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.

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.

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.

Diversity of Streptococcus salivarius ptsH mutants that can be isolated in the presence of 2-deoxyglucose and galactose and characterization of two mutants synthesizing reduced levels of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system.

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His approximately P), HPr(Ser-P), and HPr(Ser-P)(His approximately P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for alpha-galactosidase, beta-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of alpha-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).

Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure.

Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus.

Regulation of the pts operon in low G+C Gram-positive bacteria.

The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria. Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species. In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon. In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria. Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins. Their synthesis is not coordinated and is influenced by environmental factors. The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl. Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion.

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I-deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively). In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P-Ser-HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser-46-Ala or Ile-47-Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P-Ser-HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P-Ser-HPr participates also in inducer exclusion.

Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius.

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.

An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence.

Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 10(5) transformants per microg of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria.

The HPr(Ser) kinase of Streptococcus salivarius: purification, properties, and cloning of the hprK gene.

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 microM and 1 mM, respectively.

The phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius. Functional and biochemical characterization of IIABL(Man) and IIABH(Man).

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.

Replacement of isoleucine-47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine-46.

Phosphorylation of HPr on a serine residue at position 46 (Ser-46) by an ATP-dependent protein kinase has been reported in several Gram-positive bacteria, and the resulting intermediate, HPr(Ser-P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium. We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile-47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser-46, nor did it impede the phosphorylation of HPr on His-15 by EI or the transfer of the phosphoryl group from HPr(His-P) to other PTS proteins. However, the 147T substitution did perturb, in glucose-grown but not in galactose-grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His-P)(Ser-P); the levels of HPr(His-P) and HPr(Ser-P) were not affected. Growth on melibiose was virtually identical for the wild-type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2- to 1.5-fold. The preferential metabolism of PTS sugars (glucose and fructose) over non-PTS sugars (lactose and melibiose) that is observed in wild-type cells was abolished in cells of mutant G22.4. Moreover, alpha- and beta-galactosidases were derepressed in glucose- and fructose-grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non-PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr-dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.

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.

Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus suis.

HPr is a low-molecular-mass phosphocarrier protein of the bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) found in the cytoplasm or associated with the inner surface of the cytoplasmic membrane. Treatment of Streptococcus suis cells with a Sorvall Omnimixer, a technique used to extract cell surface components, resulted in the extraction of a major protein with a molecular mass of 9 kDa. Several lines of evidence suggested that this protein was HPr: (i) the S. suis protein showed homology over the first 35 N-terminal amino acid residues with the HPrs of Streptococcus salivarius and Streptococcus mutans, including the signature sequence for the site of PEP-dependent phosphorylation; (ii) it cross-reacted with the S. salivarius anti-HPr antibody preparation; (iii) it could be phosphorylated by enzyme I at the expense of PEP, and by a membrane-associated kinase at the expense of ATP; and (iv) it possessed phosphocarrier activity when used as a source of HPr in an in vitro PTS assay. The data suggested that a portion of the cellular HPr is associated with the external cell surface in S. suis, a result that was confirmed by immunogold electron microscopy. The cellular HPr of S. suis consisted of two forms that could be distinguished by the presence or the absence of the N-terminal methionine. Amino acid sequence analysis indicated that the cell-surface-associated HPr of S. suis lacked the N-terminal methionine residue.

Regulation of ptsH and ptsI gene expression in Streptococcus salivarius ATCC 25975.

The transcriptional regulation of the Streptococcus salivarius ptsH and ptsI genes coding for the general energy-coupling proteins HPr and enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system were investigated. These genes form an operon with the gene order ptsH-ptsI. Three distinct mRNA species were detected: a 0.5 kb transcript specific for ptsH, and two long transcripts (2.2 and 2.4 kb) covering the whole pts operon. Transcription of all these mRNAs initiated at the same nucleotide located 9 bp downstream from a promoter located immediately upstream from the ptsH gene. The presence of a high-energy stem-loop structure (T0) located at the beginning of ptsI was responsible for the premature transcription termination generating the 0.5 kb ptsH-specific transcript. The long transcripts ended in the poly(U) region of two rho-independent-like terminators (T1 and T2) at the 3' end of ptsI. Studies with a 2-deoxyglucose-resistant spontaneous mutant of S. salivarius (L26) that produces an HPr-EI fusion protein suggest that the regulation of HPr and EI expression involves transcriptional as well as translational mechanisms.

Distribution of proteins similar to IIIManH and IIIManL of the Streptococcus salivarius phosphoenolpyruvate:mannose-glucose phosphotransferase system among oral and nonoral bacteria.

In Streptococcus salivarius, the phosphoenolpyruvate (PEP):mannose-glucose phosphotransferase system, which concomitantly transports and phosphorylates mannose, glucose, fructose, and 2-deoxyglucose, is composed of the general energy-coupling proteins EI and HPr, the specific membrane-bound IIIMan, and two forms of a protein called IIIMan, with molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), that are found in the cytoplasm as well as associated with the membrane. Several lines of evidence suggest that IIIManH and/or IIIManL are involved in the control of sugar metabolism. To determine whether other bacteria possess these proteins, we tested for their presence in 28 oral streptococcus strains, 3 nonoral streptococcus strains, 2 lactococcus strains, 2 enterococcus strains, 2 bacillus strains, 1 lactobacillus strain, Staphylococcus aureus, and Escherichia coli. Three approaches were used to determine whether the IIIMan proteins were present in these bacteria: (i) Western blot (immunoblot) analysis of cytoplasmic and membrane proteins, using anti-IIIManH and anti-IIIManH rabbit polyclonal antibodies; (ii) analysis of PEP-dependent phosphoproteins by polyacrylamide gel electrophoresis; and (iii) inhibition by anti-IIIMan antibodies of the PEP-dependent phosphorylation of 2-deoxyglucose (a mannose analog) by crude cellular extracts. Only the species S. salivarius and Streptococcus vestibularis possessed the two forms of IIIMan. Fifteen other streptococcal species possessed one protein with a molecular weight between 35,200 and 38,900 that cross-reacted with both antibodies. In the case of 9 species, a protein possessing the same electrophoretic mobility was phosphorylated at the expense of PEP. No such phosphoprotein, however, could be detected in the other six species. A III(Man)-like protein with a molecular weight of 35,500 was also detected in Lactobacillus casei by Western blot experiments as well as by PEP-dependent phosphoprotein analysis, and a protein with a molecular weight of 38,900 that cross-reacted with anti-III(Man) antibodies was detected in Lactococcus lactis. In several cases, the involvement of these putative III(Man) proteins in the PEP-dependent phosphorylation of 2-deoxyglucose was substantiated by the inhibition of phosphorylation activity of anti-III(Man) antibodies. No proteins cross-reacting with anti-III(Man) antibodies were detected in enterococci, bacilli, and E. coli. In S. aureus, a membrane protein with a molecular weight of 50,000 reacted strongly with the antibodies. This protein, however, was not phosphorylated at the expense of PEP.

Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius.

Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.