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M H Saier

Publications and source records attributed to M H Saier.

At least 163 records · Page 9Linked to original sources
PubMed1994

Regulation of 2-deoxyglucose phosphate accumulation in Lactococcus lactis vesicles by metabolite-activated, ATP-dependent phosphorylation of serine-46 in HPr of the phosphotransferase system.

Lactococcus lactis takes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactis vesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. Intravesicular but not extravesicular wild-type HPr of Bacillus subtilis alone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DG-P. Intravesicular H15A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. Intravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The S46D mutant HPr protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km value for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli.

Repression of tryptophanase (tryptophan indole-lyase) by glucose and its non-metabolizable analogue methyl alpha-glucoside has been studied employing a series of isogenic strains of Escherichia coli lacking cyclic AMP phosphodiesterase and altered for two of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), Enzyme I and Enzyme IIAGlc. Basal activity of tryptophanase was depressed mildly by inclusion of glucose in the growth medium, but inducible tryptophanase synthesis was subject to strong glucose repression in the parental strain, which exhibited normal PTS enzyme activities. Methyl alpha-glucoside was without effect in this strain. Loss of Enzyme I decreased sensitivity to repression by glucose but enhanced sensitivity to repression by methyl alpha-glucoside. Loss of Enzyme IIAGlc activity largely abolished repression by methyl alpha-glucoside but had a less severe effect on glucose repression. The repressive effects of both sugars were fully reversed by inclusion of cyclic AMP in the growth medium. Tryptophan uptake under the same conditions was inhibited weakly by glucose and more strongly by methyl alpha-glucoside in the parental strain. Inhibition by both sugars was alleviated by partial loss of Enzyme I. Inhibition by methyl alpha-glucoside appeared to be largely due to energy competition and was not responsible for repression of tryptophanase synthesis. Measurement of net production of cyclic AMP as well as intracellular concentrations of cyclic AMP revealed a good correlation with intensity of repression. The results suggest that while basal tryptophanase synthesis is relatively insensitive to catabolite repression, inducible synthesis is subject to strong repression by two distinct mechanisms, one dependent on enzyme IIAGlc of the PTS and the other independent of this protein. Both mechanisms are attributable to depressed rates of cyclic AMP synthesis. No evidence for a cyclic-AMP-independent mechanism of catabolite repression was obtained.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria.

We have characterized a new family of proteins (the ROK family) which includes six transcriptional repressors for sugar catabolic operons, three sugar kinases, and three unidentified open reading frames. Analysis of the aligned sequences and phylogenetic tree construction allow predictions regarding the functional nature of conserved domains and residues within these proteins as well as the pathway of evolutionary divergence that gave rise to the family.

Amino Acid Sequence↗
PubMed1994

Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport.

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) includes (i) two metal-resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co-operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram-negative bacterial cell envelope.

Alcaligenes↗
PubMed1994

The bacterial phosphotransferase system: new frontiers 30 years later.

In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Thirty years later, we find that the PTS functions not only as a sugar-phosphorylating system, but also as a complex protein kinase system that regulates a wide variety of metabolic processes and controls the expression of numerous genes. As a result of recent operon- and genome-sequencing projects, novel PTS protein-encoding genes have been discovered, most of which have yet to be functionally defined. Some of them appear to be involved in cellular processes distinct from those recognized previously. Fundamental aspects of past and current PTS research are briefly reviewed, and recent advances are integrated into conceptual pictures that provide guides for future research.

Bacteria↗
PubMed1994

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.

Bacillus subtilis↗
PubMed1994

Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis.

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.

Adenosine Triphosphate↗
PubMed1994

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.

Amino Acid Sequence↗
PubMed1994

Regulation of the raffinose permease of Escherichia coli by the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system.

In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system. We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition.

Allosteric Regulation↗
PubMed1994

The mannitol repressor (MtlR) of Escherichia coli.

The mannitol operon of Escherichia coli, encoding the mannitol-specific enzyme II of the phosphotransferase system (Mt1A) and mannitol phosphate dehydrogenase (Mt1D), is here shown to contain a single additional downstream open reading frame which encodes the mannitol repressor (Mt1R). Mt1R contains 195 amino acids and has a calculated molecular weight of 21,990 and a calculated pI of 4.5. It is homologous to the product of an open reading frame (URF2D) upstream of the E. coli gapB gene but represents a novel type of transcriptional regulatory protein.

Amino Acid Sequence↗
PubMed1994

Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution.

Three-dimensional structures have been elucidated for very few integral membrane proteins. Computer methods can be used as guides for estimation of solute transport protein structure, function, biogenesis, and evolution. In this paper the application of currently available computer programs to over a dozen distinct families of transport proteins is reviewed. The reliability of sequence-based topological and localization analyses and the importance of sequence and residue conservation to structure and function are evaluated. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific transport protein families is also evaluated. Channel proteins are proposed to be functionally related to carriers. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is shown that several transport protein families have evolved independently of each other, employing different routes, at different times in evolutionary history, to give topologically similar transmembrane protein complexes. The possible significance of this apparent topological convergence is discussed.

Amino Acid Sequence↗
PubMed1994

DNA sequence of a gene in Escherichia coli encoding a putative tripartite transcription factor with receiver, ATPase and DNA binding domains.

We have sequenced downstream of the last previously sequenced gene of the glucitol operon (gutABDMRQ) in E. coli and have found that gutQ is the last gene of this operon. Downstream of the gutQ gene is found a palindromic unit (PU or REP sequence), followed by a large open reading frame of 1515 (or possibly 1590) bps transcribed in the direction opposite to that of the gut operon. This open reading frame encodes a protein of 504 (or possibly 529) amino acids with a tripartite structure. The N-terminal "receiver" domain of 187 (or possibly 212) residues is homologous to the FhlA protein of E. coli, a transcriptional activator of formate hydrogen lyase. It may possess a short domain at its extreme N-terminus exhibiting sequence similarity to carbohydrate binding proteins. The central ATPase domain (236 residues) exhibits greatest sequence similarity to the HydG protein of E. coli, a transcriptional activator of labile hydrogenase. The C-terminal DNA binding domain (81 residues) is homologous to NtrX of Azorhizobium caulinodans, a protein involved in transcriptional regulation of nitrogen fixation. Sequence comparisons with well-characterized transcription factors suggest that ORF504 encodes a protein that hydrolyzes ATP to generate the open transcriptional initiation complex of sigma 54-dependent promoters, possibly in response to redox conditions and/or ligand binding. We propose that this tripartite transcription factor arose by fusion of gene fragments encoding its three constituent modules.

Adenosine Triphosphatases↗
PubMed1993

In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium.

Evidence has been presented suggesting that the fructose repressor, FruR, is a pleiotropic transcriptional regulatory protein controlling the expression of numerous operons concerned with carbon metabolism in Escherichia coli and Salmonella typhimurium. We have conducted in vitro DNA binding studies to ascertain the nature of the DNA sequences to which FruR binds. Employing both DNA band migration retardation and DNase I footprint analyses, FruR was found to bind to two operators within the regulatory region preceding the structural genes of the fructose operon, fruB(MH)KA. These two operators, O1 and O2, comprise nearly identical palindromes of 12 bp with a half-site of TGAAAC. The binding of FruR to these inverted repeats was found to be reversed by inclusion of micromolar concentrations of fructose-1-phosphate. The two operators are located between the single putative promoter of the fructose operon and the translational initiation site of the fruB gene. Other regulated operons were shown to bind FruR to a single site upstream of the first structural gene as follows: (1) ppsA (positive regulation); (2) icd (positive regulation); (3) aceB (positive regulation); and (4) pts (negative regulation). In all cases, low concentrations of fructose-1-phosphate displaced the protein from the DNA. The binding sites were determined, and a FruR consensus sequence was established. Computer searches revealed the presence of this sequence in numerous functionally diverse operons, implying that FruR is a global transcriptional regulatory protein in enteric bacteria.

Allosteric Regulation↗
PubMed1993

Mapping of the binding interfaces of the proteins of the bacterial phosphotransferase system, HPr and IIAglc.

Enzyme IIAglc and HPr are central regulatory and phosphocarrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of bacteria. During phosphoryl transfer from phosphoenolpyruvate to glucose, phosphate is transferred from HPr to enzyme IIAglc. In order to characterize the binding interfaces of the two proteins during phosphate transfer, 15N-edited and 15N-filtered NMR experiments have been recorded for the complex of enzyme IIAglc and HPr from Bacillus subtilis. Uniformly 15N-labeled enzyme IIAglc and nonlabeled HPr were used in these studies. Residues which undergo significant chemical shift changes upon complex formation have been identified for both proteins. The binding interfaces of the two proteins, suggested by the observed chemical shift changes, involve predominantly hydrophobic surfaces near the active site His-15 of HPr and the phosphoryl acceptor His-83 of IIAglc.

Bacillus subtilis↗
PubMed1993

Introduction: protein phosphorylation and signal transduction in bacteria.

A single type of reversible protein-phosphorylating system, the ATP-dependent protein kinase/phosphatase system, is employed in signal transduction in eukaryotes. By contrast, recent work has revealed that three types of protein-phosphorylating systems mediate signal transduction in bacteria. These systems are (1) classical protein kinase/phosphatase systems, (2) sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological, structural, and mechanistic aspects of these three evolutionarily distinct systems are discussed in the papers of this written symposium.

Adenosine Triphosphate↗
PubMed1993

Regulatory interactions involving the proteins of the phosphotransferase system in enteric bacteria.

Sugar uptake and cytoplasmic inducer generation as well as cyclic AMP synthesis are regulated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Gram-negative enteric bacteria. In these organisms, the free form of the glucose-specific Enzyme IIA (IIAglc) of the PTS, which can be phosphorylated on a histidyl residue by PEP and the PTS energy coupling proteins, inhibits the activities of non-PTS carbohydrate permeases and catabolic enzymes. By contrast, the phosphorylated form of IIAglc appears to activate adenylate cyclase, the cyclic AMP biosynthetic enzyme. What is known of the molecular details of these regulatory interactions will be summarized, and a novel regulatory mechanism involving the fructose repressor, FruR, which controls the transcription of genes encoding enzymes which catalyze reactions in central pathways of carbon metabolism, will be presented.

ATP-Binding Cassette Transporters↗
PubMed1993

Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria.

We demonstrate that three integral membrane receptors of mammals--the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T-cell early activator (Tea)--are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family. We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single-celled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.

Amino Acid Sequence↗