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M H Saier

Publications and source records attributed to M H Saier.

At least 73 records · Page 4Linked to original sources
PubMed1998

Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae.

We present the complete inventory of currently recognized and putative transporters encoded within the genome of Saccharomyces cerevisiae. These 258 transporters are classified into 42 families according to phylogenetic and substrate specificity criteria. Twelve of these yeast families are found only in eukaryotic organisms, and four are so far unique to yeast. Putative yeast-specific families transport heavy metals, arsenite and calcium. The phylogenetic analyses reported allow classification of 139 functionally uncharacterized yeast transporters into families of known functions. The relative proportions of yeast transporters specific for different classes of substrates differ only slightly from those reported for Escherichia coli. However, the ratio of secondary transporters (uniporters, cation symporters and antiporters) to primary ATP-driven transporters is much higher for yeast than for bacteria.

Carrier Proteins↗
PubMed1998

The three-dimensional structure of the nitrogen regulatory protein IIANtr from Escherichia coli.

The bacterial rpoN operon codes for sigma 54, which is the key sigma factor that, under nitrogen starvation conditions, activates the transcription of genes needed to assimilate ammonia and glutamate. The rpoN operon contains several other open reading frames that are cotranscribed with sigma 54. The product of one of these, the 17.9 kDa protein IIANtr, is homologous to IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. IIANtr influences the transcription of sigma 54-dependent genes through an unknown mechanism and may thereby provide a regulatory link between carbon and nitrogen metabolism. Here we describe the 2.35 A X-ray structure of Escherichia coli IIANtr. It is the first structure of a IIA enzyme from the fructose-mannitol family of the PTS. The enzyme displays a novel fold characterized by a central mixed parallel/anti-parallel beta-sheet surrounded by six alpha-helices. The active site His73 is situated in a shallow depression on the protein surface.

Amino Acid Sequence↗
PubMed1998

High-resolution solution structure of Bacillus subtilis IIAglc.

The high-resolution solution structure of the phosphocarrier protein IIAglc from Bacillus subtilis is determined using 3D and 4D heteronuclear NMR methods. B. subtilis IIAglc contains 162 amino acid residues and is one of the larger proteins for which high-resolution solution structure has been determined by NMR methods. The structures have been calculated from a total of 2,232 conformational constraints. Comparison with the X-ray crystal structure indicates that the overall fold is the same in solution and in crystalline environments, although some local structural differences are observed. These occur largely in turns and loops, and mostly correspond to regions with high-temperature factors in the crystal structure. The N-terminus of IIAglc is disordered in solution. The active site is located in a concave region of the protein surface. The histidine, which accepts the phosphoryl group (His 83), interacts with a neighboring histidine (His 68) and is surrounded by hydrophobic residues.

Amino Acid Sequence↗
PubMed1998

Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities.

We have conducted genome sequence analyses of seven prokaryotic microorganisms for which completely sequenced genomes are available (Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Mycoplasma genitalium, Synechocystis PCC6803 and Methanococcus jannaschii). We report the distribution of encoded known and putative polytopic cytoplasmic membrane transport proteins within these genomes. Transport systems for each organism were classified according to (1) putative membrane topology, (2) protein family, (3) bioenergetics, and (4) substrate specificities. The overall transport capabilities of each organism were thereby estimated. Probable function was assigned to greater than 90% of the putative transport proteins identified. The results show the following: (1) Numbers of transport systems in eubacteria are approximately proportional to genome size and correspond to 9.7 to 10.8% of the total encoded genes except for H. pylori (5.4%), Synechocystis (4.7%) and M. jannaschii (3.5%) which exhibit substantially lower proportions. (2) The distribution of topological types is similar in all seven organisms. (3) Transport systems belonging to 67 families were identified within the genomes of these organisms, and about half of these families are also found in eukaryotes. (4) 12% of these families are found exclusively in Gram-negative bacteria, but none is found exclusively in Gram-positive bacteria, cyanobacteria or archaea. (5) Two superfamilies, the ATP-binding cassette (ABC) and major facilitator (MF) superfamilies account for nearly 50% of all transporters in each organism, but the relative representation of these two transporter types varies over a tenfold range, depending on the organism. (6) Secondary, pmf-dependent carriers are 1.5 to threefold more prevalent than primary ATP-dependent carriers in E. coli, H. influenzae, H. pylori and B. subtilis while primary carriers are about twofold more prevalent in M. genitalium and Synechocystis. M. jannaschii exhibits a slight preference for secondary carriers. (7) Bioenergetics of transport generally correlate with the primary forms of energy generated via available metabolic pathways but ecological niche and substrate availability may also be determining factors. (8) All organisms display a similar range of transport specificities with quantitative differences presumably reflective of disparate ecological niches. (9) M. jannaschii and Synechocystis have a two to threefold increased proportion of transporters for inorganic ions with a concomitant decrease in transporters for organic compounds. (10) 6 to 18% of all transporters in these bacteria probably function as drug export systems showing that these systems are prevalent in non-pathogenic as well as pathogenic organisms. (11) All seven prokaryotes examined encode proteins homologous to known channel proteins, but none of the channel types identified occurs in all of these organisms. (12) The phosphoenolpyruvate:sugar phosphotransferase system is prevalent in the large genome organisms, E. coli and B. subtilis, and is present in the small genome organisms, H. influenzae and M. genitalium, but is totally lacking in H. pylori, Synechocystis and M. jannaschii. Details of the information summarized in this article are available on our web sites, and this information will be periodically updated and corrected as new sequence and biochemical data become available.

ATP-Binding Cassette Transporters↗
PubMed1998

Molecular phylogeny as a basis for the classification of transport proteins from bacteria, archaea and eukarya.

Although enzymes catalyzing chemical reactions have long been classified according to the system developed by the Enzyme Commission (EC), no comparable system has been developed or proposed for transport proteins catalyzing transmembrane vectorial reactions. We here propose a comprehensive system, designated the Transport Commission (TC) system, based both on function and phylogeny. The TC system initially categorizes permeases according to mode of transport and energy coupling mechanism, and each category is assigned a one-component TC number (W). The secondary level of classification corresponds to the phylogenetic family (or superfamily) to which a particular permease is assigned, and each family is assigned a two-component TC number (W.X). The third level of classification refers to the phylogenetic cluster within a family (or the family within a superfamily) to which the permease belongs, and each cluster receives a three-component TC number (W.X.Y). Finally, the last level of categorization is based on substrate specificity and polarity of transport, and each entry is assigned a four component TC number (W.X.Y.Z). This system is based on the observation that mode of transport and energy coupling mechanism are fundamental properties of transport systems that very seldom transcend familial lines, but substrate specificity, being readily alterable by point mutations, is a superficial characteristic that often transcends familial lines. The proposed system has the potential to include all known permeases for which sequence data are available and has the flexibility to accommodate the multitude of permeases likely to be revealed by future genome sequencing and biochemical analysis. Major conclusions resulting from our classification efforts are described. The classification system, which will be continuously updated, is available on our World Wide Web site (http:/(/)www-biology.ucsd.edu/ approximately msaier/transport/titlepage.html).

Archaea↗
PubMed1998

A novel protein kinase that controls carbon catabolite repression in bacteria.

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.

Adenosine Triphosphate↗
PubMed1998

Evolutionary origins of multidrug and drug-specific efflux pumps in bacteria.

The available genomic sequences of three pathogenic and three nonpathogenic bacteria were analyzed to identify known and putative drug-specific and multidrug resistance transport systems. Escherichia coli was found to encode 29 such pumps, and with the exception of the archaebacterium Methanococcus jannaschii, the numbers of multidrug efflux pumps encoded within genomes of the other organisms were found to be approximately proportional to their total numbers of encoded transport systems as well as to total genome size. The similar numbers of chromosomally encoded multidrug efflux systems in pathogens and nonpathogens suggests that these transporters have not arisen recently in pathogens in response to antimicrobial chemotherapy. Phylogenetic analyses of the four transporter families that contain drug efflux permeases indicate that drug resistance arose rarely during the evolution of each family and that the diversity of current drug efflux pumps within each family arose from just one or a very few primordial systems. However, although the ability to confer drug efflux appears to have emerged on only a few occasions in evolutionary time and was stably maintained as an evolutionary trait, modulation of the substrate specificities of these systems has occurred repeatedly. A speculative model is presented that may explain the apparent capability of these multidrug transport systems to mediate drug transport from the cytoplasm or directly from the phospholipid bilayer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Unique regulation of carbohydrate chemotaxis in Bacillus subtilis by the phosphoenolpyruvate-dependent phosphotransferase system and the methyl-accepting chemotaxis protein McpC.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.

Bacillus subtilis↗
PubMed1998

CcpB, a novel transcription factor implicated in catabolite repression in Bacillus subtilis.

Recent work has shown that in Bacillus subtilis catabolite repression of several operons is mediated by a mechanism dependent on DNA-binding protein CcpA complexed to a seryl-phosphorylated derivative of HPr [HPr(Ser-P)], the small phosphocarrier protein of the phosphoenolpyruvate-sugar phosphotransferase system. In this study, it was found that a transposon insertional mutation resulted in the partial loss of gluconate (gnt) and xylose (xyl) operon catabolite repression by glucose, mannitol, and sucrose. The transposon insertion was localized to a gene, designated ccpB, encoding a protein 30% identical to CcpA, and relief from catabolite repression was shown to be due to the absence of CcpB rather than to the absence of a protein encoded by a downstream gene within the same operon. The relative intensities of CcpA- and CcpB-mediated catabolite repression depended on growth conditions. On solid media, and when cells were grown in liquid media with little agitation, CcpB and CcpA both proved to function in catabolite repression. However, when cells were grown in liquid media with much agitation, CcpA alone mediated catabolite repression. Like CcpA, CcpB appears to exert its catabolite-repressing effect by a mechanism dependent on the presence of HPr(Ser-P).

Amino Acid Sequence↗
PubMed1998

Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

Amino Acid Sequence↗
PubMed1998

Major facilitator superfamily.

The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.

Amino Acid Sequence↗
PubMed1997

A family of gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from gram-negative bacteria.

Gram-negative bacteria have evolved transport complexes that export macromolecules and toxic substances across the two membranes of the cell envelope in a single energy coupled step. The process requires (1) a cytoplasmic membrane export system, (2) a membrane fusion protein (MFP), and (3) an outer membrane factor (OMF). Families comprising the former two constituents have been described previously. We here present an analysis of the phylogenetic and structural characteristics of the OMF family. Twenty-one members of this family have been identified, and based on available evidence, they function in conjunction with ABC, RND, MFS and/or other types of cytoplasmic transport systems. OMFs exhibit fairly uniform sizes (398-495 residues with two exceptions), and based on computational analyses, they may form beta-barrel structures consisting of up to 16 beta-strands. Phylogenetic analyses reveal that while the MFPs cluster in accordance with the type of cytoplasmic membrane transport systems with which they function, OMFs do not. We conclude that OMFs probably comprise a family of outer membrane porin-type proteins of uniform structure which did not coevolve with their cognate cytoplasmic membrane transport systems.

Bacterial Outer Membrane Proteins↗
PubMed1997

Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system.

The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, the B. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity.

Alleles↗
PubMed1997

A novel family of ubiquitous heavy metal ion transport proteins.

We describe a novel diverse family of metal ion transporter (CDF) proteins (the cation diffusion facilitator (CDF) family) with members occurring in both prokaryotes and eukaryotes. Thirteen sequenced protein members of the CDF family have been identified, several of which have been shown to transport cobalt, cadmium and/or zinc. All members of the CDF family possess six putative transmembrane spanners with strongest conservation in the four N-terminal spanners, and on the basis of the analyses, we present a unified structural model. Members of the family are shown to exhibit an unusual degree of size variation, sequence divergence, and differences in cell localization and polarity. The phylogenetic tree for the CDF family reveals that prokaryotic and eukaryotic proteins cluster separately. It allows functional predictions for some uncharacterized members of this family. A signature sequence specific for the CDF family is derived.

Amino Acid Sequence↗
PubMed1997

The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases.

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

Animals↗
PubMed1997

Characterization of a novel transporter family that includes multiple Escherichia coli gluconate transporters and their homologues.

The nucleotide sequences of seven Escherichia coli genes that encode members of the gluconate permease (GntP) family have recently become available. These genes include gntP, gntU, gntW, ORf449, dsdX, and ORFo454. The deduced amino acid sequences of all seven E. coli genes are homologous to the gntP gene products from Bacillus subtilis and B. licheniformis as well as two additional gene products from Haemophilus influenzae. These 11 proteins are not demonstrably homologous to members of the major facilitator superfamily or other recognized permease families. Four of the E. coli gluconate transporter genes have been cloned and shown to encode gluconate transporters with apparent affinities ranging from 6 to 212 microM. These studies serve to characterize a novel family of bacterial permeases.

Bacillus↗