RESULTS:

The bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby Macropus eugenii was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing <97 % homology to a known bacterial species or phylotype was novel. Thus despite the close proximity of the openings of the urogenital and anal tracts within the cloaca the two sites retained a diverse range of distinct bacteria with only a small percentage of overlapping species.

Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7·0 or pH 5·0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication transcription translation protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5·0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein Ssb the transcription elongation factor GreA the RNA exonuclease polyribonucleotide nucleotidyltransferase (PnpA) and two proteinases the ATP-binding subunit ClpL of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase PepD of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments.

The recombinant primer-dependent glucosyltransferase GtfJ of Streptococcus salivarius possesses a C-terminal glucan-binding domain composed of eighteen 21 aa YG repeats. By engineering a series of C-terminal truncated proteins the position at which truncation prevented further mutan synthesis was defined to a region of 43 aa confirming that not all of the YG motifs were required for the formation of mutan by GtfJ. The role of the YG repeats in glucan binding was investigated in detail. Three proteins consisting of 3·8 7·2 or 11·0 C-terminal YG repeats were expressed in Escherichia coli. Each of the three purified proteins bound to both the 16-α-linked glucose residues of dextran and the 13-α-linked glucose residues of mutan indicating that a protein consisting of nothing but 3·8 YG repeats could attach to either substrate. Secondary structure predictions of the primary amino acid sequence suggested that 37% of the amino acids were capable of forming a structure such that five regions of β-sheet were separated by regions capable of forming β-turns and random coils. CD spectral analysis showed that the purified 3·8 YG protein possessed an unordered secondary structure with some evidence of possible β-sheet formation and that the protein maintained this relatively unordered structure on binding to dextran.

The synthesis of cell-associated and secreted proteins by Streptococcus gordonii FSS2 an infective endocarditis (IE) isolate was influenced by both environmental pH and carbon source. Controlling the pH at 7·5 in stirred batch cultures showed that cell-associated and secreted protein concentrations were increased during late exponential and stationary phase by 68% and 125% respectively compared with similar cultures without pH control. The expression of five glycosidase and eight peptidase activities were examined using fluorogen-labelled synthetic substrates. Enzyme activities were significantly down-regulated during exponential growth increasing during stationary phase (P<0·01) whether the culture pH was controlled at pH 7·5 or allowed to fall naturally to pH 4·4. Culture-supernatant activities were significantly increased (P<0·05) when the pH was maintained at 6·0 or 7·5 indicating modulation of enzyme activity by pH. Growth under nitrogen-limitation/glucose-excess conditions resulted in a significant repression of cell-associated glycosidase activities (P<0·01) whilst in the supernatant activities were generally reduced. The expression of peptidase activities in the culture supernatant did not significantly change. The results suggest a possible role for catabolite repression by glucose in regulating enzyme expression. When S. gordonii FSS2 was cultured with 50% (v/v) added heat-inactivated foetal bovine serum several cell-associated enzyme activities increased initially but were then reduced as the culture time was extended to 116 h. Culture-supernatant enzyme activities (N-acetyl-β-D-glucosaminidase N-acetyl-β-D-galactosaminidase thrombin Hageman factor collagenase and chymotrypsin) however were significantly increased (P<0·01) over the same time period. The findings indicated that most of the important glycosidases synthesized by S. gordonii FSS2 were down-regulated by acid growth conditions and may also be subject to catabolite repression by glucose but conversely may be up-regulated by growth in serum. These results may have implications for streptococcal growth in an IE vegetation and in the mouth between meals or during sleep.

Summary: Many strains of oral streptococci secrete glucosyltransferases (GTFs) that polymerize sucrose into glucans that form an integral part of the plaque matrix on the tooth surface. Recently we reported the cloning of two closely linked GTF-encoding genes (gtfJ and gtfK) from Streptococcus salivarius ATCC 25975 as well as the sequence of gtfJ which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtfK which encodes a primer-dependent GTF that synthesizes a soluble product (a GTF-S) as well as the sequence of a small downstream open reading frame of unknown function. The deduced sequence of GtfK was compared with those of seven other streptococcal Gtfs and an unrooted phylogenetic tree constructed. This analysis suggested that Gtfs with similar product specificities do not form phylogenetic clusters and was consistent with currently accepted phylogenetic schemes. The tree was tested by constructing a series of “sub-trees” from different blocks of the alignment. Evidence was obtained for recombination events involving gtfB and gtfC from S. mutans GS-5 gtfJ and gtfK from S. salivarius as well as the gtfI genes from S. downei and S. sobrinus. The recombination events between gtfB and gtfC and between the two gtfI genes were confirmed by examining divergences at silent sites.

Summary: The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage λL47.1. The inserts in the λ-clones were characterized by restriction mapping and Southern hybridization and were found to overlap implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ and that both were transcribed in the same direction.

A defined growth medium (designated AP11) in which the concentrations of Na+ and K+ could be altered independently of one another was developed for Streptococcus salivarius ATCC 25975. The addition of 100 mm-Na+ to AP11-medium containing 25 mm-K+ initially reduced the rate of expression of extracellular glucosyltransferase (GTFe). However once S. salivarius had adaptated to grow in the presence of 100 mm-Na+ the rate of GTFe expression was stimulated. In fact once adapted to the presence of Na+ in the environment the same increase in the rate of enzyme expression was observed in all batch cultures irrespective of the K+ concentration (2–50 mm). At 2 mm-K+ there was no change in the level of saturation of the membrane lipids when the Na+ concentration was increased from 0 mm to 100 mm. Na+-stimulation of GTFe expression was confirmed in non-proliferating cell suspensions at different K+ concentrations. In non-proliferating cell suspensions GTFe expression outlined a rectangular hyperbola with respect to K+ concentration when the K+ concentration was stepped up from 2 mm. The increase in GTFe synthesis and secretion was transient and was similar to that previously reported by us in Na+-rich medium though it did not reach the same high levels. The reduced transient stimulation of GTFe expression correlated both with an enrichment in the saturated fatty acids of the membrane lipids of S. salivarius and with the fact that the degree of saturation was only slightly reduced when the K+ concentration was stepped up from 2 mm to 50 mm. Needless to say the final octadecenoic to octadecanoic (C18:1/C18:0) fatty acid ratio retained its direct correlation with the transient increase in GTFe production following the step up in K+ concentration giving rise to an apparent biphasic plot when combined with that previously reported.

Summary: 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.

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30°C 37°C and 40°C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2·06 1·01 and 0·87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30°C compared to cells grown at 40°C. However cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80 but that the surfactant also had to be present.

In medium saturated with oxygen the cyclopropane synthetase (unsaturated-phospholipid methyltransferase; EC 2.1.1.16) of Proteus vulgaris was generally synthesized after the mid-exponential phase of growth. The enzyme could also be induced by rapidly limiting the oxygen supply or by initiating respiration on nitrate or thiosulphate following an initial period of growth in a highly aerobic environment. In each of these ‘step-down’ situations the specific activity of cyclopropane synthetase rose to a maximum prior to the stationary phase of growth and subsequently decreased. The cyclopropane fatty acids methylene hexadecanoic acid and methylene octadecanoic acid accumulated throughout exponential growth following the induction of the enzyme. During a 12 h period in the stationary phase there was little synthesis of either of the fatty acids despite detectable cyclopropane synthetase activity in the cells indicating that essentially all the fatty acid synthesis was complete prior to entering the stationary phase. When nitrate was used as a respiratory electron acceptor a twofold increase in octadecenoic acid was observed giving rise to an increase in methylene octadecanoic acid. This increase in octadecenoic acid was not apparent in mutants unable to respire on nitrate.