bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the archaeal, plasmid-encoded type II restriction-modification system MthTI from Methanobacterium thermoformicicum THF: homology to the bacterial NgoPII system from Neisseria gonorrhoeae
Authors: Nölling J , de Vos W M
Published: November 2016

A restriction-modification system, designated MthTI, was localized on plasmid pFV1 from the thermophilic archaeon Methanobacterium thermoformicicum THF. The MthTI system is a new member of the family of GGCC-recognizing restriction-modification systems. Functional expression of the archaeal MthTI genes was obtained in Escherichia coli. The mthTIR and mthTIM genes are 843 and 990 bp in size and code for proteins of 281 (32,102 Da) and 330 (37,360 Da) amino acids, respectively. The deduced amino acid sequence of M.MthTI showed high similarity with that of the isospecific methyltransferases M.NgoPII and M.HaeIII. In addition, extensive sequence similarity on the amino acid level was observed for the endonucleases R.MthTI and R.NgoPII. Moreover, the endonuclease and methyltransferase genes of the thermophilic MthTI system and those of the Neisseria gonorrhoeae NgoPII system show identical organizations and high (54.5%) nucleotide identity. This finding suggests horizontal transfer of restriction-modification systems between members of the domains Bacteria and Archaea.
Isolation and characterization of Pseudomonas aeruginosa mutants blocked in the synthesis of pyoverdin
Authors: Visca P , Serino L , Orsi N
Published: November 2016

We have isolated and characterized by chemical and enzymatic analyses three distinct types of pyoverdin-defective (pvd) mutants of Pseudomonas aeruginosa PAO1. The pvd-1 mutant is an L-N5-hydroxyornithine (L-N5-OH-Orn) auxotroph unable to hydroxylate L-ornithine (L-Orn) in a cell-free system and requiring L-N5-OH-Orn for pyoverdin production. The other two types of mutants appear to be blocked in further steps of the biosynthetic pathway leading to pyoverdin, namely, the acylation of L-N5-OH-Orn (pvd-2) and chromophore synthesis (pvd-3). The different pvd mutations were all found to be located in the catA1 region at 47 min of the genetic map of P. aeruginosa PAO1.
Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93))
Authors: Tseng M J , Hilfinger J M , He P , Greenberg G R
Published: November 2016

To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products. Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector. The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase. Phage T4 nrdB93, described by Wirak et al. (D. O. Wirak, K. S. Cook, and G. R. Greenberg, J. Biol. Chem. 263:6193-6201, 1988) contains a lesion in exon II of the gene. The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein. An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene.
The Caulobacter crescentus flaFG region regulates synthesis and assembly of flagellin proteins encoded by two genetically unlinked gene clusters
Authors: Schoenlein P V , Lui J , Gallman L , Ely B
Published: November 2016

At a specific time in the Caulobacter crescentus cell cycle, a single flagellar filament and multiple receptor sites for the swarmer-specific phage phi Cbk are assembled at one pole of the predivisional cell. One cluster of genes required for this morphogenesis, the flaYG region, includes the flgJKL genes, which encode structural proteins of the flagellar filament. These flagellin genes are flanked by genes required for filament assembly, the flaYE genes at one end and the flaF-flbT-flbA-flaG genes at the other. In this study, we characterized mutants carrying large chromosomal deletions within this region. Several of these strains are phi CbK resistant and produce a novel 22-kDa flagellin that is not assembled into flagella. Merodiploid strains containing either the entire flaFG region or individual fla transcription units from this region were constructed. These strains were used to correlate the presence or absence of specific gene products to changes in flagellin synthesis, filament assembly, or phage sensitivity. As a result of these studies, we were able to conclude that (i) the production of the 22-kDa flagellin results from the absence of the flbA and flaG gene products, which appear to be components of a flagellin-processing pathway common to the 25-, 27-, and 29-kDa flagellins; (ii) flbT negatively modulates the synthesis of the 27- and 25-kDa flagellins from two genetically unlinked gene clusters; (iii) flgL is the only flagellin gene able to encode the 27-kDa flagellin, and this flagellin appears to be required for the efficient assembly of the 25-kDa flagellins; (iv) flaF is required for filament assembly; and (v) phi CbK resistance results from the deletion of at least two genes in the flaFG region.
Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene
Authors: Nunoshiba T , Hidalgo E , Amábile Cuevas C F , Demple B
Published: November 2016

Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.
Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase
Authors: Eikmanns B J
Published: November 2016

To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.
New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase
Authors: Van Bruggen-Van Der Lugt A W , Kamphuis H J , De Ruiter G A , Mischnick P , Van Boom J H , Rombouts F M
Published: November 2016

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.
Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function
Authors: O'Rear J , Alberti L , Harshey R M
Published: November 2016

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.
The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli
Authors: Dai K , Lutkenhaus J
Published: November 2016

Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants. Among these, the ftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.
Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster
Authors: Jagusztyn-Krynicka E K , Hansen J B , Crow V L , Thomas T D , Honeyman A L , Curtiss R
Published: November 2016

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.
Sequence analysis and characterization of the mobilization region of a broad-host-range plasmid, pTF-FC2, isolated from Thiobacillus ferrooxidans
Authors: Rohrer J , Rawlings D E
Published: November 2016

The nucleotide sequence of a 5,317-bp fragment which includes the region required for mobilization of broad-host-range plasmid pTF-FC2 was determined. A region of approximately 3.5 kb was required for plasmid mobilization, and oriT was localized on a 138-bp fragment. Polypeptides which corresponded in size and location to several of the open reading frames were detected in an in vitro transcription-translation system. Three open reading frames essential for plasmid mobilization and two which affect the mobilization frequency were identified. There was a distinct similarity in the sizes, amino acid sequences, and locations of the proteins from the mobilization region of pTF-FC2 and the Tra1 region of IncP plasmid RP4. Similarity in the structures and sequences of the oriT regions was also apparent. A sequence with 37-of-38-bp homology to the inverted repeated sequences of Tn21 and an open reading frame with strong homology to the MerR regulatory protein was identified outside of the region required for mobilization.
DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli
Authors: Shi W , Zhou Y , Wild J , Adler J , Gross C A
Published: November 2016

The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli. Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F). Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.
Synthesis of cyclic beta-(1,2)-glucans by Rhizobium leguminosarum biovar trifolii TA-1: factors influencing excretion
Authors: Breedveld M W , Zevenhuizen L P , Zehnder A J
Published: November 2016

The synthesis of cyclic beta-(1,2)-glucans from UDP-[14C]glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum bv. trifolii TA-1 was investigated. The crude membrane system needed Mn2+, ATP, and NAD+ for optimal activity. Hardly any difference in biosynthetic activity between membrane fractions of TA-1 cells grown in the presence (200 mM) or absence of NaCl was observed. Whole TA-1 cells grown in the presence of NaCl excreted labeled, neutral cyclic beta-(1,2)-glucan during incubation with added UDP-[14C]glucose. With NaCl-free cultured TA-1 cells, no excretion was observed; however, after these cells were alternately frozen and thawed eight times, they excreted glucans. Glucan formation in vitro and glucan excretion by whole cells were strongly inhibited in the presence of 50 mg of cyclic glucan per ml (about 15 mM), indicating that biosynthesis of cyclic beta-(1,2)-glucans in strain TA-1 is controlled by end-product inhibition. These observations indicate that TA-1 cells become more permeable to cyclic glucans at high NaCl concentrations. The constant loss of glucans from cells grown in the presence of 200 mM NaCl prevented end-product inhibition and resulted in glucan accumulation of up to 1,600 mg/liter in the medium.
Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI)
Authors: González-Candelas L , Kolattukudy P E
Published: November 2016

A pectate lyase produced by Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI) was previously shown to be essential for host infection (M. S. Crawford and P. E. Kolattukudy, Arch. Biochem. Biophys. 258:196-205, 1987). Pectate lyase genes have not been cloned from any phytopathogenic fungi. A gene, designated pelA, encoding an inducible pectate lyase was isolated from F. solani f. sp. pisi. A probe was synthesized by polymerase chain reaction with oligonucleotide primers based on the known amino acid sequences of two regions of the mature protein and first-strand cDNA as template. Both cDNA and the gene were isolated and sequenced. That the cloned cDNA represents the previously purified pectate lyase is shown by the complete match of the sequences of the N-terminal 38 amino acid residues and the 20 amino acid residues of an internal peptide with the sequence deduced from the cDNA sequence. This lyase sequence shows little homology to those of other pectolytic enzymes. The pelA gene shows standard characteristics with respect to promoter, intron, and polyadenylation sequences. As determined by primer extension and nuclease S1 analysis of the origin of the transcription, there are multiple initiation sites clustered in a region of 12 nucleotides located about 55 bp upstream of the start codon. Northern (RNA) blot analysis showed a single band of mRNA at about 1 kb. The pelA gene mRNA was detected only when F. solani f. sp. pisi was grown with pectin, and there was no detectable transcript accumulation when the fungus was grown with glucose as the sole carbon source. When both carbon sources were present, the pelA gene was transcribed only after glucose was completely depleted, indicating carbon catabolite repression. Moreover, the levels of transcription decreased rapidly prior to maximal enzyme accumulation, suggesting a mechanism of self catabolite repression.
Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides
Authors: Benning C , Somerville C R
Published: November 2016

Two new mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening mutagenized cell lines for polar lipid composition by thin-layer chromatography of lipid extracts. A genomic clone which complemented the mutations in these two lines, but not the previously described sulfolipid-deficient sqdA mutant, was identified. Sequence analysis of the relevant region of the clone revealed three, in tandem open reading frames, designated sqdB, ORF2, and sqdC. One of the mutants was complemented by the sqdB gene, and the other was complemented by the sqdC gene. Insertional inactivation of sqdB also inactivated sqdC, indicating that sqdB and sqdC are cotranscribed. The N-terminal region of the 46-kDa putative protein encoded by the sqdB gene showed slight homology to UDP-glucose epimerase from various organisms. The 30-kDa putative protein encoded by ORF2 showed very striking homology to rabbit muscle glycogenin, a UDP-glucose utilizing, autoglycosylating glycosyltransferase. The 26-kDa putative protein encoded by the sqdC gene was not homologous to any protein of known function.
Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus
Authors: Priefert H , Steinbüchel A
Published: November 2016

The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54.
Escherichia coli produces a cytoplasmic alpha-amylase, AmyA
Authors: Raha M , Kawagishi I , Müller V , Kihara M , Macnab R M
Published: November 2016

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.
Two transcriptionally active OmpR mutants that do not require phosphorylation by EnvZ in an Escherichia coli cell-free system
Authors: Bowrin V , Brissette R , Inouye M
Published: November 2016

D55Q-T83A and D55Q-G94S, two pseudorevertants of the D55Q mutant OmpR, an Escherichia coli transcriptional activator, were isolated previously by R. Brissette, K. Tsung, and M. Inouye (J. Bacteriol. 173:3749-3755, 1991). These pseudorevertant OmpR proteins were purified and examined for their function as transcriptional activators in a cell-free system with an ompF DNA fragment. These proteins were transcriptionally active, even after acid treatment, whereas the wild-type OmpR was completely inactive after the same treatment. Phosphorylation of acid-treated wild-type OmpR with an EnvZ11 membrane fraction and ATP restored transcriptional activity, whereas the activities of the mutant OmpR proteins did not change after phosphorylation.
Determination of the sequence of spaE and identification of a promoter in the subtilin (spa) operon in Bacillus subtilis
Authors: Chung Y J , Hansen J N
Published: November 2016

An 851-residue open reading frame (ORF) called SpaE has been discovered in the subtilin (spa) operon. Interruption of this ORF with a chloramphenicol acetyltransferase gene destroys the ability of Bacillus subtilis LH45 delta c (a derivative of B. subtilis 168) to produce subtilin, which is an antimicrobial peptide belonging to the class of ribosomally synthesized peptide antibiotics called lantibiotics. SpaE shows strong homology to NisB, which is in the nisin (nis) operon in Lactococcus lactis ATCC 11454. Despite the strong sequence homology between SpaE and NisB, the spaE and nisB genes occupy very different locations in their respective operons, indicating that they have been evolving separately for a long time. Primer extension analysis was employed to identify a promoter upstream from the spaE gene, which appears to define the 5' end of the spa operon, which contains four other ORFs (Y. J. Chung, M. T. Steen, and J. N. Hansen, J. Bacteriol. 174:1417-1422, 1992).
Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases
Authors: von Eichel-Streiber C , Sauerborn M , Kuramitsu H K
Published: November 2016

The homologous C-terminal repeats of Clostridium difficile toxins (ToxA and ToxB) and streptococcal glucosyltransferases appear to mediate protein-carbohydrate interactions at cellular binding sites with sugar moieties as substrates. A consensus sequence of 134 repeating units from gram-positive bacteria indicates that these repeats have a modular design with (i) a stretch of aromatic amino acids proposed to be involved in the primary carbohydrate-protein interaction, (ii) an amplification of this interaction by repetition of the respective sequences, and (iii) a second domain, not characterized, that is responsible for carbohydrate specificity.
The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein
Authors: Hauser J , Levine A S , Ennis D G , Chumakov K M , Woodgate R
Published: November 2016

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.
Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli
Authors: Nelson K , Selander R K
Published: November 2016

Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.
Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans
Authors: Sakurada K , Ohta T , Hasegawa M
Published: November 2016

We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.
Resolution of chromosomes III and VI of Aspergillus nidulans by pulsed-field gel electrophoresis shows that the penicillin biosynthetic pathway genes pcbAB, pcbC, and penDE are clustered on chromosome VI (3.0 megabases)
Authors: Montenegro E , Fierro F , Fernandez F J , Gutiérrez S , Martín J F
Published: November 2016

An improved electrophoretic molecular karyotype of Aspergillus nidulans ATCC 28901 has been obtained by contour-clamped electric field gel electrophoresis, which separates seven chromosomal bands and allows resolution of chromosomes III and VI. The three genes of the penicillin biosynthetic pathway, pcbAB, pcbC, and penDE, encoding alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase, and isopenicillin N acyltransferase, respectively, are clustered together on a chromosome of 3.0 Mg, corresponding to linkage group VI, whereas the argB gene was located on a chromosome of 3.4 Mb, corresponding to linkage group III. Three other strains of A. nidulans contained a modified chromosome III of about 3.1 Mb that overlaps with chromosome VI, forming a doublet. Resolution of chromosomes III and VI in strain ATCC 28901 allowed unequivocal mapping of the penicillin gene cluster on chromosome VI of A. nidulans.
Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions
Authors: Eaton R W , Chapman P J
Published: November 2016

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.
Mechanism of glutamate uptake in Zymomonas mobilis
Authors: Ruhrmann J , Krämer R
Published: November 2016

The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.
Autoregulation of the stability operon of IncFII plasmid NR1
Authors: Tabuchi A , Min Y N , Womble D D , Rownd R H
Published: November 2016

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.
Carbon and energy metabolism of atp mutants of Escherichia coli
Authors: Jensen P R , Michelsen O
Published: November 2016

The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis.
In vitro transcription from the Escherichia coli ilvIH promoter
Authors: Willins D A , Calvo J M
Published: November 2016

Lrp (leucine-responsive regulatory protein) activates the expression of the Escherichia coli ilvIH operon in vivo and mediates the repression of the operon by exogenous leucine. In previous studies, operon expression in vivo was measured with transcriptional fusions of lacZ to the ilvIH promoter. Here, ilvIH mRNA was measured directly by primer extension. The steady-state level of ilvIH mRNA was 11-fold higher in a wild-type parent strain than in a derivative lacking Lrp. A two-step procedure was developed for measuring ilvIH mRNA synthesized in vitro. RNA was synthesized with plasmid templates and purified RNA polymerase, and then ilvIH mRNA was measured by primer extension. In vitro, mRNA synthesis was initiated at two sites, one corresponding to the in vivo site (promoter P1) and the other corresponding to a site about 60 bp further upstream (promoter P2). Purified Lrp stimulated transcription two- to fivefold from promoter P1, whereas it decreased transcription more than fivefold from promoter P2. Transcription from promoter P1 was stimulated by Lrp with templates containing the wild-type ilvIH promoter but not with templates containing mutations in an Lrp binding site. Furthermore, under at least some conditions, leucine reversed the stimulatory effect of Lrp. Taken together with the results of mutational analyses, these results establish that Lrp acts directly to stimulate transcription from the ilvIH promoter. Furthermore, they suggest that the ilvIH promoter is recognized by a sigma 70 RNA polymerase.
Characterization of a high-affinity iron transport system in Acinetobacter baumannii
Authors: Echenique J R , Arienti H , Tolmasky M E , Read R R , Staneloni R J , Crosa J H , Actis L A
Published: November 2016

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.
Alginate synthesis in Pseudomonas aeruginosa: environmental regulation of the algC promoter
Authors: Zielinski N A , Maharaj R , Roychoudhury S , Danganan C E , Hendrickson W , Chakrabarty A M
Published: November 2016

The exopolysaccharide alginate is a major virulence factor of Pseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity of P. aeruginosa within the environment of the cystic fibrosis lung. The gene algC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation of algC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgR1. AlgR1 was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgR1 specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgR1 protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the algC promoter, which has significant homology with the RNA polymerase sigma-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.
Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium
Authors: Swenson D L , Clegg S
Published: November 2016

Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.