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M H Saier

Publications and source records attributed to M H Saier.

At least 91 records · Page 5Linked to original sources
PubMed1997

Multidrug-resistant transport proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames with the major facilitator superfamily.

Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae reveals that 28 open reading frames (ORFs) are homologous to each other and to established bacterial members of the drug-resistant subfamily of the major facilitator superfamily. The phylogenesis of these protein sequences shows that they fall into three major clusters. Cluster I contains 12 ORFs, cluster II contains ten ORFs and cluster III contains six ORFs. Hydropathy analyses indicate that in cluster II and III ORFs, 14 transmembrane spans are predicted whereas only 12 transmembrane spans are predicted in cluster I ORFs. Three ORFs that have known functions as multidrug-resistance pumps in other yeast species such as Schizosaccharomyces pombe (CAR1), Candida albicans (BMRP) or C. maltosa (CYHR), also fall into cluster I. Two S. cerevisiae ORFs of known multidrug-resistance function (ATR1, SGE1) fall into cluster II. Cluster III consists exclusively of ORFs of unknown function but binary sequence comparisons show homology to ORFs from cluster II. Analysis of the multiple alignment for these proteins leads to the identification of characteristic signature sequences for each of the three clusters.

Amino Acid Sequence↗
PubMed1997

The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.

Amino Acid Sequence↗
PubMed1997

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.

Bacillus subtilis↗
PubMed1997

Nonplastid eukaryotic response regulators have a monophyletic origin and evolved from their bacterial precursors in parallel with their cognate sensor kinases.

We have analyzed all currently sequenced eukaryotic proteins containing either a kinase module or a receiver module, corresponding to those found in bacterial sensor kinases or response regulators, respectively, of the so-called two-component regulatory systems. We demonstrate that the eukaryotic receiver modules belong to a single subfamily of the bacterial receiver modules. Moreover, the cognate eukaryotic kinase modules exhibit a similar clustering pattern on the sensor kinase phylogenetic tree, suggesting that they evolved in parallel with the receiver modules from a common ancestral source that bore both modules. Multiple alignments of the sequences corresponding to these modules are presented and discussed, and eukaryotic-specific signature sequences are derived.

Amino Acid Sequence↗
PubMed1997

Modular multidomain phosphoryl transfer proteins of bacteria.

Recent phylogenetic and structural analyses of multidomain phosphoryl transfer proteins of bacteria have revealed that interdomain (but not intradomain) splicing and fusion, as well as domain duplication and deletion, have occurred frequently during evolution. These events have been found to be exceedingly rare in certain other protein families. Domain-shuffling events are illustrated by examples from the superfamilies of phosphoenolpyruvate-dependent sugar phosphotransferase systems, their transcriptional regulatory protein targets of phosphorylation, sensor autokinase/response regulator signal transduction systems, and permeases of the ATP-binding-cassette type.

Bacterial Proteins↗
PubMed1997

A bacterial model system for understanding multi-drug resistance.

Mankind stands at the crossroads, recognizing the need for a radical change in bacterial disease management. The development of several antimicrobial agents in the 1940s and 1950s allowed man to gain the upper hand in controlling these diseases. However, the horizon is now clouded by the activation in bacteria of cryptic multi-drug resistance (MDR) genes and the spread of plasmid- and integron-born MDR genes through bacterial populations. Unless remedial measures are taken, nearly all currently available antimicrobial agents are likely to soon lose their efficacies. We briefly review the bacterial MDR phenomenon and focus on a recently emerging family of small multi-drug resistance (SMR) pumps which may provide an ideal model system for understanding the MDR phenomenon in general.

Amino Acid Sequence↗
PubMed1997

Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases.

We have characterized a novel family of response regulator aspartyl-phosphate (RAP) phosphatases found exclusively in gram-positive bacteria. The family consists of 15 members, 12 of which are from Bacillus subtilis. The N-terminal domains proved to be more highly conserved than the C-terminal domains, and a signature sequence for the family was derived from the former domains. Phylogenetic analyses revealed clustering patterns showing that all Bacillus proteins are closely related. Most of the Bacillus RAP phosphatase genes are followed by and are translationally coupled to small nonhomologous phosphatase regulator (phr) genes that encode exported peptides with regulatory functions. Most of the paralogous RAP phosphatases of B. subtilis may serve related functions in signal transduction systems. They appear to have arisen by relatively recent gene duplication events that occurred after the divergence of major groups within the gram-positive bacterial kingdom. We suggest that the N-terminal domains of the RAP phosphatases function in catalysis, whereas the C-terminal domains function in regulation.

Amino Acid Sequence↗
PubMed1996

Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain.

Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome. We here report the presence of a third E. coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr. Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins [(127 amino acids (aa)] joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa). Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized. We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon. It may thereby provide a link between carbon and nitrogen assimilatory pathways.

Animals↗
PubMed1996

In vitro reconstitution of transcriptional antitermination by the SacT and SacY proteins of Bacillus subtilis.

Expression of the sacPA and sacB genes of Bacillus subtilis is positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively. Previous genetic studies led to the suggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes. Here we report the overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.

Amino Acid Sequence↗
PubMed1996

Cyclic AMP-independent catabolite repression in bacteria.

Until recently, only one mechanism of catabolite repression in bacteria, a mechanism dependent on the cyclic AMP receptor protein of Escherichia coli, was understood in molecular detail. Two cyclic AMP-independent catabolite repression mechanisms are currently under study. One such mechanism, found in E. coli, involves the catabolite repressor/activator (Cra) protein (formerly designated the fructose repressor FruR) which represses sugar catabolic systems and activates sugar anabolic systems. When catabolites bind to Cra, Cra dissociates from the DNA causing catabolite activation and catabolite repression, respectively. The second such mechanism, found in Bacillus subtilis, involves a catabolite-activated, ATP-dependent protein kinase that phosphorylates a specific seryl residue in the small phosphocarrier protein, HPr, of the phosphotransferase system. HPr(ser-P) binds to a transcription factor, CcpA, to promote DNA binding. DNA binding of the complex in turn promotes catabolite repression or catabolite activation, depending on the target operon. The characterization of these novel mechanisms establishes that cyclic AMP-independent catabolite control is operative in bacteria, and that multiple mechanisms of catabolite control evolved independently of each other.

Bacillus subtilis↗
PubMed1996

Function of the duplicated IIB domain and oligomeric structure of the fructose permease of Escherichia coli.

The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated IIB domain. The protein therefore consists of three distinct domains, B', B, and C (N-terminal to C-terminal), joined by flexible linkers and is thus designated FruB'BC. The N-terminal B' domain was removed using molecular genetic techniques, and the truncated Enzyme II (FruBC) was characterized relative to the wild-type enzyme both in vivo and in vitro. In vivo, FruBC exhibited depressed fermentation characteristics at low fructose concentrations. [14C]Fructose uptake measurements revealed reduced rates only when the permease was rate-limiting for transport. In vitro, FruBC exhibited a 10-fold lower affinity for its phosphoryl donating protein, the IIA-FPr diphosphoryl transfer protein (DTP), than was observed with the wild-type enzyme, and the maximal velocity of fructose phosphorylation was 7-fold depressed. Because the fructose-1-phosphate:[14C]fructose transphosphorylation reaction appeared normal, we conclude that the loss of the B' domain primarily affected phosphoryl transfer between the IIA and IIB domains of the permease. A mutant FruBC derivative with cysteine 112 replaced by serine (C112S FruBC) was inactive as a phosphoryl carrier and a sugar transport protein. Expression of the plasmid-encoded mutant protein inhibited the in vivo activity of the chromosomally encoded wild-type fructose permease, but it did not observably affect the activities of the mannitol or glucitol PTS permeases or of non-PTS sugar permeases. Further, the presence of the detergent extracted mutant protein inhibited the activity of the detergent solubilized wild-type or FruBC enzyme. In contrast, the wild-type FruB BC permease was apparently epistatic over the truncated FruBC permease in vivo. The experiments reported 1) show that the B' domain of the fructose permease functions to facilitate phosphoryl transfer between DTP and the permease, 2) establish the essentiality of cysteine 112 in the B domain of the permease, 3) provide evidence that a functional fructose permease consists of an oligomer in which both IIB domains must be active for the enzyme to catalyze normal rates of phosphoryl transfer and transport, 4) suggest that a single B' domain in the oligomeric Enzyme II is sufficient to allow high efficiency phosphoryl transfer between the IIA domain of DTP and the IIB domain of the permease, and 5) show that the B' domain is not important for oligomerization.

Bacterial Proteins↗
PubMed1996

Phylogenetic, structural and functional characteristics of the Na-K-Cl cotransporter family.

Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane alpha-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggest that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom.

Amino Acid Sequence↗
PubMed1996

Phylogenetic characterization of the MIP family of transmembrane channel proteins.

The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity for water and/or neutral solutes. We have identified 84 fully or partially sequenced members of this family, have multiply aligned over 50 representative, divergent, fully sequenced members, have used the resultant multiple alignment to derive current MIP family-specific signature sequences, and have constructed a phylogenetic tree. The tree reveals novel features relevant to the evolutionary history of this protein family. These features plus an evaluation of functional studies lead to the postulates: (i) that all current MIP family proteins derived from two divergent bacterial paralogues, one a glycerol facilitator, the other an aquaporin, and (ii) that most or all current members of the family have retained these or closely related physiological functions.

Amino Acid Sequence↗
PubMed1996

Cooperative interaction between Cra and Fnr in the regulation of the cydAB operon of Escherichia coli.

In vivo and in vitro experiments are reported demonstrating that the catabolite repressor-activator (Cra) protein (formerly designated FruR) regulates expression of the cydAB operon of Escherichia coli encoding cytochrome d oxidase. The Fnr protein is required for Cra-mediated transcriptional control, but the ArcA protein antagonizes the response to Cra. The results establish that Fnr, ArcA, and Cra exert their effects in an interdependent fashion.

Bacterial Outer Membrane Proteins↗
PubMed1996

Novel PTS proteins revealed by bacterial genome sequencing: a unique fructose-specific phosphoryl transfer protein with two HPr-like domains in Haemophilus influenzae.

The completely sequenced genome of Haemophilus influenzae has been analysed for proteins of the phosphoenolpyruvate: sugar phosphotransferase system (PTS). We show that within the fructose PTS H. influenzae possesses a novel multi-domain phosphoryl transfer protein, not previously recognized, that includes two fructose-specific HPr domains fused in tandem in a single polypeptide chain.

Bacterial Proteins↗