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M H Saier

Publications and source records attributed to M H Saier.

At least 127 records · Page 7Linked to original sources

Differential codon usage: a safeguard against inappropriate expression of specialized genes?

Recent work has suggested that rare codons are sometimes used for the regulation of specialized gene expression in bacteria. Moreover, the cellular levels of certain tRNAs may fluctuate with growth conditions. Evidence implicating such mechanisms in the control of photosynthesis in Rhodobacter, solventogenesis in Clostridium, sporulation in Streptomyces, and fimbrial phase variation in E. coli is summarized. It is suggested that such mechanisms will prove applicable to the control of numerous additional specialized functions, and that the empirical tools for testing this possibility are currently available.

Bacteria↗

Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant.

Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

Amino Acid Sequence↗

Effect of the FruR regulator on transcription of the pts operon in Escherichia coli.

The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRP.cAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRP.cAMP-binding sites. The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRP.cAMP, and restore P1a transcription, which is repressed by CRP.cAMP. FruR can displace CRP.cAMP from its binding site in the presence of RNA polymerase even though FruR and CRP.cAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR- mutant cells as in cells grown in the presence of glucose. These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.

Bacterial Proteins↗

Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.

Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell.

Allosteric Regulation↗

Phosphorylation of serine-46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure.

The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N-cap of alpha-helix-B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a delta delta G of 0.7-0.8 kcal mol-1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.

Amino Acid Sequence↗

Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution.

Response regulators of bacterial sensory transduction systems generally consist of receiver module domains covalently linked to effector domains. The effector domains include DNA binding and/or catalytic units that are regulated by sensor kinase-catalyzed aspartyl phosphorylation within their receiver modules. Most receiver modules are associated with three distinct families of DNA binding domains, but some are associated with other types of DNA binding domains, with methylated chemotaxis protein (MCP) demethylases, or with sensor kinases. A few exist as independent entities which regulate their target systems by noncovalent interactions. In this study the molecular phylogenies of the receiver modules and effector domains of 49 fully sequenced response regulators and their homologues were determined. The three major, evolutionarily distinct, DNA binding domains found in response regulators were evaluated for their phylogenetic relatedness, and the phylogenetic trees obtained for these domains were compared with those for the receiver modules. Members of one family (family 1) of DNA binding domains are linked to large ATPase domains which usually function cooperatively in the activation of E. coli sigma 54-dependent promoters or their equivalents in other bacteria. Members of a second family (family 2) always function in conjunction with the E. coli sigma 70 or its equivalent in other bacteria. A third family of DNA binding domains (family 3) functions by an uncharacterized mechanism involving more than one sigma factor. These three domain families utilize distinct helix-turn-helix motifs for DNA binding. The phylogenetic tree of the receiver modules revealed three major and several minor clusters of these domains. The three major receiver module clusters (clusters 1, 2, and 3) generally function with the three major families of DNA binding domains (families 1, 2, and 3, respectively) to comprise three classes of response regulators (classes 1, 2, and 3), although several exceptions exist. The minor clusters of receiver modules were usually, but not always, associated with other types of effector domains. Finally, several receiver modules did not fit into a cluster. It was concluded that receiver modules usually diverged from common ancestral protein domains together with the corresponding effector domains, although domain shuffling, due to intragenic splicing and fusion, must have occurred during the evolution of some of these proteins. Multiple sequence alignments of the 49 receiver modules and their various types of effector domains, together with other homologous domains, allowed definition of regions of striking sequence similarity and degrees of conservation of specific residues. Sequence data were correlated with structure/function when such information was available.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗

Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-alpha and ETF-beta.

Electron transfer flavoproteins (ETF) are alpha beta-heterodimers found in eukaryotic mitochondria and bacteria. We have identified currently sequenced protein members of the ETF-alpha and ETF-beta families. Members of these two families include (a) the ETF subunits of mammals and bacteria, (b) homologous pairs of proteins (FixB/FixA) that are essential for nitrogen fixation in some bacteria, and (c) a pair of carnitine-inducible proteins encoded by two open reading frames in Escherichia coli (YaaQ and YaaR). These three groups of proteins comprise three clusters on both the ETF-alpha and ETF-beta phylogenetic trees, separated from each other by comparable phylogenetic distances. This fact suggests that these two protein families evolved with similar overall rates of evolutionary divergence. Relative regions of sequence conservation are evaluated, and signature sequences for both families are derived.

Animals↗

Phylogenetic analyses of the ATP-binding constituents of bacterial extracytoplasmic receptor-dependent ABC-type nutrient uptake permeases.

Thirty-eight ATP-binding cassette (ABC) protein constituents of bacterial extracytoplasmic receptor-dependent nutrient uptake systems, including one homologous chloroplast protein were analysed for sequence conservation and phylogenetic relatedness. The proteins were generally found to cluster in accordance with the clustering patterns previously observed for the extracytoplasmic receptors and the transmembrane channel-forming constituents of these permeases. The results suggest that these transport systems evolved from a single primordial system with minimal shuffling of the three dissimilar protein constituents of the systems.

ATP-Binding Cassette Transporters↗

Protein phosphorylation and regulation of carbon metabolism in gram-negative versus gram-positive bacteria.

Bacteria impose regulatory mechanisms on metabolic processes to ensure that the needs of the cell are met but not exceeded. Here, we discuss the basic features of a mechanism by which carbohydrate catabolism in Gram-positive bacteria is regulated. Although the physiological consequences of this regulation are the same as in Gram-negative bacteria, the mechanism is entirely different. These regulatory processes evidently evolved late, after the divergence of Gram-negative bacteria, even though the targets of regulation are universal.

Bacterial Proteins↗

Identification and characterization of phosphoenolpyruvate:fructose phosphotransferase systems in three Streptomyces species.

Streptomyces lividans, S. coelicolor and S. griseofuscus were examined for the presence of the enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). All three species were shown to possess Enzyme I, HPr and fructose-specific Enzyme II (IIFru) activities. In S. lividans and S. coelicolor, all three PTS enzymes were fructose-inducible, but in S. griseofuscus the system was expressed constitutively. These organisms apparently lack the HPr(Ser) kinase and HPr(Ser-P) phosphatase that characterize low-GC Gram-positive bacteria.

Bacillus subtilis↗

Novel phosphotransferase system genes revealed by bacterial genome analysis--a gene cluster encoding a unique Enzyme I and the proteins of a fructose-like permease system.

Previous publications have demonstrated the presence of a cryptic gene encoding a novel Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Recent Escherichia coli genome sequencing revealed a gene (ptsA) encoding a new Enzyme I homologue in the 89.1-89.3 centisome region. We have analysed this region, and here describe and characterize open reading frames (ORFs) encoding (1) a fused PTS Enzyme I-IIAFru homologue, (2) a glycerol dehydrogenase, (3) a transaldolase homologue, (4) two PTS IIBFru homologues, (5) a PTS IICFru homologue, and (6) homologues of pyruvate formate-lyase and its activating enzyme. Binary comparison scores, multiple alignments and phylogenetic trees establish the families of proteins to which each of the relevant ORFs belong. Identification of the putative products of this gene cluster leads to the proposal that several of the proteins encoded in this region function in anaerobic carbon metabolism.

Acetyltransferases↗

Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms.

Streptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme IIs for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl beta-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlc rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant.

'Sphingomonas (formerly Pseudomonas) elodea' produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from 'S. elodea' and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.

Amino Acid Sequence↗

The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli.

The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator-containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR-binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.

Bacterial Proteins↗

Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion.

Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion.

Bacterial Proteins↗

Allosteric regulation of the glucose:H+ symporter of Lactobacillus brevis: cooperative binding of glucose and HPr(ser-P).

Lactobacillus brevis transports glucose and the nonmetabolizable glucose analog 2-deoxyglucose via a proton symport mechanism that is allosterically inhibited by the seryl-phosphorylated derivative of HPr, the small phosphocarrier protein of the phosphotransferase system. We have demonstrate that S46DHPr, a mutant analog of HPr which conformationally resembles HPr(ser-P) but not free HPr, specifically binds to membranes derived from glucose-grown L. brevis cells if and only if a substrate of the glucose permease is also present.

Allosteric Regulation↗