Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains.

Summary The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EII^Nag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia colt K12. The deduced EII^Nag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.     

Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation

Summary Four genes, nagR, A, B and E, clustered in the nag locus of Escherichia coli K12 and Klebsiella pneumoniae, were cloned and physically mapped, and the corresponding gene products involved in amino sugar metabolism identified. Expression of the nag genes was also analysed using a series of lacZ fusions. In both bacteria, the genes are arranged in two divergent operons and controlled by a common NagR repressor. The corresponding gene nagR was found to map in the first operon together with the promoter proximal gene nagB, encoding the enzyme d-glucosamine isomerase (deaminase) (NagB) and the middle gene nagA, coding for N-acetyl-glucosamine deacetylase (NagA). Polar mutations in nagB and nagA prevent the efficient expression of nagR and cause constitutive expression of all nag genes. This includes the gene nagE encoding Enzyme II^Nag of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), encoded in the second divergently transcribed operon. No further gene is found in this operon which in both organisms is directly adjacent to the gene glnS. It is interesting that the NagR repressor also affects the mannose PTS (genes manX, Y, Z), the second transport system involved in amino sugar uptake and phosphorylation.     

Cloning of a novel human NHEDC1 (Na+/H+ exchanger like domain containing 1) gene expressed specifically in testis

The Na⁺/H⁺ exchangers (NHEs) catalyze the transport of Na⁺ in exchange for H⁺ across membranes in organisms and are required for numerous physiological processes. Here we report the cloning and characterization of a novel human NHEDC1 (Na⁺/H⁺ exchanger like domain containing 1) gene, which was mapped to human chromosome 4p24. This cDNA is 1859 bp in length, encoding a putative protein of 515 amino acids. The NHEDC1 proteins are highly conserved in mammals including human, mouse, rat, and Macaca fascicularis. One remarkable characteristic of human NHEDC1 gene is that it is exclusively expressed in the testis by RT-PCR analysis. Western blot analysis showed that the molecular weight of NHEDC1 is about 56 KDa. Guangming Ye and Cong Chen contributed equally to this work.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII^Mtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [³H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII^Mtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.     

Structural characterization of the PTS IIA and IIB proteins associated with pneumococcal fucose utilization

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EII^fuc) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three‐dimensional structures of IIA^fuc and IIB^fuc providing evidence that this PTS system belongs to the EII^man family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EII^fuc transports the H‐disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963–968. © 2016 Wiley Periodicals, Inc.     

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EII^man), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (K _m=11 M) and a low-affinity proton motive force driven glucose permease (GP) (K _m=170 M). (ii) Both 9R-4 and X-160 had only the low-affinity system (K _m=181 M for 9R-4, 278 M for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EII^man) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.Abbreviations GCR glucose-mediated catabolite repression - FCR fructose-mediated catabolite repression - PEP phosphoenolpyruvate - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - glc:PTS phosphoenolpyruvate:glucose phosphotransferase system - GP glucose permease - CCCP carbonylcyanide mchlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - P proton motive force - G-6-P glucose-6-phosphate - 2DG 2-deoxyglucose - IAA iodoacetic acid - EII^man enzyme II component of man:PTS - EIII^man enzyme III component of man:PTS - EII^glc enzyme II component of glc:PTS - EIII^glc enzyme III component of glc:PTS     

Isolation of Escherichia coli Mannitol Permease,EIImtl, Trapped in Amphipol A8-35 and Fluorescein-Labeled A8-35

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII^mtl, a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII^mtl, some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII^mtl soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII^mtl mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII^mtl using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.     

Molecular analysis of thescrA andscrB genes fromKlebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme IIScr of the phosphotransferase system and a sucrose-6-phosphate invertase

TheKlebsiella pneumoniae genesscrA andscrB are indispensable for sucrose (Scr) utilisation. GenescrA codes for an Enzyme II^Scr (II^Scr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), whilescrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscrAB DNA fragment has been cloned fromK. pneumoniae and expressed inEscherichia coli. Its nucleotide sequence was determined and the coding regions forscrA (1371 bp) andscrB (1401 bp) were identified by genetic complementation, enzyme activity tests and radiolabelling of the gene products. In addition, the nucleotide sequence of thescrB gene from the conjugative plasmid pUR400 isolated fromSalmonella typhimurium was also determined and errors in the previously published sequence of thescrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven II^Scr proteins, a hypothetical model of the secondary structure of II^Scr is proposed.     

Identification and phylogenetic classification of eleven putative P-type calcium transport ATPase genes in the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe

Calcium is an essential second messenger in yeast metabolism and physiology. So far, only four genes coding for calcium translocating ATPases had been discovered in yeast. The recent completion of the yeastSaccharomyces cerevisiae genome allowed us to identify six new putative Ca⁺⁺-ATPases encoding genes. Protein sequence homology analysis and phylogenetic classification of all putative Ca⁺⁺-ATPase gene products from the yeastsSaccharomyces cerevisiae andSchizosacchraomyces pombe reveal three clusters of homologous proteins. Two of them comprises seven proteins which might belong to a new class of P-type ATPases of unknown subcellular location and of unknown physiological function.     

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca²⁺/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca²⁺/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca²⁺-signaling in C. cinerea.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.