in an Spp+ strain resulted in strain DS512, which displayed an Spp(-)-like phenotype and had 3% of wild-type GTF activity. A plasmid containing the rgg gene was able to complement the DS512 phenotype and significantly increase GTF expression above wild-type levels. Sequence analysis and other data showed that the S. gordonii GTF determinant, designated gtfG, is located 66 bp downstream of the rgg gene. The sequence also revealed interesting inverted repeats which may play a role in the regulation of gtfG. We conclude that rgg positively regulates the expression of GTF and influences expression of the Spp phenotype.
The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.
Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.
The Enterococcus faecalis plasmid pAD1 conjugatively transfers in response to a sex pheromone, cAD1, excreted by potential recipient cells. A key determinant responsible for regulation of pAD1 transfer is traA, which encodes a negative regulator also believed to function in signal sensing. In this study, we analyzed the nucleotide sequence and transcription of traA. A protein of 319 amino acids with a molecular weight of 37,856 was inferred and found to exhibit limited homology with several DNA-binding proteins. Analysis of Tn917-lac insertions resulting in transcriptional lacZ fusions within the 3' end of the traA transcript showed that it overlaps slightly with a convergently-transcribed C-region transcript. Insertional mutations affecting TraA repressor function and signal sensing functions were localized.
RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.
Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pAD1 is enhanced by the sex pheromone cAD1, which is excreted from recipient cells. A membrane-anchored 137 kDa protein is a pAD1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS): it indicates that asal is cleaved specifically between K-510 and G-511.
Aeration of plasmid-free Enterococcus faecalis strains resulted in an 8- to 16-fold decrease in sex pheromone cAD1 activity in culture filtrates. Levels of two unrelated pheromones, cPD1 and cAM373, were unaffected by culture aeration. Aeration also resulted in a decrease in the expression of conjugative transfer functions observed in cells containing pAD1 traB mutations, verifying a link between traB function and pheromone "shutdown." Tests with a series of pAD1 mini-plasmids indicated that the product of the traB gene was involved in, but not sufficient for, pheromone shutdown; the cooperation of one or more other gene products encoded within the pheromone response control region was required.
A nucleotide sequence correction on the Enterococcus faecalis transposon Tn917 indicates that what was formerly thought to be two open reading frames (ORF5 and ORF6) is actually one reading frame (ORF5). The latter exhibits homology with the Tn3-family transposase determinants known as tnpA.
Enterococcus faecalis cells carrying the conjugative plasmid pAD1 undergo several related changes when induced by the sex pheromone cAD1. Included are the production of novel surface proteins, the formation of cellular aggregates in broth cultures, the ability to transfer the plasmid at high frequency in broth matings, and the change from a soft to a "dry" colony morphology. Spontaneous, constitutively dry colony (Dryc) variants of E. faecalis (pAD1) were found to arise at a frequency of 10(-4)-10(-2). Dryc phase variants constitutively expressed aggregation and plasmid transfer functions typically expressed only under cAD1-inducing conditions. Reversion of Dryc variants to a cAD1-inducible phenotype (Dry+) occurred at a similar frequency. Tn917-lac mutagenesis of regions of pAD1 previously shown to be involved in plasmid transfer revealed that in Dry+ cells these regions were transcribed only when the inducer, cAD1, was present. In Dryc variants the regions were transcribed constitutively. A pAD1 miniplasmid containing determinants regulating cAD1 inducible plasmid transfer and a cAD1-inducible lacZ transcriptional fusion displayed phase variation in LacZ expression at a rate similar to the Dry+/Dryc phase variation. These results suggest that the site of mutation(s) resulting in the Dryc phenotype is within the regulation-related region of pAD1. Complementation tests showed that this region, when supplied in trans, complemented the Dryc phenotype. Phase variation affecting mating functions represents an alternative (pheromone independent) method of regulating pAD1 transfer.
When sucrose is provided as a substrate for glucosyltransferase (GTF), Spp+ cells of the oral bacteria Streptococcus gordonii grow embedded in an insoluble glucan mass associated with surfaces. Spp- phase variants with lower GTF activity, which either arise from or are grown with Spp+ cells, segregate preferentially as unattached cells in the culture supernatants. Conversely, Spp+ revertants preferentially accumulate on surfaces. GTF phase variation, therefore, may facilitate the dispersion of S. gordonii cells throughout the oral cavity.
The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.
Streptococcus gordonii exhibits a phase variation involving expression of high (Spp+) or low (Spp-) glucosyltransferase activity. The related bacterial accumulation on hydroxyapatite (HA) and saliva-coated HA surfaces was examined and found to be significant. Spp+ cells growing anaerobically in a defined medium utilize about 30% of the glucose available from sucrose to make insoluble glucans. These glucans formed cohesive masses on HA beads, which contained 80 to 90% of the total bacteria. The bacterial polymer mass had a volume of about 40 microns3 and contained more than 5 x 10(10) viable cells per cm3. In the absence of sucrose, the beads were saturated by 1 x 10(8) to 2 x 10(8) Spp+ cells. Spp- bacteria, which make 30-fold less glucan than do Spp+ bacteria, did not accumulate on surfaces in numbers significantly above the saturation level of 1 x 10(8) to 2 x 10(8) cells in the presence or absence of sucrose. Insoluble glucan synthesized by Spp+ cells from sucrose also enabled these bacteria to accumulate on saliva-coated HA seven times more effectively than the Spp- cells and 10 times more effectively than the Spp+ cells grown in medium without sucrose.