bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H
Authors: Gorkovenko A , Roberts M F
Published: November 2016

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris.
Use of the "blue halo" assay in the identification of genes encoding exported proteins with cleavable signal peptides: cloning of a Borrelia burgdorferi plasmid gene with a signal peptide
Authors: Giladi M , Champion C I , Haake D A , Blanco D R , Miller J F , Miller J N , Lovett M A
Published: November 2016

We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasmic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inner membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos. Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions. In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B. burgdorferi. One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.
Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis
Authors: Teufel P , Götz F
Published: November 2016

The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metalloprotease SepP1, was cloned into pT181mcs. DNA sequencing revealed an open reading frame of 1,521 nucleotides encoding a 507-amino-acid protein with an M(r) of 55,819. The sepA-containing DNA fragment did not hybridize with Staphylococcus hyicus or Staphylococcus carnosus DNA. Expression of sepA in the protease-negative S. carnosus (pT181mcsP1) resulted in overproduction of a 33-kDa protease found in the culture medium. The first 15 N-terminal amino acids of the partially purified protease completely matched the deduced DNA sequence starting at GCA (Ala-208). This finding indicated that SepP1 is synthesized as a preproenzyme with a 28-amino-acid signal peptide, a 179-amino-acid hydrophilic pro region, and a 300-amino-acid extracellular mature form with a calculated M(r) of 32,739. In activity staining, the mature protease prepared from S. carnosus (pT181mcsP1) corresponded to the extracellular S. epidermidis Tü3298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Bacillus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.
The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus
Authors: McDowall K J , Hernandez R G , Lin-Chao S , Cohen S N
Published: November 2016

Two temperature-sensitive mutations, ams-1 and rne-3071, in the ams (altered mRNA stability) gene have been used extensively to investigate the processing and decay of RNA in Escherichia coli. We have sequenced these temperature-sensitive alleles and found that the mutations are separated by only 6 nucleotides and cause conservative amino acid substitutions next to a possible nucleotide-binding site within the N-terminal domain of the Ams protein. Computer analysis revealed that the region altered by the mutations has extensive sequence similarity to a predicted gene product from the mre (murein pathway cluster e) locus of E. coli, which has been implicated previously in determining bacterial cell shape.
Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes
Authors: Goldfine H , Johnston N C , Knob C
Published: November 2016

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.
The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat
Authors: Shrader T E , Tobias J W , Varshavsky A
Published: November 2016

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins.
Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase
Authors: Wolgel S A , Dege J E , Perkins-Olson P E , Jaurez-Garcia C H , Crawford R L , Münck E , Lipscomb J D
Published: November 2016

Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJ1b has been purified to homogeneity for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from O2. The holoenzyme has a mass of 143 +/- 7 kDa as determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits with M(r)s of 35,500, each containing one iron atom. Mössbauer spectroscopy of 57Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and substrate-bound enzyme. However, the quadrupole splitting, delta EQ, and isomer shift, delta, of the Mössbauer spectrum changed from delta EQ = 2.57 mm/s and delta = 1.29 mm/s to delta EQ = 2.73 mm/s and delta = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The enzyme was also found to bind variable and substoichiometric amounts of Mn2+, but this metal could be removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe2+ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe2+. Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was inactivated by reagents that oxidize the Fe2+, such as H2O2 and K3FE(CN)6; full activity was restored after reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before O2. The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases. All substrates have vicinal hydroxyl groups on the aromatic ring except 4-NH2-3-hydroxybenzoate. This is the first substrate lacking vicinal hydroxyl groups reported for catecholic extradiol dioxygenases. 2,3-PCD is the final member of the PCA dioxygenase family to be purified. It is compared with other members of this family as well as other catecholic dioxygenases.
Repression of the traM gene of plasmid R100 by its own product and integration host factor at one of the two promoters
Authors: Abo T , Ohtsubo E
Published: November 2016

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated pM1 and pM2. Promoter pM2 lies upstream of pM1 and overlaps the sbmC-sbmD region. Promoter pM1 is constitutively expressed, while pM2 is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near pM2. The traM gene is likely to be expressed from pM2 when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from pM2 could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.
Regulation of kdp operon expression in Escherichia coli: evidence against turgor as signal for transcriptional control
Authors: Asha H , Gowrishankar J
Published: November 2016

Kdp, an inducible high-affinity K+ transporter in Escherichia coli, is encoded by genes of the kdpABC operon, and its expression is regulated by the products of kdpD and kdpE. Loss of cell turgor has been proposed to be the signal which induces kdp expression (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464-468, 1981). We reexamined kdp expression during steady-state growth under a variety of conditions and were able to confirm earlier observations which had indicated that it is primarily affected by the concentration of K+ in the medium and by mutations in genes encoding various K+ transporters in E. coli. Changes in pH of the culture also altered kdp expression. In all of these cases, an increase in [K+] of the medium repressed the operon. Several ionic solutes induced steady-state kdp expression (but to differing extents), whereas nonionic solutes had no effect, indicating that kdp expression is not determined by osmolarity of the growth medium. kdp expression during steady-state growth was shown also to be unaffected by the accumulation of other intracellular compatible solutes such as trehalose or glycine betaine, which would be expected to restore cell turgor during growth in high-osmolarity media. Two mutants that are defective in perception of the signal regulating kdp were isolated, and the mutation in each of them was mapped to the kdpDE regulatory locus. Analysis of kdp expression in one of these mutants provided additional evidence against the turgor regulation model. On the basis of these data, we discuss alternative candidates that might serve as the signal for control of kdp operon transcription.
Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling
Authors: Gill R E , Karlok M , Benton D
Published: November 2016

The bsgA gene of Myxococcus xanthus plays an essential role in the regulation of early gene expression during fruiting body formation and sporulation. bsgA mutants behave as though unable to initiate a required cell-cell interaction and consequently fail to transcribe normal levels of many developmentally induced genes. We determined the nucleotide sequence of bsgA, which predicts a single gene encoding a 90.4-kDa protein. The deduced BsgA protein shares 45 and 48% amino acid identity with the lon genes of Escherichia coli and Bacillus brevis, respectively. The cloned bsgA gene was expressed in E. coli, and the BsgA protein was partially purified and found, like its E. coli homolog, to be an ATP-dependent protease. Thus, the basis for the phenotype of bsgA mutants is likely to be a defect in intracellular proteolysis.
The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus
Authors: Tojo N , Inouye S , Komano T
Published: November 2016

Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coli lon, and Bacillus brevis lon genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.
Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707
Authors: Gibson D T , Cruden D L , Haddock J D , Zylstra G J , Brand J M
Published: November 2016

Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.
Protein D, a putative immunoglobulin D-binding protein produced by Haemophilus influenzae, is glycerophosphodiester phosphodiesterase
Authors: Munson R S , Sasaki K
Published: November 2016

Protein D of Haemophilus influenzae is 67% identical to the periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli. Extracts prepared from E. coli expressing recombinant protein D had an 8- to 22-fold-higher specific activity of glycerophosphodiester phosphodiesterase compared with extracts of E. coli not expressing protein D.
Relative expression of the products of glyoxylate bypass operon: contributions of transcription and translation
Authors: Chung T , Resnik E , Stueland C , LaPorte D C
Published: November 2016

Although the genes of the aceBAK operon are expressed from the same promoter, the relative cellular levels of their products are approximately 0.3:1:0.003. Gene and operon fusions with lacZ were constructed to characterize this differential expression. The upshift in expression between aceB and aceA resulted from differences in translational efficiency. In contrast, inefficient translation and premature transcriptional termination contributed to the downshift in expression between aceA and aceK. Premature transcriptional termination occurred within aceK and appears to result from inefficient translation. Deletion of repetitive extragenic palindromic elements between aceA and aceK had little effect on the relative expression of these genes. Rather, the sequences responsible for inefficient expression of aceK lie within the aceK ribosome binding site.
Characterization of a periplasmic 3':5'-cyclic nucleotide phosphodiesterase gene, cpdP, from the marine symbiotic bacterium Vibrio fischeri
Authors: Dunlap P V , Callahan S M
Published: November 2016

Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.
Regulation of the urea active transporter gene (DUR3) in Saccharomyces cerevisiae
Authors: ElBerry H M , Majumdar M L , Cunningham T S , Sumrada R A , Cooper T G
Published: November 2016

The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined. The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins. Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function. Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway. High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes. Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product. DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.
The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex
Authors: Rossi M , Pollock W B , Reij M W , Keon R G , Fu R , Voordouw G
Published: November 2016

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.
Physiological and biochemical characterization of the soluble formate dehydrogenase, a molybdoenzyme from Alcaligenes eutrophus
Authors: Friedebold J , Bowien B
Published: November 2016

Organoautotrophic growth of Alcaligenes eutrophus on formate was dependent on the presence of molybdate in the medium. Supplementation of the medium with tungstate lead to growth cessation. Corresponding effects of these anions were observed for the activity of the soluble, NAD(+)-linked formate dehydrogenase (S-FDH; EC 1.2.1.2) of the organism. Lack of molybdate or presence of tungstate resulted in an almost complete loss of S-FDH activity. S-FDH was purified to near homogeneity in the presence of nitrate as a stabilizing agent. The native enzyme exhibited an M(r) of 197,000 and a heterotetrameric quaternary structure with nonidentical subunits of M(r) 110,000 (alpha), 57,000 (beta), 19,400 (gamma), and 11,600 (delta). It contained 0.64 g-atom of molybdenum, 25 g-atom of nonheme iron, 20 g-atom of acid-labile sulfur, and 0.9 mol of flavin mononucleotide per mol. The fluorescence spectrum of iodine-oxidized S-FDH was nearly identical to the form A spectrum of milk xanthine oxidase, proving the presence of a pterin cofactor. The molybdenum-complexing cofactor was identified as molybdopterin guanine dinucleotide in an amount of 0.71 mol/mol of S-FDH. Apparent Km values of 3.3 mM for formate and 0.09 mM for NAD+ were determined. The enzyme coupled the oxidation of formate to a number of artificial electron acceptors and was strongly inactivated by formate in the absence of NAD+. It was inhibited by cyanide, azide, nitrate, and Hg2+ ions. Thus, the enzyme belongs to a new group of complex molybdo-flavo Fe-S FDH that so far has been detected in only one other aerobic bacterium.
Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties
Authors: Romeo T , Gong M , Liu M Y , Brun-Zinkernagel A M
Published: November 2016

Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.
Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification
Authors: Rainey F A , Ward N L , Morgan H W , Toalster R , Stackebrandt E
Published: November 2016

Small subunit rDNA sequences were determined for 20 species of the genera Acetogenium, Clostridium, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobium, and Thermobacteroides, 3 non-validly described species, and 5 isolates of anaerobic thermophilic bacteria, providing a basis for a phylogenetic analysis of these organisms. Several species contain a version of the molecule significantly longer than that of Escherichia coli because of the presence of inserts. On the basis of normal evolutionary distances, the phylogenetic tree indicates that all bacteria investigated in this study with a maximum growth temperature above 65 degrees C form a supercluster within the subphylum of gram-positive bacteria that also contains Clostridium thermosaccharolyticum and Clostridium thermoaceticum, which have been previously sequenced. This supercluster appears to be equivalent in its phylogenetic depth to the supercluster of mesophilic clostridia and their nonspore-forming relatives. Several phylogenetically and phenotypically coherent clusters that are defined by sets of signature nucleotides emerge within the supercluster of thermophiles. Clostridium thermobutyricum and Clostridium thermopalmarium are members of Clostridium group I. A phylogenetic tree derived from transversion distances demonstrated the artificial clustering of some organisms with high rDNA G+C moles percent, i.e., Clostridium fervidus and the thermophilic, cellulolytic members of the genus Clostridium. The results of this study can be used as an aid for future taxonomic restructuring of anaerobic sporogenous and asporogenous thermophillic, gram-positive bacteria.
Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli
Authors: Schiller N L , Monday S R , Boyd C M , Keen N T , Ohman D E
Published: November 2016

Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.
Isolation and sequencing of the gene coding for an antigenic 34-kilodalton protein of Mycobacterium paratuberculosis
Authors: Gilot P , De Kesel M , Machtelinckx L , Coene M , Cocito C
Published: November 2016

The gene coding for an antigenic 34-kDa protein of Mycobacterium paratuberculosis was isolated and sequenced. The 897-bp open reading frame coded for a novel protein containing specific B epitopes. The occurrence of well-defined hydrophobic and hydrophilic portions suggests the wall location of the protein.
Interplay between the membrane-associated UhpB and UhpC regulatory proteins
Authors: Island M D , Kadner R J
Published: November 2016

Expression of the Escherichia coli uhpT gene, encoding the sugar phosphate transport protein, is induced by extracellular glucose-6-phosphate and requires the function of the uhpABC regulatory genes. The UhpA and UhpB proteins are related to the response-regulator and sensor-kinase proteins of two-component regulatory systems, whereas the UhpC protein is related to UhpT and homologous transport proteins. To investigate the role of segments of the membrane-associated UhpB and UhpC regulatory proteins, a series of mutations were constructed in vitro by insertion of a 12- or 24-bp oligonucleotide linker at 44 sites within the uhpABCT locus. The effect of these mutations on regulation of a uhpT-lacZ transcriptional reporter was assayed with the mutated uhp alleles in single copy on the chromosome. All but one of the insertions in uhpA or uhpT were inactive for transcription activation or transport, respectively. In contrast, about half of the insertions in uhpB and uhpC retained Uhp expression, and insertions at four sites in uhpB and at one site in uhpC conferred high-level constitutive expression. The constitutive mutants in UhpB resulted from insertions in the nonpolar amino-terminal half of the protein, and all insertions in that half of UhpB affected Uhp expression in some manner, which suggests that the transmembrane segments of UhpB might negatively regulate the kinase activity of the carboxyl portion. The constitutive behavior of all but one of these uhpB alleles was dependent on the presence of active forms of both UhpA and UhpC, which suggests that UhpB and UhpC act jointly as a complex in the signaling process.
The Rhizobium meliloti rhizopine mos locus is a mosaic structure facilitating its symbiotic regulation
Authors: Murphy P J , Trenz S P , Grzemski W , De Bruijn F J , Schell J
Published: November 2016

The Rhizobium meliloti L5-30 mos locus, encoding biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine, is shown to be a mosaic structure. The mos locus consists of four open reading frames (ORFs) (ORF1 and mosABC) arranged in an operon structure. Within this locus, several domains of homology with other prokaryotic symbiotic genes (nifH, fixA, fixU, and nifT) are present, suggesting that this locus may represent a hot spot for rearrangement of symbiotic genes. Unusually, these domains are present in the coding as well as noncoding regions of the mos locus. Proteins corresponding to those encoded by mosABC, but not ORF1, have been detected in nodule extracts by using antibodies. As ORF1 shows extensive homology with the 5' region of the nifH gene (P.J. Murphy, N. Heycke, S.P. Trenz, P. Ratet, F.J. de Bruijn, and J. Schell, Proc. Natl. Acad. Sci. USA 85:9133-9137, 1988) and a frameshift mutation indicates that expression of this ORF is not required for mos activity, we propose that the mos locus has acquired a duplicated copy of nifH, including the promoter region, in order to become symbiotically regulated. Surprisingly, since the functions are likely different, MosA has an amino acid sequence similar to that of the DapA protein of Escherichia coli. The central domain of MosB has extensive homology with a range of diverse proteins involved with carbohydrate metabolism in either antibiotic or outer-cell-wall biosynthesis. This region is also common to the regulatory proteins DegT and DnrJ, suggesting a regulatory role for MosB. The structure of MosC is consistent with its being a membrane transport protein.
Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure
Authors: Thorstenson Y R , Kuldau G A , Zambryski P C
Published: November 2016

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.
Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone
Authors: Tanimoto K , An F Y , Clewell D B
Published: November 2016

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.
Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein
Authors: Gornicki P , Scappino L A , Haselkorn R
Published: November 2016

Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases. The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases. Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.
Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules
Authors: Gerngross T U , Reilly P , Stubbe J , Sinskey A J , Peoples O P
Published: November 2016

Antibodies raised against the Alcaligenes eutrophus poly-beta-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules. The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation. From these basic observations and data from the recent literature, a model of granule assembly is proposed.
Escherichia coli produces linoleic acid during late stationary phase
Authors: Rabinowitch H D , Sklan D , Chace D H , Stevens R D , Fridovich I
Published: November 2016

Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.
Cloning and characterization of a gene whose product is a trans-activator of anthrax toxin synthesis
Authors: Uchida I , Hornung J M , Thorne C B , Klimpel K R , Leppla S H
Published: November 2016

The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted M(r) of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis. The cloned atxA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.
Subunit association in acetohydroxy acid synthase isozyme III
Authors: Sella C , Weinstock O , Barak Z , Chipman D M
Published: November 2016

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.
Comparison of genetic maps for two Leptospira interrogans serovars provides evidence for two chromosomes and intraspecies heterogeneity
Authors: Zuerner R L , Herrmann J L , Saint Girons I
Published: November 2016

Genetic maps were constructed for Leptospira interrogans serovars icterohaemorrhagiae and pomona. Previously we independently constructed physical maps of the genomes for these two serovars. The genomes of both serovars consist of a large replicon (4.4 to 4.6 Mb) and a small replicon (350 kb). Genes were localized on the physical maps by using Southern blot analysis with specific probes. Among the probes used were genes encoding a variety of essential enzymes and genes usually found near bacterial chromosomal replication origins. Most of the essential genes are on the larger replicon of each serovar. However, the smaller replicons of both serovars contain the asd gene. The asd gene encodes aspartate beta-semialdehyde dehydrogenase, an enzyme essential in amino acid and cell wall biosyntheses. The finding that both L. interrogans replicons contain essential genes suggests that both replicons are chromosomes. Comparison of the genetic maps of the larger replicons of the two serovars showed evidence of large rearrangements. These data show that there is considerable intraspecies heterogeneity in L. interrogans.