Complete genome sequence of the ureolytic Streptococcus salivarius strain 57.I

Streptococcus salivarius 57.I is one of the most abundant and highly ureolytic bacteria in the human mouth. It can utilize urea as the sole nitrogen source via the activity of urease. Complete genome sequencing of S. salivarius 57.I revealed a chromosome and a phage which are absent in strain SK126.     

Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.     

Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity

We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Deltappc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with L-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of L-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p(ureI)-gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.     

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products

AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.     

Molecular genetics of Streptococcus thermophilus

Abstract The metabolism and genetics of Streptococcus thermophilus (presently Streptococcus salivarius ssp. thermophilus ) have only been investigated recently despite its widespread use in milk fermentation processes. The development of recombinant DNA technology has allowed impressive progress to be made in the knowledge of thermophilic dairy streptococci. In particular, it has permitted a careful analysis of phenotypically altered variants which were derived from a mother strain by plasmid or chromosomal DNA reorganization. While natural phage defense mechanisms of S. thermophilus remain poorly documented, information on the bacteriophages responsible for fermentation failures has accumulated. The lysogenic state of two S. thermophilus strains has also been demonstrated for the first time. Gene transfer techniques for this species have been established and improved to the point that targeted manipulation of their chromosomal determinants is now feasible. Cloning and expression vectors have been constructed, and a few heterologous genes were successfully expressed in S. thermophilus . The first homologous genes, involved in carbohydrate utilization, have been cloned and sequenced, shedding some light on the molecular organization of key metabolic steps.     

Dual functions of Streptococcus salivarius urease

A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at the ureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivarius could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.     

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).     

A species-specific DNA probe obtained from Streptococcus salivarius subsp. thermophilus detects strain restriction polymorphism

Genomic polymorphism in Streptococcus salivarius subsp. thermophilus was revealed by DNA restriction pattern analysis. A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP. Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe. Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S. salivarius subsp. salivarius, confirming a close relationship between S. salivarius and S. thermophilus.     

Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk

Aim:  The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant.
Methods and results:  The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617^T and its derivative, carbamoylphosphate synthetase-negative mutant A17( ΔcarB ), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO₂ depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l -arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine.
Conclusions:  Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk.
Significance and impact of the study:  The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

G arvie , E.I. C ole , C.B., F uller , R. & H ewitt , D. 1984. The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat. Journal of Applied Bacteriology 56 , 237–245
Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro . Two enzyme systems (β-galactosidase and lactase) were studied using, respectively, o -nitrophenyl-β-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates. increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly hetero-fermentative ( Lact. reuteri ) to one which was predominantly homofermentative ( Lact. salivarius ).     

A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius

Fibrillar and fimbriate strains of Streptococcus salivarius were compared for their ability to adhere to buccal epithelial cells and saliva-coated hydroxyapatite beads, and for their ability to coaggregate with Veillonella strains. The fibrillar Lancefield group K strains adhered statistically significantly better to both buccal epithelial cells and saliva-coated hydroxyapatite beads than the fimbriate strains, which lacked the Lancefield group K antigen. After 1 h the fibrillar strains coaggregated statistically significantly better than the fimbriate strains with V. parvula strain V1, but after 24 h, coaggregation both of fibrillar and of fimbriate strains reached approximately 90%. Freshly isolated Veillonella strains all coaggregated with the S. salivarius strains, but the percentage coaggregation varied considerably after 1 h depending on the Veillonella strain. Coaggregation was independent of the presence of Ca2+. S. salivarius strain HB-V5, a mutant of strain HB that had lost the Veillonella-binding protein, coaggregated weakly with V. parvula strain V1, but coaggregated very well with other wild-type veillonellae, suggesting the presence of an alternative mechanism for Veillonella-binding for strain HB. Fibrillar strains were, therefore, more adhesive to oral surfaces and coaggregated with veillonellae after 1 h better than the fimbriate S. salivarius strains. Both fibrillar and fimbriate strains were highly hydrophobic in the hexadecane-buffer partition assay.     

Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity

Arginase of the Helicobacter pylori urea cycle hydrolyzes L-arginine to L-ornithine and urea. H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in H. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori gene hp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 and rocF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was approximately 1, 000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of L-arginine, in contrast to the WT, and yielded a approximately 10, 000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylori mouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log(10) CFU) were 6.1, 5.5, and 4.1, respectively. Thus, H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.