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

ABSTRACT The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major carbohydrate transport system in oral streptococci. The mannose-PTS of Streptococcus mutans, which transports mannose and glucose, is involved in carbon catabolite repression (CCR) and regulates the expression of known virulence genes. In this study, we investigated the role of EIIGlc and EIIABMan in sugar metabolism, gene regulation, biofilm formation, and competence. The results demonstrate that the inactivation of ptsG, encoding a putative EIIGlc, did not lead to major changes in sugar metabolism or affect the phenotypes of interest. However, the loss of EIIGlc was shown to have a significant impact on the proteome and to affect the expression of a known virulence factor, fructan hydrolase (fruA). JAM1, a mutant strain lacking EIIABMan, had an impaired capacity to form biofilms in the presence of glucose and displayed a decreased ability to be transformed with exogenous DNA. Also, the lactose- and cellobiose-PTSs were positively and negatively regulated by EIIABMan, respectively. Microarrays were used to investigate the profound phenotypic changes displayed by JAM1, revealing that EIIABMan of S. mutans has a key regulatory role in energy metabolism, possibly by sensing the energy levels of the cells or the carbohydrate availability and, in response, regulating the activity of transcription factors and carbohydrate transporters.

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Proteomic analysis of the colistin-resistant E. coli clinical isolate: Explorations of the resistome

Protein and Peptide Letters ◽

10.2174/0929866528666211129095001 ◽

2021 ◽

Vol 28 ◽

Author(s):

Divakar Sharma ◽

Manisha Aswal ◽

Nayeem Ahmad ◽

Manish Kumar ◽

Asad U Khan

Keyword(s):

Clinical Isolate ◽

Protein Interaction ◽

Functional Annotation ◽

Drug Targets ◽

Bacterial Infections ◽

Sugar Metabolism ◽

Colistin Resistance ◽

Phosphotransferase System ◽

E Coli ◽

Protein Protein Interaction

Background: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. Objective: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. Methods: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. Results: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. Conclusion: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

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Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

Applied and Environmental Microbiology ◽

10.1128/aem.01244-06 ◽

2006 ◽

Vol 73 (2) ◽

pp. 465-476 ◽

Cited By ~ 35

Author(s):

Konstantinos Papadimitriou ◽

Harris Pratsinis ◽

Gerhard Nebe-von-Caron ◽

Dimitris Kletsas ◽

Effie Tsakalidou

Keyword(s):

Single Cell ◽

Cell Sorting ◽

De Novo ◽

Acid Tolerance ◽

Logarithmic Phase ◽

Oral Streptococci ◽

Phosphotransferase System ◽

Lethal Challenge ◽

Fluorescence Characteristics

ABSTRACT An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC4(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.

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Phosphoenolpyruvate-Dependent Glucose Transport in Oral Streptococci

Journal of Dental Research ◽

10.1177/00220345730520060801 ◽

1973 ◽

Vol 52 (6) ◽

pp. 1209-1215 ◽

Cited By ~ 37

Author(s):

Charles F. Schachtele ◽

John A. Mayo

Keyword(s):

Glucose Uptake ◽

Streptococcus Mutans ◽

Glucose Transport ◽

Enzyme System ◽

Cell Suspensions ◽

Oral Streptococci ◽

Phosphotransferase System ◽

Resting Cell

Streptococcus mutans, S sanguis, and S salivarius use a phosphoenolpyruvate (PEP)-dependent phosphotransferase system that results in phosphorylation of glucose at carbon 6. This enzyme system is not sensitive to fluoride. Glucose uptake into resting cell suspensions is sensitive to fluoride because of inhibition of intracellular PEP production. The glucose phosphotransferase system is constitutive in oral streptococci.

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Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms

Applied and Environmental Microbiology ◽

10.1128/aem.00637-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3671-3682 ◽

Cited By ~ 17

Author(s):

Lin Zeng ◽

Tanaz Farivar ◽

Robert A. Burne

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Sugar Metabolism ◽

Amino Sugar ◽

Amino Sugars ◽

Mrna Levels ◽

Oral Streptococci ◽

Streptococcus Gordonii ◽

Carbohydrate Source ◽

Content Type

ABSTRACTBiochemical and genetic aspects of the metabolism of the amino sugarsN-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogenStreptococcus mutanswere explored. MultipleS. mutanswild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms betweenS. mutansandStreptococcus gordoniishowed thatS. gordoniiwas particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced inS. mutansthan inS. gordonii. Exposure to H2O2, which is produced byS. gordoniiand antagonizes the growth ofS. mutans, led to reduced mRNA levels ofnagAandnagBinS. mutans. When the gene for the transcriptional regulatory NagR was deleted inS. gordonii, the strain produced constitutively high levels ofnagA(GlcNAc-6-P deacetylase),nagB(GlcN-6-P deaminase), andglmS(GlcN-6-P synthase) mRNA. Similar to NagR ofS. mutans(NagRSm), theS. gordoniiNagR protein (NagRSg) could bind to consensus binding sites (dre) in thenagA,nagB, andglmSpromoter regions ofS. gordonii. Notably, NagRSgbinding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm. This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries.IMPORTANCEAmino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much greater capacity to utilize amino sugars than the dental pathogenStreptococcus mutansand that the ability of the model commensalStreptococcus gordoniito compete againstS. mutansis substantively enhanced by the presence of amino sugars commonly found in the oral cavity. The mechanisms underlying the greater capacity and competitive enhancements of the commensal are shown to depend on how the genes for the catabolic enzymes are regulated, the role of the allosteric modulators affecting such regulation, and the ability of amino sugars to enhance certain activities of the commensal that are antagonistic toS. mutans.

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Difference in Amounts between Titratable Acid and Total Carboxylic Acids Produced by Oral Streptococci during Sugar Metabolism

Journal of Dental Research ◽

10.1177/00220345890680010101 ◽

1989 ◽

Vol 68 (1) ◽

pp. 16-19 ◽

Cited By ~ 8

Author(s):

Y. Iwami ◽

S. Hata ◽

N. Takahashi ◽

T. Yamada

Keyword(s):

Carboxylic Acid ◽

Carboxylic Acids ◽

Sugar Metabolism ◽

Resting Cells ◽

Oral Streptococci ◽

Titratable Acid ◽

Ph Stat ◽

Carbonic Acids ◽

The Difference ◽

Pyruvic Acids

The acid produced by the resting cells of Streptococcus mutans NCTC 10449 and HS 6 and S. sanguis ATCC 10556 during sugar metabolism was estimated with a pH-stat and a carboxylic acid analyzer. Lactic, formic, acetic, pyruvic, and carbonic acids were detected in the reaction mixtures, but propionic, citric, succinic, iso-butyric, butyric, iso-valeric, and valeric acids were not detected. The amount of titratable acid estimated by alkaline titration with the pH-stat was larger than the amount of total carboxylic acids estimated with the carboxylic acid analyzer. The difference in quantity between the titratable and the total carboxylic acids increased significantly with an increase in the period of incubation with sugar. Moreover, the value of the alkaline titration of standard lactic, formic, acetic, and pyruvic acids was equal to the amount analyzed with the carboxylic acid analyzer. The results indicated that these two streptococci produced not only these carboxylic acids but also other acid(s), possibly non-carboxylic acid(s), during their sugar metabolism.

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Changes in Glycolytic Activity of Lactococcus lactis Induced by Low Temperature

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3686-3691.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3686-3691 ◽

Cited By ~ 56

Author(s):

Jeroen A. Wouters ◽

Henrike H. Kamphuis ◽

Jeroen Hugenholtz ◽

Oscar P. Kuipers ◽

Willem M. de Vos ◽

...

Keyword(s):

Low Temperature ◽

Lactococcus Lactis ◽

Low Temperatures ◽

Cold Adaptation ◽

Cold Shock ◽

Protein A ◽

Sugar Metabolism ◽

Regulatory Function ◽

Phosphotransferase System ◽

Glycolytic Activity

ABSTRACT The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30Ã¯Â¿Â½C increased approximately 2.5-fold following a shift from 30 to 10Ã¯Â¿Â½C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The lasoperon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

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Two Closely Related ABC Transporters in Streptococcus mutans Are Involved in Disaccharide and/or Oligosaccharide Uptake

Journal of Bacteriology ◽

10.1128/jb.01509-07 ◽

2007 ◽

Vol 190 (1) ◽

pp. 168-178 ◽

Cited By ~ 44

Author(s):

Alexander J. Webb ◽

Karen A. Homer ◽

Arthur H. F. Hosie

Keyword(s):

Streptococcus Mutans ◽

Abc Transporters ◽

Sole Carbon Source ◽

Binding Protein ◽

Sugar Metabolism ◽

System Component ◽

Phosphotransferase System ◽

Atpase Subunits ◽

Membrane Components ◽

Distinct Membrane

ABSTRACT Streptococcus mutans has a large number of transporters apparently involved in the uptake of carbohydrates. At least two of these, the multiple sugar metabolism transporter, MsmEFGK, and the previously uncharacterized MalXFGK, are members of the ATP-binding cassette (ABC) superfamily. Mutation analysis revealed that the MsmEFGK and MalXFGK transporters are principally involved in the uptake of distinct disaccharides and/or oligosaccharides. Furthermore, the data also indicated an unusual protein interaction between the components of these two related transporters. Strains lacking msmE (which encodes a solute binding protein) can no longer utilize raffinose or stachyose but grow normally on maltodextrins in the absence of MalT, a previously characterized EIImal phosphotransferase system component. In contrast, a mutant of malX (which encodes a solute binding protein) cannot utilize maltodextrins but grows normally on raffinose or stachyose. Radioactive uptake assays confirmed that MalX, but not MsmE, is required for uptake of [U-14C]maltotriose and that MalXFGK is principally involved in the uptake of maltodextrins with as many as 7 glucose units. Surprisingly, inactivation of the corresponding ATPase components did not result in an equivalent abolition of growth: the malK mutant can grow on maltotetraose as a sole carbon source, and the msmK mutant can utilize raffinose. We propose that the ATPase domains of these ABC transporters can interact with either their own or the alternative transporter complex. Such unexpected interaction of ATPase subunits with distinct membrane components to form complete multiple ABC transporters may be widespread in bacteria.

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Galactose Metabolism by Streptococcus mutans

Applied and Environmental Microbiology ◽

10.1128/aem.70.10.6047-6052.2004 ◽

2004 ◽

Vol 70 (10) ◽

pp. 6047-6052 ◽

Cited By ~ 36

Author(s):

Jacqueline Abranches ◽

Yi-Ywan M. Chen ◽

Robert A. Burne

Keyword(s):

Streptococcus Mutans ◽

Wild Type Strain ◽

Sugar Metabolism ◽

Growth Defect ◽

Wild Type ◽

Phosphotransferase System ◽

Galactose Metabolism ◽

Gene Encoding ◽

Leloir Pathway ◽

In The Wild

ABSTRACT The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.

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Involvement of an Inducible Fructose Phosphotransferase Operon in Streptococcus gordonii Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.185.21.6241-6254.2003 ◽

2003 ◽

Vol 185 (21) ◽

pp. 6241-6254 ◽

Cited By ~ 53

Author(s):

C. Y. Loo ◽

K. Mitrakul ◽

I. B. Voss ◽

C. V. Hughes ◽

N. Ganeshkumar

Keyword(s):

Biofilm Formation ◽

Transcriptional Repressor ◽

Oral Streptococci ◽

Streptococcus Gordonii ◽

Galactosidase Activity ◽

Phosphotransferase System ◽

Reading Frame ◽

Transposon Insertion ◽

Catabolite Control

ABSTRACT Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3′ end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation. Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm. Expression of fruK, as measured by the β-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in β-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms.

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