Constitutive competence for genetic transformation in Streptococcus pneumoniae caused by mutation of a transmembrane histidine kinase.

Competence for DNA uptake and genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system. A competence-stimulating polypeptide (CSP) is secreted by the bacteria and acts back on the cells via a transmembrane histidine kinase. This enzyme phosphorylates a response regulator that activates synthesis of a SigH-like protein. The new sigma factor enables expression of a set of proteins transcribed from a novel promoter. A mutation called trt had been found that circumvented this regulation. The mutant cells are constitutively competent; that is, they can be transformed at low cell densities, in the presence of proteases that attack CSP, or during growth at low pH. In this work, cells containing trt were shown to be competent even in the presence of a comAB mutation that blocks secretion of CSP. The trt mutation was localized to comD, the gene encoding the transmembrane histidine kinase. A DNA segment of the trt mutant corresponding to comCDE was cloned, and it was shown to contain the trt mutation by its ability to confer constitutive competence. A two-step assay, which was based on transfer of trt to a wild strain and screening for transformability in the presence of trypsin, served to locate the trt mutation precisely. It corresponds to a GC-->AT transition, which changes Asp299 in the histidine kinase to Asn. This alteration in the carboxyl terminal half of the protein, which is cytoplasmically located and contains the phosphorylase activity, presumably alters the enzyme conformation so that it is permanently activated, independent of signals from the transmembrane domain. These results may help illuminate the mechanism by which external signals affect kinase action in two-component regulatory systems, and they may be of practical value in facilitating genetic studies by rendering pneumococcal strains permanently competent.

Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system.

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of streptococcus pneumoniae bound to S-adenosylmethionine.

BACKGROUND: . Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. RESULTS: . The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. CONCLUSIONS: . DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.

Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme.

The spi gene of Streptococcus pneumoniae was cloned and its nucleotide sequence was determined. It encodes a protein of 204 amino acids that is homologous to bacterial signal peptidase I proteins. The S. pneumoniae protein contains all of the conserved amino acid sequence motifs previously identified in this enzyme from both prokaryotic and eukaryotic sources. Sequence comparisons revealed several additional motifs characteristic of the enzyme. The cloned S. pneumoniae gene complemented an Escherichia coli mutant defective in its leader peptidase gene. Expression of the spi gene in S. pneumoniae appeared to be essential for viability. The cloned gene was shown to produce a polypeptide of approximately 20 kDa. Overproduction of the S. pneumoniae spi gene in an E. coli expression system gave a native protein product, soluble in the presence of a non-ionic detergent, which should be amenable to structural determination.

The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes.

A single RNase H enzyme was detected in extracts of Streptococcus pneumoniae. The gene encoding this enzyme was cloned and expressed in Escherichia coli, as demonstrated by its ability to complement a double-mutant rnhA recC strain. Sequence analysis of the cloned DNA revealed an open reading frame of 290 codons that encodes a polypeptide of 31.9 kDa. The predicted protein exhibits a low level of homology (19% identity of amino acid residues) to RNase HII encoded by rnhB of E. coli. Identification of the S. pneumoniae RNase HII translation start site by amino-terminal sequencing of the protein and of mRNA start sites by primer extension with reverse transcriptase showed that the major transcript encoding rnhB begins at the protein start site. Comparison of the S. pneumoniae and E. coli RNase HII sequences and sequences of other, putative bacterial rnhB gene products surmised from sequencing data revealed three conserved motifs. Use of these motifs to search for homologous genes in eucaryotes demonstrated the presence of rnhB genes in a yeast and a roundworm. Partial rnhB gene sequences were detected among expressed sequences of mouse and human cells. From these data, it appears that RNase HII is universally present in living cells.

Cloning and expression of pneumococcal genes in Streptococcus pneumoniae.

An overview of gene cloning in Streptococcus pneumoniae is presented. The advantages of such cloning, especially for pneumococcal genes, are enumerated. The molecular fate of DNA in transformation of S. pneumoniae, in particular, the conversion of DNA to single-strand segments on entry, determines the mechanisms for plasmid establishment and interaction with the chromosome. One of these mechanisms, the chromosomal facilitation of plasmid establishment, is useful for obtaining recombinant plasmids and for introducing an allele from the chromosome into a plasmid. The difference between linear and circular synapsis of donor DNA strands with the chromosome is illustrated. Circular synapsis can give rise to circular integration, which is useful for insertional mutagenesis of chromosomal genes, for coupled cloning in Escherichia coli, and for sequential cloning of DNA along the pneumococcal chromosome. Cloning in S. pneumoniae is not notably affected by DNA mismatch repair or restriction systems in the host cell. Unusual features of gene expression in S. pneumoniae are discussed. Transcription begins most often at promoters with extended -10 sequences, and in a small but significant number of cases, translation does not require a ribosome-binding site with a Shine-Dalgarno sequence.

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.

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.

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.

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.

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.

A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities.

A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.

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.

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.

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.

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.

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.

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.

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.

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.