Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus.

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

Galactose metabolism and capsule formation in a recombinant strain of Streptococcus thermophilus with a galactose-fermenting phenotype.

The capsule-producing, galactose-negative Streptococcus thermophilus MR-1C strain was first transformed with a low-copy plasmid containing a functional galK gene from Streptococcus salivarius to generate a recombinant galactose-fermenting Strep. thermophilus strain named MR-AAC. Then, we compared the functional properties of Strep. thermophilus MR-AAC with those of the parent MR-1C strain when used as starter for fermented products and cheese. In lactose-supplemented laboratory medium, MR-AAC metabolized galactose, but only when the amount of lactose was less than 0.1% (wt/vol). After 7 h of fermentation, the medium was almost depleted of galactose. The parent strain, MR-1C, showed the same pattern, except that the concentration of galactose decreased by only 25% during the same period. It was found that, during milk fermentation and Mozzarella cheese production, the galactose-fermenting phenotype was not expressed by MR-AAC and this strain expelled galactose into the medium at a level similar to the parent MR-1C strain. In milk and in lactose-supplemented medium, capsular exopolysaccharide production occurred mainly during the late exponential phase and the stationary growth phase with similar kinetics between MR-1C and MR-AAC.

Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes.

The aim of this work was to use fluorescently labeled lectins to develop a convenient and reliable method to determine the relative abundance of capsular polysaccharides (CPS) at the surface of Streptococcus thermophilus MR-1C cells. Fluorescein isothiocyanate-labeled peanut agglutinin isolated from Arachis hypogaea was found to interact specifically with the CPS of Strep. thermophilus MR-1C. This labeled lectin was then used as an effective probe to detect and quantify CPS. A fluorescence-based lectin-binding assay was successfully applied to follow the accumulation of CPS during the growth of Strep. thermophilus MR-1C in milk and in M17 broth supplemented with lactose. Our results showed that in both media, CPS production by Strep. thermophilus MR-1C began during the exponential phase of growth and continued for several hours after the culture reached the stationary growth phase.

The phosphoenolpyruvate:sugar phosphotransferase system of oral streptococci and its role in the control of sugar metabolism.

Oral streptococci are sugar-fermentative bacteria comprising at least 19 distinct species and are a significant proportion of the normal microbial population of the mouth and upper respiratory tract of humans. These streptococci transport several sugars by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which concomitantly catalyzes the phosphorylation and translocation of mono- and disaccharides via a chain of enzymic reactions that transfer a phosphate group from phosphoenolpyruvate to the incoming sugar. A number of PTS components, including HPr, Enzyme I and some Enzymes II, have been studied at the biochemical and/or genetical level in Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus. Moreover, compelling evidence indicates that the oral streptococcal PTS is involved in the regulation of sugar metabolism. Results are accumulating suggesting that a protein called IIABMan, as well as the phosphocarrier protein HPr, are key regulatory components that allow these bacteria to select rapidly metabolizable sugars, such as glucose or fructose, over less readily utilizable carbohydrates. Circumstantial evidence suggests that the molecular mechanisms by which oral streptococcal PTS exert their regulatory functions differ from mechanisms in other Gram-negative or Gram-positive bacteria.

Use of an alpha-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus.

The alpha-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the alpha-galactosidase of L. raffinolactis is thermolabile and inoperative at 42 degrees C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this alpha-galactosidase at 42 degrees C was only 3% of that at 30 degrees C, whereas the enzyme retained 23% of its activity at 37 degrees C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37 degrees C or below. The transformed cells possessed 6-fold more alpha-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.

Phosphotransferase system of Streptococcus salivarius: characterization of the ptsH gene and its product.

The Streptococcus salivarius ptsH gene encoding histidine-containing phosphocarrier protein (HPr) of the phosphotransferase system (PTS) has been cloned, sequenced, and found to be part of a ptsH, ptsI operon. Upstream from ptsH, putative -35 and -10 boxes and a Shine-Dalgarno sequence highly similar to the Escherichia coli consensus regulatory elements were identified. A second promoter, located in the ptsH coding sequence was also observed and is sufficient for the expression of the S. salivarius ptsI gene, encoding enzyme I of the PTS in E. coli [Gagnon et al., Gene 121 (1992) 71-78]. The amino acid sequence of S. salivarius HPr, inferred from the ptsH sequence, shared identity varying between 37 and 76% with known HPr from other bacteria. Moreover, the S. salivarius HPr shared 78% identity with an HPr-like protein of Aspergillus fumigatus, a eukaroytic mold that does not possess a functional PTS. Expression analysis of S. salivarius HPr in E. coli demonstrated that (i) S. salivarius ptsH is expressed in E. coli under the control of its own promoter, (ii) S. salivarius HPr synthesized by E. coli is completely processed by methionine aminopeptidase, and (iii) S. salivarius HPr is phosphorylated in vivo by E. coli enzyme I. It was also observed that, in E. coli, the copy number of pUC18 bearing S. salivarius ptsH was reduced more than 25-fold, as compared to pUC18 without an insertion.

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

HPr polymorphism in oral streptococci is caused by the partial removal of the N-terminal methionine.

HPr is a small phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system involved in the transport and phosphorylation of sugars. It has recently been reported that streptococci possess two forms of HPr having identical biochemical properties. In this communication, we show by N-terminal amino-acid sequencing and by ion-spray mass spectroscopy that these two forms differ by the presence or the absence of the N-terminal methionine.

Altered expression of several genes in IIIManL-defective mutants of Streptococcus salivarius demonstrated by two-dimensional gel electrophoresis of cytoplasmic proteins.

Mannose, glucose and fructose are transported in Streptococcus salivarius by a phosphoenolpyruvate:mannose phosphotransferase system (PTS) which consists of a membrane-bound Enzyme II (EII) and two forms of IIIMan having molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), respectively. We have previously reported the isolation of spontaneous mutants lacking IIIManL and showed that they exhibit higher beta-galactosidase activity than the parental strain after growth on glucose, and that some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. In an attempt to determine whether the expression of other genes is affected by the mutation and what the physiological link is between them, we examined three S. salivarius IIIManL-defective mutants (strains A37, B31 and G29) and the parental strain using two-dimensional gel electrophoresis after growth of the cells on a variety of sugars. After growth on glucose, five new proteins were detected in the cytoplasm of the three mutants. Two of these proteins were induced in the parental strain by galactose or oligosaccharides containing galactose, and one was specifically induced by melibiose. The other two proteins were not detected in the parental strain under any of the growth conditions tested. Two other proteins were only detected in glucose-grown cells of mutant A37, and a protein associated with the metabolism of fructose was constitutively expressed in mutants B31 and G29. Moreover, we have found that under identical growth conditions the amounts of several other proteins which were detected in the parental strain were either increased or decreased in the mutants. Globally, our results have indicated that (1) the expression of several genes was affected in the spontaneous IIIManL-defective mutants; (2) some of the proteins abnormally produced in the mutants were specifically induced in the parental strain by sugars; (3) the phenotypic modifications observed in the mutants were of two types: most were observed solely after growth of the cells on glucose whereas the others were glucose-independent; and (4) the mutants shared common phenotypic traits, but also exhibited idiosyncratic characteristics.

Amino-terminal methionine processing of the protein HPr in Streptococcus salivarius grown in continuous culture.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Streptococci possess two forms of HPr which differ by the presence or the absence of the N-terminal methionine (Met). These forms are called HPr-1 (without Met) and HPr-2 (with Met). In order to determine whether the ratio of these two forms varies with growth conditions, we measured the amount of HPr-1 and HPr-2 present in Streptococcus salivarius grown in continuous culture at pH 7.5. The results indicated that the HPr-1/HPr-2 ratio: 1) was not related to the cellular amount of total HPr; 2) was highest (10.2 +/- 3.5) under glucose (a PTS sugar) limitation (10 mM) and low dilution rate (D = 0.1 h-1; g = 6.9 h); 3) was decreased 2.4- to 5.7-fold when the amount of glucose and/or D was increased; 4) was not influenced by D when cells were cultured on galactose (a non-PTS sugar) but was two-fold higher under conditions of galactose excess (200 mM). We suggest that the cleavage of the N-terminal HPr Met is not a stochastic phenomenon but is dictated by growth conditions.

Properties of the cytoplasmic glutamyl-tRNA synthetase in high molecular weight complexes from bovine brain.

The glutamyl-tRNA synthetase purified 300-fold from calf brain is associated with other aminoacyl-tRNA synthetases in a complex whose molecular weight is about 2,000,000. However, in a less purified state, the enzyme is present in a complex larger than 5,000,000. The properties of the enzyme are the same in both complexes except for the pH optimum of the aminoacylation reaction. The presence of 2-mercaptoethanol protects and increases the enzymatic activity. gamma-Methyl-L-glutamate and salicylate show competitive inhibition with respect to glutamate but kainic acid and taurine have no effect on the rate of aminoacylation of tRNAGlu.

A comparative study of enzymes involved in glucose phosphorylation in oral streptococci.

The properties of two enzymes involved in the phosphorylation of glucose were studied in three oral streptococci species. The glucokinase of Streptococcus mutans had a lower affinity for glucose and ATP than did those from S. salivarius and S. sanguis. The enzyme had an identical pH optimum (pH 8.0) in all three bacteria. However, the result from the phosphoenolpyruvate phosphotransferase system showed a different pattern when its activity was measured using 2-deoxyglucose with toluenized cells. Uptake studies of 2-deoxyglucose also revealed that the three microorganisms had different affinities for this compound. This glucose analogue strongly inhibited the acid production of S. salivarius, but did not affect the glycolysis of the other two bacteria.

Effect of gramicidin D on the acidogenic properties of oral streptococci and human dental plaque.

The effect of gramicidin D, Gramicidin S, and carbonylcyanide p-trifluoromethoxyphenyl hydrazone on the glycolysis of various oral streptococci was investigated. Gramicidin D was the most effective and inhibited the glycolysis of all the streptococci studied in the presence of the most common sugars found in man's diet (sucrose, lactose, glucose, and fructose). A concentration of 2.5 X 10(-6) M gramicidin D was sufficient to decrease significantly the rate of glycolysis of human dental plaque in the presence of sucrose. The use of this ionophore as a caries preventive agent is discussed.

Effect of xylitol on the growth and glycolysis of acidogenic oral bacteria.

We investigated the effect of xylitol on the growth of different oral bacteria in the presence of glucose. Xylitol inhibited the growth of all but one of ten strains of S. mutans and failed to inhibit the growth of the lactobacilli, actinomycetes, and other streptococci tested except S. sanguis 10556, which was slightly inhibited. However, the rate of acid production of the sensitive S. mutans strains was not equally affected by xylitol. These data, obtained with pure cultures of acidogenic oral bacteria, may explain the lack of an in vitro inhibitory effect of xylitol on dental plaque samples.

Heterofermentative glucose metabolism by glucose transport-impaired mutants of oral streptococcal bacteria during growth in batch culture.

Spontaneous mutants defective in a membrane component of the phosphoenolpyruvate-glucose phosphotransferase system were isolated by plating cells of Streptococcus sanguis 10556, Streptococcus mutans GS5-2 and NCTC 10449 on agar containing lactose and 2-deoxyglucose. Toluenized cells of these mutants were defective in their ability to catalyse the phosphoenolpyruvate-dependent phosphorylation of 2-deoxyglucose. The parental strains were mainly homofermentative when grown in batch culture in the presence of various sugars. Nevertheless, the mutants produced acetate, formate and ethanol when cultured in the presence of glucose but were homofermentative when grown in the presence of lactose or maltose. Analysis of one mutant isolated from Strep. sanguis (mutant GS26) revealed normal levels of glucokinase, glucose-6-phosphate dehydrogenase, puruvate kinase and lactate dehydrogenase. This last enzyme was dependent on fructose 1,6-diphosphate for catalytic activity. The determination of the intracellular level of fructose 1,6-diphosphate (FDP) during growth of the cells in batch culture showed that the mutant strains contained 2 to 15 times less FDP than the parental strains. Growth experiments performed at pH 6.0 and 7.0 with Strep. sanguis and its PTS-negative mutant GS26 suggested that the regulation of pyruvate metabolism in this bacterium include the intracellular level of FDP and the initial hydrogen concentration of the growth medium. The results also suggested that, in these bacteria, an active PTS is required to maintain the intracellular concentration of FDP high enough to keep the cell homofermentative during growth in batch culture.

Control of sugar utilization in the oral bacteria Streptococcus salivarius and Streptococcus sanguis by the phosphoenolpyruvate: glucose phosphotransferase system.

Three different Strep. salivarius (G2, G5 and G29) and two Strep. sanguis (GS3 and GS12) mutants affected in the phosphoenolpyruvate: glucose phosphotransferase system were selected on agar plates containing lactose and 2-deoxyglucose. All 5 were defective in a membrane-bound component of the transport system and grew less rapidly than the parent strain in 5 mM glucose-containing medium. Mutants G2 and G29 grew poorly in the presence of 5 mM mannose. Growth on mixed substrates revealed that the mutants and wild-type parents behaved differently. Wild-type strains in medium containing glucose plus another sugar (lactose, galactose, melibiose, raffinose or trehalose for Strep. salivarius and lactose, galactose or trehalose for Strep. sanguis) always exhausted most of the glucose before utilizing the other sugar. The mutants used the second sugar concurrently or preferentially to glucose. In medium containing glucose plus fructose or mannose, the wild types consumed both sugars concurrently whereas the mutants utilized the second sugar before glucose. Mutants G2 and G5 were insensitive to repression by fructose and released glucose into the medium when grown in the presence of 0.4 per cent lactose. Mutant G5 also released galactose. Sugar release was not detected with the wild types. The Strep. salivarius mutants contained normal levels of glucokinase and beta-galactosidase but G5 was almost totally devoid of galactokinase activity after growth on lactose. On galactose, the activity was restored. It seems that the phosphoenolpyruvate: glucose phosphotransferase system is involved in the regulation of sugar utilization in these two streptococci.

Gastrointestinal mucormycosis causing an acute abdomen in the immunocompromised pediatric patient--three cases.

Mucormycosis is an infection caused by a ubiquitous fungus in immunocompromised individuals. Typically, it invades blood vessels, producing thrombosis and tissue infarction. This infection spans all pediatric age groups and can lead to hollow viscus perforation and bowel obstruction. A 30-month old male with large cell anaplastic lymphoma had a bowel obstruction. During emergency laparotomy, an ileoileal intussusception was identified, which required resection and anastomosis. In the pathological specimen, fungi of the Mucorales order were found to be associated with tissue necrosis. On the eighth day of life, a premature infant had abdominal distension secondary to bowel perforation. Partial gastric resection and multiple intestinal stomas were performed. Death occurred soon after, secondary to multiorgan failure. The autopsy and surgical specimens showed widespread mucormycosis. An adolescent had meningococcemia-induced septic shock. During recovery, hemorrhagic colitis developed, which led to perforation. The subtotal colectomy specimen showed widespread mucormycosis. The laparotomy findings are typical (black necrotic tissue involving the bowel), and when seen in the immunocompromised patient, should make one suspect gastrointestinal mucormycosis. Aggressive surgical debridement of devitalized tissue augmented by intravenous antifungal medication is the mainstay of treatment.

A paediatric palliative care programme in development: trends in referral and location of death.

PURPOSE: To describe the formation of a paediatric palliative care programme providing care in hospital, at home or in hospice, ensuring continuity of care where the child and family desire. STUDY DESIGN: Descriptive analysis was performed on referral source, diagnosis and reason for discharge for patients referred to the Palliative Care Team at the Children's Hospital of Eastern Ontario in Ottawa, Ontario, Canada from 1999 to 2007. RESULTS: 341 children were referred. 24% had a neurological condition, 21% had genetic disorders or congenital anomalies, 20% had cancer, 18% had metabolic or neurodegenerative conditions and 17% had another diagnosis. The major sources of referral included paediatricians, neonatologists, oncologists and intensivists. 55% of the children have died. 58% of these died in hospital, 27% at home and 15% in hospice. Of the remaining 152 children, 7% were discharged from the programme after clinical improvement, 4% were moved to another geographic location or an adult centre, 2% were not eligible, 1% declined services and 4% were lost to follow-up. The remaining 90 children continue to be followed-up. In the hospitalised patients who died, the annual referral rate increased from 20% to >50%. IMPLICATIONS: Referral to the palliative care team has increased over time in all diagnostic categories and from all sources. Most children died in hospital; however, a significant number of families chose end-of-life care at home or in a hospice.