Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

Adaptation of the membrane fatty acid composition by growth in the presence of n-alkanols influences glycosyltransferase expression in Streptococcus salivarius

Growth of Streptococcus salivarius ATCC 25975 in the presence of n-alkanols in the series methanol to decan-1-ol led to a decrease in the unsaturated to saturated fatty acid ratio. Each member of the set of n-alkanols which was examined over a range of concentrations possessed a point at which extracellular glucosyltransferase (GTF) production was minimal; increasing the concentration of the n-alkanol past this point stimulated GTF production. This effect was greatest with hexan-1-ol although it was observed to a lesser extent with pentan-1-ol and heptan-1-ol. Reduced cell-associated fructosyltransferase activity was observed with increasing concentrations of each n-alkanol. Growth in the presence of 25 mM-propan-1-ol gave rise to a fatty acid profile in which 55% of the fatty acids were of an odd chain length. S. salivarius ATCC 25975 was shown to be able to utilize ethanol in a similar manner to propan-1-ol by growing it in the presence of 400 mM-[14C]ethanol. Analysis of the membrane lipids at the stationary phase of growth indicated that 17.6% of the carbon of the fatty acids was derived from ethanol. A leaky adh mutant, S. salivarius MJ 37501, was isolated. The leaky nature of the mutant enabled it to incorporate reduced levels of odd-chain-length fatty acids into its membrane lipids when grown in the presence of 100 mM-propan-1-ol, but not when grown in the presence of 25 mM-propan-1-ol. S. salivarius ATCC 25975 therefore metabolized propan-1-ol (and ethanol) via a constitutive alcohol dehydrogenase.     

Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.     

The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system.

The ftf gene, coding for the cell-bound beta-D-fructosyltransferase (FTF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FTF of Streptococcus mutans and the levansucrases (SacBs) of Bacillus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivarius ftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host.     

Functional Analysis of Glycosyltransferase Genes from Lactococcus lactis and Other Gram-Positive Cocci: Complementation, Expression, and Diversity

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.     

Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans

Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.     

Identification of a gene, rgg, which regulates expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii Challis.

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.     

Sequence and phylogenetic analyses of the water‐soluble glucan synthesizing‐glucosyltransferase genes of Streptococcus dentirousetti

Two tandemly aligned glucosyltransferase (GTF) genes whose gene products are responsible for water‐soluble glucan synthesis were isolated from Streptococcus dentirousetti NUM1303 and sequenced. One of the GTF genes of S. dentirousetti consisted of a 4110 bp open reading frame (ORF) that encoded for a 1369 amino acid protein and was revealed to be a S. sobrinus gtfS homolog. The percent similarity of amino acid sequences of the GTF‐S from S. dentirousetti compared to those from S. sobrinus was 99%. In addition, a putative gtfT was found in tandem in the downstream region of the S. dentirousetti gtfS. The gtfT of S. dentirousetti consisted of a 4527 bp ORF encoding for 1508 amino acids. The similarity of amino acid sequences of the GTF‐T from S. dentirousetti and S. sobrinus was 94%. Phylogenetic analysis based on amino acid sequences from other related streptococcal GTFs suggested that both GTF‐S and GTF‐T of S. dentirousetti are closely related to S. sobrinus.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.     

Proper expression of myosin genes in transgenic nematodes.

Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product.