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M H Saier

Publications and source records attributed to M H Saier.

At least 109 records · Page 6Linked to original sources
PubMed1996

Structural and phylogenetic analysis of the MotA and MotB families of bacterial flagellar motor proteins.

MotA and MotB are two well-characterized proteins in Escherichia coli which are believed to function as the proton channel and the anchor, respectively, of the motor component of the bacterial flagellum. We have identified and analysed all currently sequenced members of the MotA and MotB families. Members of these families include (1) these E. coli proteins, (2) their pmf-interacting motor homologues in other bacteria, (3) two ORFs which map downstream of the gene encoding the catabolite repression-mediating CepA protein in Bacillus species and (4) unidentified open reading frames. With one exception (the MotB protein of Rhodobactec sphaeroides), members of the MotB family exhibit a C-terminal domain that is homologous to peptidoglycan-interaction domains of numerous sequenced lipoproteins and outer membrane proteins. Multiple alignments, average hydropathy and similarity plots, and phylogenetic trees have allowed (1) identification of regions of relative conservation, (2) definition of signature sequences for these protein families and (3) determination of relative phylogenetic distances relating all members of each family. The phylogenies of these proteins do not follow those of the organisms from which they were isolated, suggesting the presence of divergent isoforms in many bacteria. Phylogenetic analyses of the peptidoglycan-interaction domains of MotB proteins indicated that, except for MotB of R. sphaeroides, these domains became associated with the MotB proteins early during evolutionary history, before members of the MotB family or members of the outer membrane protein family diverged from each other.

Amino Acid Sequence↗
PubMed1996

Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system.

Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae, regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzae ptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild-type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP-dependent sugars was restored by addition of cAMP. In wild-type cells the non-metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250-fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose-specific PTS.

Adenylyl Cyclases↗
PubMed1996

A simple flexible program for the computational analysis of amino acyl residue distribution in proteins: application to the distribution of aromatic versus aliphatic hydrophobic amino acids in transmembrane alpha-helical spanners of integral membrane transport proteins.

We describe a simple, flexible program (AAD) with a primary function of depicting the distribution of aliphatic and aromatic amino acid residues along the linear aligned sequence of a family of homologous proteins and a secondary function of depicting the distribution of all amino acids along the same linear sequence. The program is used to examine the distribution of aromatic versus aliphatic residues in representative well-characterized families of polytopic membrane proteins. Many but not all such protein families are shown to exhibit a predominance of aliphatic residues in the central regions of their transmembrane spanners but a predominance of aromatic residues at the peripheries of their spanners. We propose that this distribution stabilizes the hydrophobic-hydrophilic interface and renders the centers of these integral membrane proteins more fluid than their peripheries.

Amino Acids↗
PubMed1996

Phylogenetic approaches to the identification and characterization of protein families and superfamilies.

With the advent of megabase genome sequencing, the need for computational analyses increases exponentially. Sequencing errors must be corrected, encoded proteins must be identified, functions must be assigned to these proteins, and distant phylogenetic relationships must be recognized in order to maximize the yield of information obtainable from genome sequencing projects. Both the computer and the human brain have their limitations, but using them in combination, the biologist can vastly extend his or her analytic capabilities. Computer techniques can be used to estimate protein structure, function, biogenesis, and evolution. In this review, the application of available computer programs to several protein families, particularly transport, receptor, and transcriptional regulatory protein families, illustrate our current capabilities and limitations. Although some multidomain protein families are evolutionarily homogeneous, others have mosaic origins. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific protein families is evaluated. It is shown that specific families of enzymes, receptors, transport proteins, and transcriptional regulatory proteins share a common evolutionary origin, frequently diverging in function because of domain splicing and ligation. Some large families arose gradually over evolutionary time, whereas others developed suddenly, due to bursts of intragenic or intergenic (or both) duplication events occurring over relatively short periods of time. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is also 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.

Bacterial Proteins↗
PubMed1996

Novel phosphotransferase system genes revealed by bacterial genome analysis: the complete complement of pts genes in mycoplasma genitalium.

The complete sequence of the Mycoplasma genitalium chromosome has recently been determined. We here report analyses of the genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system, PTS. These genes encode (1) Enzyme I, (2) HPr, (3) a glucose-specific Enzyme IICBA, (4) an inactive glucose-specific Enzyme IIB, lacking the active site cysteyl residue, and (5) a fructose-specific Enzyme IIABC. Some of the unique features of these genes and their enzyme products are as follows. (1) Each of the genes is encoded within a distinct operon. (2) Both Enzyme I and HPr have basic isoelectric points. (3) The glucose-specific Enzyme IIC bears a centrally located, hydrophilic, 200 amino acyl residue insert that lacks sequence similarity with any protein in the current database. (4) The fructose-specific Enzyme II has a domain order (IIABC), different from those of previously characterized fructose permeases, and its IIA domain more closely resembles the IIANtr protein of Escherichia coli than other fructose-specific IIA domains. The potential significance of these novel features is discussed.

Bacterial Proteins↗
PubMed1996

The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs.

The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic alpha-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.

Amino Acid Sequence↗
PubMed1996

Identification of an anaerobically induced phosphoenolpyruvate-dependent fructose-specific phosphotransferase system and evidence for the Embden-Meyerhof glycolytic pathway in the heterofermentative bacterium Lactobacillus brevis.

Heterofermentative gram-positive bacteria are believed to metabolize sugars exclusively via the pentose phosphoketolase pathway following uptake via sugar:cation symport. Here we show that anaerobic growth of one such bacterium, Lactobacillus brevis, in the presence of fructose induces the synthesis of a phosphotransferase system and glycolytic enzymes that allow fructose to be metabolized via the Embden-Meyerhof pathway.

Aerobiosis↗
PubMed1996

Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr.

By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion.

Adenosine Triphosphate↗
PubMed1996

Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.

Bacillus subtilis↗
PubMed1996

Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.

Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.

Allosteric Regulation↗
PubMed1996

Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.

Acetylglucosamine↗
PubMed1995

Phylogenetic, structural and functional analyses of the LacI-GalR family of bacterial transcription factors.

Phylogenetic tree construction for 25 sequenced members of the LacI-GalR family (LGF) of transcription factors revealed that almost all branches are similar in length, radiating essentially from a single point. This observation suggests that most of these proteins arose by duplication events which occurred at a specific time in evolutionary history, and that further duplication events were rare. Analyses of the multiple alignment of the LGF proteins lead to suggestions regarding structure-function relationships and reveal that the helix-turn-helix DNA-binding motif of LGF proteins is similar in sequence to those of numerous non-homologous DNA-binding proteins.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a small membrane-associated sugar phosphate phosphatase that is allosterically activated by HPr(Ser(P)) of the phosphotransferase system in Lactococcus lactis.

In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate. The enzyme exhibits a Ka for S46D HPr of 15 microM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.

Allosteric Regulation↗
PubMed1995

In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of gram-positive bacteria.

Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the cis-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.

Bacillus megaterium↗