Putative Permease Research Articles

Cloning of the pepX gene of Lactobacillus helveticus IF03809 encoding salt-tolerant X-prolyl dipeptidyl aminopeptidase and characterization of the enzyme

X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactobacillus helveticus IF03809 expressed nearly full activity under high salt conditions, such as 2 M NaCl. We cloned and sequenced the pepX gene for X-PDAP. The calculated M, of deduced X-PDAP (803 amino acids) was 90,847 and the protein was distantly related (35 to 44% identity) to known X-PDAPs of Lactobacillus sp. including L. helveticus CNRZ32 (40% identity). Native and recombinant X-PDAP were purified to homogeneity from both L. helveticus IF03809 and Escherichia coli DH5α harboring the pepX gene on a plasmid, respectively. The native enzyme appeared to be a dimer of 220 kDa, as estimated by gel filtration column chromatography. It hydrolyzed an X-prolyl-linkage, but not prolyl- or X-prolyl-X-peptide bonds, and tolerated up to 2 M NaCl as well as some other chlorides of monovalent cations. Determination of the flanking sequences revealed two divergent genes. The upstream region of the pepX gene encodes oppA gene for a putative oligopeptide permease, while the downstream region encodes tnp gene specifying a possible transposase of the IS3 family. The oppA gene shares a 176 bp-promoter region with pepX in the intergenic region, implying a relationship between this oligopeptide transport system and X-PDAP.

Molecular and Biochemical Analysis of Two β-Galactosidases fromBifidobacterium infantisHL96

Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).

Cloning and Characterization of a Putative Human Glycerol 3-Phosphate Permease Gene (SLC37A1 or G3PP) on 21q22.3: Mutation Analysis in Two Candidate Phenotypes, DFNB10 and a Glycerol Kinase Deficiency

Using multiple exons trapped from human chromosome 21 (HC21)-specific cosmids with homology to a putative Arabidopsis thaliana glycerol 3-phosphate permease, we have determined the full-length cDNA sequence of a novel HC21 gene encoding a putative sugar-phosphate transporter (HGMW-approved symbol SLC37A1, aka G3PP). The predicted protein has 12 putative transmembrane domains and is also highly homologous to bacterial glpT proteins. The transcript was precisely mapped to 21q22.3 between D21S49 and D21S113. Comparison of the SLC37A1 cDNA to genomic sequence revealed that the gene encompasses 82 kb, and it is split into 19 coding exons and 7 untranslated exons, which are alternatively spliced in a complex and tissue-specific manner. Glycerol 3-phosphate (G3P) is produced by glycerol kinase (GK) and is found in several biochemical pathways in different cellular compartments, such as the glycerol phosphate shuttle and glycerophospholipid synthesis. Thus SLC37A1 mutations may cause a phenotype similar to GK deficiency. Mutational analyses of SLC37A1 in seven patients with no mutations in the GK gene and low GK activity revealed only nonpathogenetic sequence variants, excluding SLC37A1 as the gene for the phenotype in these patients. SLC37A1 maps in the refined critical region of the autosomal recessive deafness locus, DFNB10, on 21q22.3. Mutation analyses also excluded SLC37A1 as the gene for DFNB10.

Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression.

Bacillus subtilis grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the lctEP locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of lctE results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the alsSD operon. Mutation of alsSD has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by pta. Similar to the case for lctEP, mutation of pta significantly reduces anaerobic fermentative and respiratory growth. The expression of both lctEP and alsSD is strongly induced under anaerobic conditions. Anaerobic lctEP and alsSD induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed fnr dependence might be the result of Fnr-induced arfM (ywiD) transcription and subsequent lctEP and alsSD activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by resDE is also involved in anaerobic lctEP induction. No direct resDE influence on the redox regulation of alsSD was observed. The alternative electron acceptor nitrate represses anaerobic lctEP and alsSD transcription. Nitrate repression requires resDE- and fnr-dependent expression of narGHJI, encoding respiratory nitrate reductase. The gene alsR, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic lctEP and alsSD expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent pta regulation was observed. A model for the regulation of the anaerobic fermentation genes in B. subtilis is proposed.

The Bacillus subtilis cysP gene encodes a novel sulphate permease related to the inorganic phosphate transporter (Pit) family.

Sulphate permeases in the plasma membrane are responsible for uptake of environmental sulphate used in the sulphate assimilation pathway in bacteria and plants. Here it is reported that the ORF designated cysP, located on the Bacillus subtilis chromosome between cysH and five putative genes involved in sulphate assimilation, encodes a sulphate permease. cysP is able to complement Escherichia coli cysteine auxotrophs with mutations affecting either the membrane or periplasmic components of the sulphate-thiosulphate permease. Transport studies with cell suspensions of a cysA97 E. coli strain transformed with a plasmid expressing the B. subtilis cysP gene indicated that CysP catalyses sulphate uptake. Analysis of the primary sequence showed that CysP (354 amino acids, estimated molecular mass 24 kDa) is a highly hydrophobic protein which has 11 putative transmembrane helices. Sequence comparisons revealed that CysP, together with the phosphate permease of Neurospora crassa, Pho-4, and E. coli PitA, belongs to the family of related transporters, the inorganic phosphate transporter (Pit) family. Among the putative phosphate permeases, CysP shows a similar size and the same domain organization as the archaeal transporters. This is the first report of a sulphate permease in a Gram-positive organism.

The multiply-regulated gabA gene encoding the GABA permease of Aspergillus nidulans: a score of exons.

We describe the cloning, sequence and expression of gabA, encoding the gamma-amino-n-butyrate (GABA) permease of the fungus Aspergillus nidulans. Sequence changes were determined for three up-promoter (gabI ) and six gabA loss-of-function mutations. The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease. Structural predictions favour 11-12 transmembrane domains. Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene. In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, omega-amino acid induction and regulation in response to ambient pH (being acid-expressed). In agreement with this, we report consensus binding sites 5' to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.

Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island.

Upon entry into the host, Salmonella enterica strains are presumed to encounter an iron-restricted environment. Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment. We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island of Salmonella typhimurium. The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of the S. typhimurium chromosome. This locus, designated sit, consists of four open reading frames which encode polypeptides with extensive homology to the yfe ABC iron transport system of Yersinia pestis, as well as other ABC transporters. The sitA gene encodes a putative periplasmic binding protein, sitB encodes an ATP-binding protein, and sitC and sitD encode two putative permeases (integral membrane proteins). This operon is capable of complementing the growth defect of the enterobactin-deficient Escherichia coli strain SAB11 in iron-restricted minimal medium. Transcription of the sit operon is repressed under iron-rich growth conditions in a fur-dependent manner. Introduction of a sitBCD deletion into wild-type S. typhimurium resulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype. These results further support the existence of redundant iron uptake systems in S. enterica.

Novel organization of the genes for phthalate degradation from Burkholderia cepacia DBO1.

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (ophA1) is present by itself, while the genes for an inactive transporter (ophD) and 4,5-dihydroxyphthalate decarboxylase (ophC) are linked and the genes for phthalate dioxygenase oxygenase (ophA2) and cis-phthalate dihydrodiol dehydrogenase (ophB) are linked. ophA1 and ophDC are adjacent to each other but are transcribed in opposite directions, while ophA2B is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for ophD are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.

Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily.

Uptake of iron from various siderophores by a deltafet3deltafet4 strain of Saccharomyces cerevisiae was investigated. The catecholate enterobactin and the hydroxamate coprogen were taken up by the cells by passive diffusion, whereas the hydroxamates ferrioxamine B (FOB) and ferricrocin (FC) were taken up via a high-affinity energy-dependent mechanism. The kinetics of FOB and FC uptake showed reciprocal competitive inhibition. The transport was regulated by iron availability, but was independent of the Aft1p and Mac1p transcriptional activators. Mutants affected in the transport of FOB were isolated. The transport of FC was not impaired in these mutants. Functional complementation of one mutant allowed the identification of the SIT1 gene (Siderophore Iron Transport) encoding a putative permease belonging to the major facilitator superfamily. The Sit1 protein is probably a permease specific for the transport of ferrioxamine-type siderophores. The evidence suggests that the uptake of ferrichrome-type siderophores like FC involves other specific permease(s), although there seems to be a common handling of FOB and FC following their internalization by the cell.

Homologs of the vancomycin resistance d-Ala- d-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: attributes of catalytic efficiency, stereoselectivity and regulation with implications for function

Background: Vancomycin-resistant enterococci are pathogenic bacteria that have altered cell-wall peptidoglycan termini ( d-alanyl- d-lactate [ d-Ala- d-lactate] instead of d-alanyl- d-alanine [ d-Ala- d-Ala]), which results in a 1000-fold decreased affinity for binding vancomycin. The metallodipeptidase VanX (EntVanX) is a key enzyme in antibiotic resistance as it reduces the cellular pool of the d-Ala- d-Ala dipeptide. Results: A bacterial genome search revealed vanX homologs in Streptomyces toyocaensis (StoVanX), Escherichia coli (EcoVanX), and Synechocystis sp. strain PCC6803 (SynVanX). Here, the d, d-dipeptidase catalytic activity of all three VanX homologs is validated, and the catalytic efficiencies and diastereoselectivity ratios for dipeptide cleavage are reported. The ecovanX gene is shown to have an RpoS ( σ s)-dependent promoter typical of genes turned on in stationary phase. Expression of ecovanX and an associated cluster of dipeptide permease genes permitted growth of E. coli using d-Ala- d-Ala as the sole carbon source. Conclusions: The key residues of the EntVanX active site are strongly conserved in the VanX homologs, suggesting their active-site topologies are similar. StoVanX is a highly efficient d-Ala- d-Ala dipeptidase; its gene is located in a vanHAX operon, consistent with a vancomycin-immunity function. StoVanX is a potential source for the VanX found in gram-positive enterococci. The catalytic efficiencies of d-Ala- d-Ala hydrolysis for EcoVanX and SynVanX are 25-fold lower than for EntVanX, suggesting they have a role in cell-wall turnover. Clustered with the ecovanX gene is a putative dipeptide permease system that imports d-Ala- d-Ala into the cell. The combined action of EcoVanX and the permease could permit the use of d-Ala- d-Ala as a bacterial energy source under starvation conditions.

The uracil permease of Schizosaccharomyces pombe: a representative of a family of 10 transmembrane helix transporter proteins of yeasts.

The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w. A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696.

SacY, a transcriptional antiterminator from Bacillus subtilis, is regulated by phosphorylation in vivo.

SacY antiterminates transcription of the sacB gene in Bacillus subtilis in response to the presence of sucrose in the growth medium. We have found that it can substitute for BglG, a homologous protein, in antiterminating transcription of the bgl operon in Escherichia coli. We therefore sought to determine whether, similarly to BglG, SacY is regulated by reversible phosphorylation in response to the availability of the inducing sugar. We show here that two forms of SacY, phosphorylated and nonphosphorylated, exist in B. subtilis cells and that the ratio between them depends on the external level of sucrose. Addition of sucrose to the growth medium after SacY phosphorylation in the cell resulted in its rapid dephosphorylation. The extent of SacY phosphorylation was found to be proportional to the cellular levels of SacX, a putative sucrose permease which was previously shown to have a negative effect on SacY activity. Thus, the mechanism by which the sac sensory system modulates sacB expression in response to sucrose involves reversible phosphorylation of the regulator SacY, and this process appears to depend on the SacX sucrose sensor. The sac system is therefore a member of the novel family of sensory systems represented by bgl.

Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae

On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans. Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins. Binary sequence comparison made it possible to cluster 149 of them into 23 families. Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport. Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins. Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members. The latter resulted in the identification of 21 subclusters with presumably tightly related permease function. No functional clues were predicted for a total of 41 clustered or unclustered proteins.

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005

Derived structure of the putative sialic acid transporter from Escherichia coli predicts a novel sugar permease domain

Catabolism of sialic acids by Escherichia coli requires the genes nanA and nanT, which were previously mapped between argG and rpoN (E.R. Vimr and F.A. Troy, J. Bacteriol. 164:845-853, 1985). This organization is confirmed and extended by physical mapping techniques. An open reading frame beginning 135 bp from the nanA translational stop codon could code for a 53,547-Da hydrophobic polypeptide predicted to contain 14 transmembrane segments. Complementation analysis confirmed that nanT is required for sialic acid uptake when expressed in trans. NanT is homologous to a putative permease encoded by open reading frame 425, which maps between leuX and fecE in the E. coli chromosome. However, unlike this hypothetical permease or previously reported monosaccharide transporters, NanT contains a centrally located domain with two additional potential membrane-spanning segments plus one amphiphilic alpha-helix that may be important for the structure and function of sialic acid-permease.

Active Transport by the CscB Permease in Escherichia coli K-12

The cscB gene encoding a putative sucrose permease from Escherichia coli EC3132 [Bockmann, J., Heuel, H. & Lengeler, J. W. (1992) Mol. Gen. Genet. 235, 22-32] was placed under control of the lacZ promoter/operator and overexpressed. Cells of E. coli K-12 expressing cscB gene product from a high copy number plasmid accumulate sucrose against a concentration gradient with an apparent Km of about 1 mM and a Vmax of approximately 23 nmol/min/mg protein. In contrast, transport of lactose or melibiose is not observed. Epitope-tagging and immunological analysis reveal that concomitant with increased transport activity, a protein with an apparent Mr of 33-34 kDa appears in the cytoplasmic membrane. The results provide direct support for the contention that the cscB gene encodes a sucrose permease and provide the basis for further molecular analysis.

Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-1 in heart. Overexpression of Ram-1 severely repressed Glvr-1 synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-1 and Glvr-1 expression 3- to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy.

Cloning and chromosomal organization of a gene encoding a putative amino-acid permease from Saccharomyces cerevisiae

A new member of the yeast amino acid (aa) permease gene family has been cloned, mapped and sequenced. The sequence of the PAP1 (putative aa permease 1) gene contains an open reading frame of 566 aa corresponding to a polypeptide with a calculated M r of 62704. Its hydropathy profile suggests the presence of 13 membrane-spanning regions and a charged N-terminal domain. It does not resemble hydrophobic signal sequences found in secreted proteins. Hence, PAP1 encodes a protein with characteristics typical of integral membrane proteins translocating ligands across cellular membranes. Sequence comparisons indicate strong homology to the five known aa permeases of Saccharomyces cerevisiae and to an aa transporter in Trichoderma harzianum. Primer extension analysis revealed one major and one minor transcription start point located 121 and 125 nucleotides upstream from the ATG start codon, corresponding to a 2.1-kb transcript. PAP1 was mapped in a contig of three known ( DBF4, TPI and HEM12), but so far unlinked, genes on chromosome IV.

Gene structure of the ?large? sialidase isoenzyme fromClostridium perfringens A99 and its relationship with other clostridialnanH proteins

Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the 'large' form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs ('Asp-boxes'), whereas the remaining 3'-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resulting E. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino acids, from which a molecular weight of 72,956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3'-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the 'large' isoenzyme is very similar to the sialidase of Clostridium septicum (55% identical amino acids), whereas the homology with the 'small' form of the same species is comparatively low (26%).

Novel phosphotransferase system genes revealed by bacterial genome analysis: Unique, putative fructose‐ and glucoside‐specific systems

Analyses of sequences made available through the Escherichia coli genome project in the 87.2-89.2-min and 81.5-84.5-min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582, ORFf147 and ORFf485 comprise an Enzyme IIA-Enzyme IIBC pair of the PTS. The sequence similarity of ORFf485 to previously characterized fructose-specific Enzymes IIBC suggests that ORFf485 may be specific for fructose. ORFf147 encodes a protein with comparable degrees of sequence similarity to fructose and mannitol-specific Enzymes IIA as well as homologous proteins implicated in sigma 54-dependent transcriptional regulation. Unique features of this system include a detached IIA protein and the absence of a IIB domain duplication. ORFf356 and ORFf582 are functionally unidentified and nonhomologous to other ORFs in the current protein databases, but ORFf582 contains 2 N-terminal helix-turn-helix motifs, suggestive of a role in frv operon transcriptional regulation. The second putative operon, designated glv, includes 3 ORFs, ORFf455, ORFf161, and ORFf212. We suggest that ORFf455 was incorrectly assigned and should be designated ORFf368. ORFf368 and ORFf161 encode an Enzyme IIC and IIB pair of the PTS showing greatest sequence similarity to Enzymes II specific for sugars of the gluco configuration. ORFf212 encodes a protein with sequence similarity to a phospho-beta-glucosidase and an alpha-galactosidase. No putative transcriptional regulator of the glv operon was found. This operon is the first one encoding a putative PTS permease with detached Enzymes IIB and IIC and lacking an Enzyme IIA. It is suggested that both the frv and glv operons are cryptic in E. coli and that additional genes encoding novel PTS-related proteins will be revealed by bacterial genome sequence analyses.