Overproduction of a dextranase inhibitor by Streptococcus sobrinus mutants.

An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex- mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex- Adh- mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei). Dei was heat stable but trypsin sensitive. By adding excess dextranase following BD-SDS-PAGE, Dei was detected as blue bands with apparent molecular masses of 43, 40, 37, 27, and 23 kDa. Dei competitively inhibits dextranase activity and is synthesized by wild-type S. sobrinus strains, with the amount varying depending upon growth medium and stage in the growth cycle. R. M. Hamelik and M. M. McCabe (Biochem. Biophys. Res. Commun. 106:875-880, 1982) previously described a Dei in a wild-type S. sobrinus strain.     

Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.     

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.     

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.     

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.     

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.     

Construction of scrA::lacZ gene fusions to investigate regulation of the sucrose PTS of Streptococcus mutans

The scrA gene coding for sucrose EnzymeII of the phosphoenolpyruvate dependent phosphotransferase system previously isolated from Streptococcus mutans was fused in vitro to the promoterless lacZ' gene to monitor the expression of the scrA gene. The scrA::lacZ gene fusion was introduced back into S. mutans GS-5IS3 by two independent transformation procedures involving either linear or plasmid DNA to produce both scrA and scrA+ mutants. These mutants should prove useful for analyzing the regulation of sucrose transport in S. mutans.     

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system

We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.     

Cloning and DNA sequencing of the dextranase inhibitor gene (dei) from Streptococcus sobrinus.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.     

Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.     

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.     

Substrate specificity of hydrolase activity of the primer-dependent glucosyltransferases from Streptococcus sobrinus.

Four kinds of glucosyltransferases, P1, P2, P3 and P4, were separately purified from the culture supernatant of Streptococcus sobrinus. Their dependencies on primer were analysed. There were two primer-dependent glucosyltransferases (P3 and P4). In the absence of primer 1,6-alpha-D-glucan, P3 was not able to produce glucan from sucrose. However, P3 showed sucrose hydrolase activity, whereas P4 was still able to produce glucan without primer 1,6-alpha-D-glucan. Consequently, glucosyltransferase activity of P4 was incompletely primer-dependent. Both P3 and P4 showed high substrate specificity for sucrose, failing to use melezitose, raffinose, or stachyose as the substrates.     

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.     

Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene

Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1 B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1 B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology.No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.     

Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene.

The product of the spoIIM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spoIIM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilitates the use of a genetic technique employed by Illing et al to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spoIIM must be expressed in the forespore. Biochemical analysis of forespore and mother cell fractions suggests that spoIIM is also expressed in the mother cell. The conditional integrational vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of alpha-complementation and thus should be useful for other kinds of genetic manipulations in B subtilis.     

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.