Biomedical subjects

S A Lacks

Publications and source records attributed to S A Lacks.

At least 19 recordsLinked to original sources

PubMed1995

An extended -10 promoter alone directs transcription of the DpnII operon of Streptococcus pneumoniae.

The genetic cassette encoding the DpnII restriction-modification system of Streptococcus pneumoniae gave transcription products of approximately 2.7 and 1.8 kilobases. The larger, mRNA1, covered both of the methylase genes, dpnM and dpnA, and the endonuclease gene dpnB; the smaller, mRNA2, covered only the dpnA and dpnB genes. Transcription of mRNA1 was shown to begin at the translation start site for dpnM, thereby producing an mRNA without any apparent ribosome-binding site for translation of the DpnM methylase. The promoter for mRNA1 was shown by base substitution and deletion analysis to consist of an extended -10 site, TaTGgTATAAT, with no required -35 site. A possible promoter further upstream with close matches to a -35 site and a nonextended -10 site was not used. A survey of 36 proven and putative promoters used by S. pneumoniae revealed that 61% of them contained the full -10 extension, although, other than the dpnM promoter, they matched at a -35 site, as well. It appears that, unlike those found in Escherichia coli, S. pneumoniae promoters frequently require an extended -10 site, and such a site can function naturally without a -35 site.

Base Sequence↗

PubMed1995

Possible regulation of DNA methyltransferase expression by RNA processing in Streptococcus pneumoniae.

Atypical ribosome-binding sites lacking Shine-Dalgarno sequences appear to be used for translation of the DpnM and DpnA DNA methyltransferases of the DpnII restriction system. Preliminary results indicate that the 5'-endpoints of DpnII system mRNAs result from degradation of the original transcript. These tentative findings serve as the basis for a possible regulatory model that would accommodate the DpnII cassette either as a single copy in the chromosome or on a multicopy plasmid.

Base Sequence↗

PubMed1995

A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae.

Two genes, sulB and sulC, in a folate biosynthetic gene cluster of Streptococcus pneumoniae were identified after determination of the DNA sequence between two previously reported genes, sulA and sulD, in a cloned segment of chromosomal DNA containing a mutation to sulfonamide resistance. The gene products, SulB and SulC, correspond to polypeptides of 49 and 21 kDa, respectively. SulC has GTP cyclohydrolase activity and catalyzes the first step in the folate biosynthetic pathway. SulB apparently has dihydrofolate synthetase activity in that it complements a folC mutant of Escherichia coli and thus catalyzes the last step in the pathway. Prior work showed that SulA, a dihydropteroate synthase, and SulD, a bifunctional enzyme, catalyze three intervening steps. Mapping of the mRNA transcribed from the operon was consistent with its beginning at a promoter with a -35 site (gTGtCc) and an extended -10 site (T-TG-TAaAAT) and its termination at the end of a hairpin structure, which would give a transcript 3,745 nucleotides in length. SulC showed a considerable conservation of sequence by comparison with proven or putative GTP cyclohydrolases from four unrelated species, with 38 to 53% of the residues being identical. A similar comparison of SulB with dihydrofolate synthetases showed an identity of only 26 to 37%. Overall, comparisons of the five folate biosynthetic enzymes in different species suggest that S. pneumoniae is related more closely to other gram-positive bacteria, less closely to eucaryotes, and least closely to the gram-negative E. coli. The varied arrangements of folate biosynthetic genes in different species imply an early evolutionary period of fluidity in genomic rearrangement.

Amino Acid Sequence↗

PubMed1995

Lethal and mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine potentiated by oxidized glutathione, a seemingly harmless substance in the cellular environment.

Both the lethal and the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on cells of Streptococcus pneumoniae were greatly potentiated by a component of yeast extract added to the cellular environment. This component was found to be an oxidation product of glutathione, glutathione disulfide (GSSG). At low concentrations in the medium, both GSSG and glutathione potentiated MNNG action, but at high concentrations, glutathione (and other sulfhydryl compounds) abolished the effect. Point mutations in a cellular gene conferred resistance to the potentiating effect, and they blocked uptake of either GSSG or glutathione into the cells as well. This gene apparently encodes a component of the system for glutathione transport in S. pneumoniae. The mechanism by which GSSG, an apparently innocuous substance in the environment, renders low levels of MNNG genotoxic and cytotoxic thus depends on its transport into the cell, where it is reduced by glutathione reductase and then activates intracellular MNNG. Also, it was observed that mutants of S. pneumoniae defective in DNA mismatch repair are more resistant to MNNG than are wild-type cells by a factor of 2.5.

Biological Transport↗

PubMed1994

Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1.

The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5'-to-3' exonuclease activities of DNA polymerase I function in plasmid replication.

DNA Polymerase I↗

PubMed1992

The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae.

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.

DNA Mutational Analysis↗

PubMed1992

Streptococcus pneumoniae DNA polymerase I lacks 3'-to-5' exonuclease activity: localization of the 5'-to-3' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.

Bacterial Proteins↗

PubMed1991

The polymerase domain of Streptococcus pneumoniae DNA polymerase I. High expression, purification and characterization.

The 3'-terminal two-thirds of the Streptococcus pneumoniae polA gene was cloned in an Escherichia coli genefusion vector with inducible expression. The resulting recombinant plasmid (pSM10) directs the hyperproduction of a polypeptide of 70.6 kDa corresponding to the C-terminal fragment of pneumococcal DNA polymerase I. Induced cells synthesized catalytically active protein to the extent of 7% of the total soluble protein in the cells. The polymerase fragment was purified to greater than 90% homogeneity with a yield of 1.5 mg pure protein/l culture. The protein has DNA polymerase activity, but no exonuclease activity. The enzyme requires a divalent cation (MgCl2 or MnCl2) for polymerization of DNA. Comparison of the mutant and wild-type pneumococcal polymerases shows that the construction did not affect the enzymatic affinity for the various substrates. The mutant protein, like its parent DNA polymerase I, exhibited an intermediate level of activity with primed single-stranded DNA. At high molar ratio of enzyme/DNA substrate, the polymerase fragment catalyzes strand displacement and switching after completing the replication of a primed single-stranded M13 DNA molecule.

Amino Acid Sequence↗

PubMed1991

Sequential cloning by a vector walking along the chromosome.

A procedure was devised for sequential cloning of chromosomal DNA by cyclical integration and excision of a plasmid vector so that slightly overlapping chromosomal segments are successively cloned. The method depends on circular integration of the vector into the chromosome of a host nonpermissive for its replication, and on excision and reduction of a recombinant plasmid by use of an appropriately designed set of restriction enzyme sites in the vector. A vector suitable for cloning in Escherichia coli was constructed by combining a segment of pBR322 with a gene encoding chloramphenicol resistance expressible in many species. Sequential cloning was demonstrated in Streptococcus pneumoniae by extending a previously cloned segment of the region of the chromosome encoding maltosaccharide utilization by 8 kb in three cycles of cloning. Accuracy of the method was confirmed by hybridization of cloned DNA with chromosomal restriction fragments. It is pointed out that the similarity of the requisite genetic processes in bacteria and yeasts should allow use of the method for sequential cloning of yeast chromosomal DNA and of human or other mammalian DNA in artificial chromosomes of yeast.

Animals↗

PubMed1991

Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae.

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A.T----G.C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G.C----A.T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G.C----A.T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A.T----G.C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.

Base Composition↗

PubMed1990

Genetic and structural characterization of endA. A membrane-bound nuclease required for transformation of Streptococcus pneumoniae.

The endA gene encoding the membrane nuclease of Streptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying the endA+ gene complemented cells with endA- in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end. In vitro synthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity of endA and the effect of various insertions and deletions, it appears that endA is the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.

Amino Acid Sequence↗

PubMed1990

DNA sequence of folate biosynthesis gene sulD, encoding hydroxymethyldihydropterin pyrophosphokinase in Streptococcus pneumoniae, and characterization of the enzyme.

A cloned segment of the chromosome of Streptococcus pneumoniae, in which mutations to sulfonamide resistance occur, contains several genes encoding enzymes for folate biosynthesis. Determination of the DNA sequence of parts of this segment and identification of a putative promoter and terminator of transcription indicate an operon composed of four genes. The first, sulA, encodes the enzyme dihydropteroate synthase. The functions of the second and third possible genes, sulB and sulC, are not known. The last gene, sulD, encodes a 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase. The product of this enzyme is the substrate for dihydropteroate synthetase. The enzyme protein was partially purified and shown to consist of a single subunit of 31 kilodaltons, encoded by sulD. On the basis of gel filtration behavior, the native protein appears to be a trimer or tetramer. Subcloning of the sulD gene in an Escherichia coli expression vector increased expression of the pyrophosphokinase 1,000-fold over the level produced by a single copy of the chromosomal gene.

Amino Acid Sequence↗

PubMed1989

Crystallization of the DpnM methylase from the DpnII restriction system of Streptococcus pneumoniae.

Three proteins, two DNA methylases and an endonuclease, from the DpnII restriction system of Streptococcus pneumoniae recognize the DNA sequence 5' GATC 3' but have very different amino acid sequences, which make them interesting subjects for structural determination. A purification procedure was developed that conveniently yields milligram amounts of the DpnM methylase. The DpnM protein tends to precipitate at reduced ionic strength, and this property was exploited to yield well-formed bipyramidal crystals. By X-ray diffraction, the crystals of DpnM were found to be orthorhombic, with cell dimensions a = 56.9 A, b = 68.2 A, c = 84.5 A; systematic absences identify the space group as P2(1)2(1)2(1). Diffraction extends beyond 3 A, so the crystals may allow structural determination at atomic resolution.

Crystallization↗

PubMed1989

Characterization of the polA gene of Streptococcus pneumoniae and comparison of the DNA polymerase I it encodes to homologous enzymes from Escherichia coli and phage T7.

The DNA sequence of the polA gene of Streptococcus pneumoniae was determined, and the DNA polymerase I encoded by the gene was purified to homogeneity. Determination of the amino-terminal amino acid sequence of the protein showed it to correspond to the Mr 99,487 polypeptide predicted from the nucleotide sequence. The mRNA transcript was mapped with respect to its sites of initiation and termination in the DNA. Inasmuch as the mRNA begins only two nucleotides before the first codon, it lacks a typical ribosome binding site. Nevertheless, 500 molecules of the protein are produced per cell. Like the Escherichia coli DNA polymerase I, the protein from S. pneumoniae has 5'- and 3'-exonuclease as well as polymerase activities, and it also undergoes a single cleavage on mild proteolysis. Alignment of the two different polymerase I proteins shows 40% of their amino acid residues to be identical. Homology is evident also with the DNA polymerase encoded by phage T7 gene 5. In addition, the amino-terminal regions of the bacterial polymerase I proteins are homologous to the separate 5'-exonuclease protein encoded by phage T7 gene 6. Analysis of the patterns of homology suggests that the bacterial polymerase I may represent the accretion of at least six separate genetic regions.

Base Sequence↗

PubMed1989

DpnA, a methylase for single-strand DNA in the Dpn II restriction system, and its biological function.

The two DNA-adenine methylases encoded by the Dpn II restriction gene cassette were purified, and their activities were compared on various DNA substrates. DpnA was able to methylate single-strand DNA and double-strand DNA, whereas DpnM methylated only double-strand DNA. Although both enzymes act at 5'-GATC-3' in DNA, DpnA can also methylate sequences altered in the guanine position, but at a lower rate. A deletion mutation in the dpnA gene was constructed and transferred to the chromosome. Transmission by way of the transformation pathway of methylated and unmethylated plasmids to dpnA mutant and wild-type recipients was examined. The mutant cells restricted unmethylated donor plasmid establishment much more strongly than did wild-type cells. In the wild type, the single strands of donor plasmid DNA that enter by the transformation pathway are apparently methylated by DpnA prior to conversion of the plasmid to a double-strand form, in which the plasmid would be susceptible to the Dpn II endonuclease. The biological function of DpnA may, therefore, be the enhancement of plasmid transfer to Dpn II-containing strains of Streptococcus pneumoniae.

Base Sequence↗

PubMed1989

Transformation-deficient mutants of piliated Neisseria gonorrhoeae.

Seven transformation-deficient mutants of piliated, competent Neisseria gonorrhoeae were isolated by screening them for their inability to be transformed by chromosomal DNA after chemical mutagenesis. Three distinct classes of mutants were obtained, each of which was piliated, as determined by electron microscopy. One class exhibited abnormal colony morphology and was unable to take up DNA into a DNase-resistant state. A second class was morphologically normal and took up DNA into a DNase-resistant state normally, but was deficient in both chromosomal and plasmid transformation; mutations in these mutants may affect entry of DNA into the cell proper. A third class was similar to the second but was fully competent for plasmid transformation, suggesting that there was a defect in a late stage of chromosomal transformation.

Chromosomes, Bacterial↗

PubMed1989

Plasmid structural instability associated with pC194 replication functions.

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.

Bacterial Proteins↗

PubMed1989

The exoA gene of Streptococcus pneumoniae and its product, a DNA exonuclease with apurinic endonuclease activity.

The gene encoding the major DNA exonuclease of Streptococcus pneumoniae, exoA, was cloned in a streptococcal host vector system. Its location was determined by subcloning and by insertion mutations. Transfer of a DNA segment containing the gene to an Escherichia coli expression vector showed that exoA was the structural gene for the enzyme and that it was adjacent to its promoter. DNA sequence determination indicated that the gene encoded a protein, ExoA, of molecular weight 31,263. Under hyperexpression conditions, the ExoA protein constituted 10% of total cellular protein. In addition to previously demonstrated 3' to 5' exonuclease and 3'-phosphatase activities, ExoA was shown to make single-strand breaks at apurinic sites in DNA. Its enzymatic activities are thus similar to those of exonuclease III of E. coli and other gram-negative bacteria. The nucleotide sequence of exoA revealed it to be homologous to xth of E. coli, with 26% identity of amino acid residues in the predicted proteins. So far, no null chromosomal mutants of exoA have been obtained, and the biological function of ExoA remains unknown.

Amino Acid Sequence↗