bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis
Authors: Engelmann S , Lindner C , Hecker M
Published: November 2016

A sigma B-dependent stress gene of Bacillus subtilis was localized downstream of the licS gene. The predicted amino acid sequence exhibited a significant similarity to the sequence of the katE-encoded catalase HPII of Escherichia coli, and we designated it the open reading frame katE. In a B. subtilis katE mutant, catalase 2 could not be detected. The amount of katE-specific mRNA was increased after heat, salt, or ethanol stress or after glucose starvation in a sigma B-dependent manner. As in E. coli, the transcription of the katE gene in B. subtilis was unaffected by the addition of H2O2 to exponentially growing cells. In contrast, the katA gene encoding catalase 1 of B. subtilis showed an induction pattern different from that of katE; katA expression was strongly increased by oxidative stress. The similarity between E. coli sigma S-dependent genes and B. subtilis sigma B-dependent genes suggests that both may confer multiple stress resistance to stationary-phase cells.
The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene
Authors: Hernando Y , Palacios J M , Imperial J , Ruiz-Argüeso T
Published: November 2016

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.
Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC7942 required for maximum efficiency of nitrogen assimilation
Authors: Suzuki I , Horie N , Sugiyama T , Omata T
Published: November 2016

Two nitrogen-regulated genes were found in the genomic DNA region upstream of the nirA operon involved in uptake and utilization of nitrate in Synechococcus sp. strain PCC7942. The two genes (nirB and ntcB) are transcribed divergently from nirA and encode proteins of 349 and 309 amino acid residues, respectively. The levels of nirB and ntcB transcripts were low in cells growing on ammonium and increased upon transfer of ammonium-grown cells to nitrate-containing medium. The deduced NirB protein sequence has no similarities to other known proteins, whereas the deduced NtcB protein sequence is homologous to bacterial transcriptional activators of the LysR family. Defined mutants constructed by interrupting nirB or ntcB with a drug resistance marker grew as fast as the wild-type strain on ammonium but grew slower than the wild-type strain on nitrate or nitrite. The nirB mutant had higher activities of nitrate reductase, glutamine synthetase, and glutamate synthase than the wild-type strain, but its nitrite reductase activity was 40% of the wild-type levels. The mutant excreted nitrite into the medium during growth on nitrate, showing that nitrite reductase limits nitrate assimilation. These findings suggested that nirB is required for expression of maximum nitrite reductase activity. When grown on ammonium, the nirB mutant grew normally but cultures of the ntcB mutant still showed a yellowish-green color typical of nitrogen-limited cells. NtcB seems to regulate utilization of fixed nitrogen by controlling the expression of a certain gene(s) involved in nitrogen metabolism.
Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions
Authors: Sarsero J P , Pittard A J
Published: November 2016

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.
Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination
Authors: Kaschabek S R , Reineke W
Published: November 2016

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH. The properties of the halogen substituents influenced the affinity to the enzyme in the following manner. Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding). The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position > 5 position. A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed.
Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase
Authors: Pucci M J , Thanassi J A , Ho H T , Falk P J , Dougherty T J
Published: November 2016

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).
Genetic and physical mapping of genes involved in pyoverdin production in Pseudomonas aeruginosa PAO
Authors: Tsuda M , Miyazaki H , Nakazawa T
Published: November 2016

Pseudomonas aeruginosa PAO was mutagenized with Tn1737KH, a type I transcription probe transposon containing a promoterless lacZ (beta-galactosidase) gene, and 24 insertion mutants that did not grow under iron-deficient conditions were isolated. None of the culture supernatants from any mutants contained pyoverdin, a low-molecular-weight siderophore able to sequester ferric iron at very high affinity, and the growth defects of the mutants were all phenotypically recovered by the addition of the culture supernatant from the wild-type strain. These phenotypes led to the inference that all the mutants had defects in the genes (pvd genes) for production of pyoverdin. In some pvd::Tn1737KH mutants, high levels of beta-galactosidase activities were observed, and such activities were drastically reduced by the addition of ferric ion in the culture media, indicating that the expression of at least some pvd genes is regulated at the transcriptional level. Molecular cloning and physical analysis of the chromosomal fragments with Tn1737KH insertions allowed us to allocate all the mutations within a 103-kb region, referred to as the pvd region, that was found to locate at 47 min on the genetic map of PAO. Further physical mapping and Southern analysis showed that there is a 10-kb overlap between the pvd region and the 125-kb catA region described by Zhang and Holloway (C. Zhang and B. W. Holloway, J. Gen. Microbiol. 138:1097-1107, 1992). We could hence illustrate the physical map of the P. aeruginosa chromosome with a size of 218 kb.
Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome
Authors: Wilkinson S R , Young M
Published: November 2016

A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin.
D-serine deaminase is a stringent selective marker in genetic crosses
Authors: Maas W K , Maas R , McFall E
Published: November 2016

The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source. The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed.
A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli
Authors: Alksne L E , Keeney D , Rasmussen B A
Published: November 2016

The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation.
Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations
Authors: Huang R , Reusch R N
Published: November 2016

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.
Genetics in methanogens: transposon insertion mutagenesis of a Methanococcus maripaludis nifH gene
Authors: Blank C E , Kessler P S , Leigh J A
Published: November 2016

We designed a transposon insertion mutagenesis system for Methanococcus species and used it to make mutations in and around a nifH gene in Methanococcus maripaludis. The transposon Mudpur was constructed with a gene for puromycin resistance that is expressed and selectable in Methanococcus species. A 15.6-kb nifH region from M. maripaludis cloned in a lambda vector was used as a target for mutagenesis. A series of 19 independent Mudpur insertions spanning the cloned region were produced. Four mutagenized clones in and around nifH were introduced by transformation into M. maripaludis, where each was found to replace wild-type genomic DNA with the corresponding transposon-mutagenized DNA. Wild-type M. maripaludis and a transformant containing a Mudpur insertion upstream of nifH grew on N2 as a nitrogen source. Two transformants with insertions in nifH and one transformant with an insertion downstream of nifH did not grow on N2. The transposon insertion-gene replacement technique should be generally applicable in the methanococci for studying the effects of genetic manipulations in vivo.
Transaldolase B of Escherichia coli K-12: cloning of its gene, talB, and characterization of the enzyme from recombinant strains
Authors: Sprenger G A , Schörken U , Sprenger G , Sahm H
Published: November 2016

A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or sugars with the L configuration at C(2) (L-glyceraldehyde, D-arabinose-5-phosphate).
The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter
Authors: Segal G , Ron E Z
Published: November 2016

The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons. Transcription of this operon in A. tumefaciens was stimulated by heat shock as well as by exposure to ethanol and hydrogen peroxide. There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing only the dnaK gene. Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells. The upstream regulatory region of the dnaKJ operon of A. tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons. Sequence analysis of the promoter region of several groESL and dnaK operons from alpha-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor. This promoter may constitute the heat shock promoter of these alpha-purple proteobacteria.
TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii
Authors: Golderer G , Dlaska M , Gröbner P , Piendl W
Published: November 2016

The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.
Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers
Authors: Van Dyk T K , Reed T R , Vollmer A C , LaRossa R A
Published: November 2016

Escherichia coli strains carrying transcriptional fusions of four sigma 32-controlled E. coli heat shock promoters to luxCDABE or lacZ reporter genes were stressed by chemicals added singly or in pairs. Much more than additive induction resulted from combinations of cadmium chloride, copper sulfate, ethanol, formamide, 4-nitrophenol, and pentachlorophenol.
Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids
Authors: Rodríguez M , Holcík M , Iyer V N
Published: November 2016

The transmission of plasmid pCU1 (or other IncN group plasmid) into a population of Klebsiella oxytoca cells reduces the viability of the population. A 2,400-bp region adjacent to traA is responsible for this phenotype and includes two regions, called kikA and kikC. Klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. To obtain insight into the mode of this apparent lethality, an appropriate pCU1lacZ derivative was constructed. It could be introduced with high efficiency into Klebsiella cells. Analyses of the resultant colonies indicate that the loss of viability is not a consequence of the death of plasmid-free segregants. On the contrary and unlike postsegregational killing by plasmids, cells survived by losing the plasmid or by acquiring, secondarily, a chromosomal mutation which confers immunity to killing.
BvgAS is sufficient for activation of the Bordetella pertussis ptx locus in Escherichia coli
Authors: Uhl M A , Miller J F
Published: November 2016

BvgA and BvgS, which regulate virulence gene expression in Bordetella pertussis, are members of the two-component signal transduction family. The effects of growth conditions on the ability of BvgAS to activate transcription of fhaB (encoding filamentous hemagglutinin) and ptxA (encoding the S1 subunit of pertussis toxin) were assessed in Escherichia coli by using chromosomal fhaB-lacZYA and ptxA-lacZYA fusions. Although it had previously been reported that a ptxA-lacZYA transcriptional fusion was not activated by bvgAS in E. coli (J. F. Miller, C. R. Roy, and S. Falkow, J. Bacteriol. 171:6345-6348, 1989), we now present evidence that ptxA is activated by bvgAS in E. coli in a manner that is highly dependent on the growth conditions. Higher levels of beta-galactosidase were produced by ptxA-lacZYA in the presence of bvgAS during growth in Stainer-Scholte medium or M9 minimal salts medium with glucose than in Luria-Bertani medium. In contrast, the level of fhaB-lacZYA expression was high during growth in all media. Addition of modulating stimuli which inhibit BvgAS function eliminated expression of ptxA-lacZYA. Levels of beta-galactosidase expressed from the ptx-lacZYA fusion correlated with growth rate and with the final optical density at 600 nm, suggesting that the lower growth rate in M9-glucose and Stainer-Scholte media was responsible for greater accumulation of beta-galactosidase than was seen in Luria-Bertani medium. Overproduction of BvgA was not sufficient for activation of ptxA expression but was sufficient for fhaB expression. However, overproduction of a constitutive BvgA allele (bvgA-Cl) or overproduction of BvgA in the presence of BvgS was able to activate ptxA. Our results demonstrate Bvg-dependent activation of a ptxA-lacZYA fusion in E. coli and indicate that bvg is the only Bordetella locus required for ptxA activation in this heterologous system.
sigma E changed to sigma B specificity by amino acid substitutions in its -10 binding region
Authors: Tatti K M , Moran C P
Published: November 2016

The association of a sigma factor (sigma) with RNA polymerase in bacteria determines its specificity of promoter utilization. To identify amino acid residues in sigma E from Bacillus subtilis that determine the specificity of its interaction with the nucleotides at the -10 region of its cognate promoters, we tested whether base pair substitutions in the -10 region of a sigma B-dependent promoter could signal its utilization by sigma E-RNA polymerase. We found that a combination of base pair substitutions at positions -15 and -14 of the sigma B-dependent ctc promoter resulted in its utilization by sigma E-RNA polymerase in vivo. We also found that the combination of two amino acid substitutions at positions 119 and 120 in sigma E changed its specificity for promoter utilization, resulting in a sigma factor that directed transcription from the sigma B-dependent ctc promoter, but not from sigma E-dependent promoters. These results suggest that amino acid residues at positions 119 and 120 determine, at least in part, the specificity of interactions between sigma E and the nucleotides in the -10 region of its cognate promoters.
Sequence and mutational analysis of a tartrate utilization operon from Agrobacterium vitis
Authors: Crouzet P , Otten L
Published: November 2016

The grapevine is the natural host of the tumorigenic bacterium Agrobacterium vitis. Most of the A. vitis isolates can use tartrate, an unusually abundant compound in grapevine. The nopaline strain, AB4, contains a 170-kb conjugative plasmid (pTrAB4) encoding tartrate utilization. A 5.65-kb pTrAB4 region which enables non-tartrate-utilizing Agrobacterium tumefaciens to grow on tartrate was sequenced and mutagenized with the transcriptional fusion transposon Tn5-uidA1. This DNA fragment contains four intact open reading frames (ORFs) (ttuABCD) required for tartrate-dependent growth. The mutant phenotypes of each ORF, their homologies to published sequences, and their induction patterns allowed us to propose a model for tartrate utilization in A. vitis. ttuA encodes a LysR-like transcriptional activator and is transcribed in the absence of tartrate. ttuB codes for a protein with homology to transporter proteins and is required for entry of tartrate into bacteria. ttuC codes for a tartrate dehydrogenase, while ttuD lacks homology to known sequences; the growth properties of ttuD mutants suggest that TtuD catalyzes the second step in tartrate degradation. A fifth incomplete ORF (ttuE) encodes a pyruvate kinase which is induced by tartrate and required for optimal growth. Although the ttuABCD fragment allows growth of A. tumefaciens on tartrate, it does not provide full tartrate utilization in the original A. vitis background.
Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence
Authors: Chang J T , Green C B , Wolf R E
Published: November 2016

Previous research has indicated that the growth rate-dependent regulation of Escherichia coli gnd expression involves the internal complementary sequence (ICS), a negative control site that lies within the 6-phosphogluconate dehydrogenase coding sequence. To determine whether the ICS acts as a transcriptional operator or attenuator, we measured beta-galactosidase-specific activities in strains carrying gnd-lac operon and protein fusions containing or lacking the ICS. Whereas the presence of the ICS repressed beta-galactosidase expression from a protein fusion by 5-fold during growth on acetate and by 2.5-fold during growth on glucose, it had no effect on beta-galactosidase expression from an operon fusion. In vitro ribosome binding experiments employing the primer extension inhibition (toeprint) assay demonstrated that the presence of the ICS in gnd mRNA reduces both the maximum extent and the rate of ternary complex formation. Moreover, the effects of deletions scanning the ICS on in vivo gene expression were highly correlated with the effects of the deletions on ribosome binding in vitro. In addition, the distal end of the ICS element was found to contribute more to ICS function than did the proximal portion, which contains the complement to the Shine-Dalgarno sequence. Finally, RNA structure mapping experiments indicated that the presence of the ICS in gnd mRNA reduces the access of the nucleotides of the ribosome binding site to the single-strand-specific chemical reagents dimethyl sulfate and kethoxal. Taken together, these data support the hypothesis that the role of the ICS in the growth rate-dependent regulation of gnd expression is to sequester the translation initiation region into a long-range mRNA secondary structure that blocks ribosome binding and thereby reduces the frequency of translation initiation.
Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level
Authors: Misawa N , Satomi Y , Kondo K , Yokoyama A , Kajiwara S , Saito T , Ohtani T , Miki W
Published: November 2016

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.
Specific binding of the activator Mga to promoter sequences of the emm and scpA genes in the group A streptococcus
Authors: McIver K S , Heath A S , Green B D , Scott J R
Published: November 2016

Transcription of the surface-associated virulence factors of the group A streptococcus (GAS) Streptococcus pyogenes, M protein (emm) and the C5a peptidase (scpA), is activated by a protein called Mga (formerly Mry or VirR). To determine whether Mga binds directly to the promoters of the genes it regulates, a protein resulting from the fusion of Mga to the C-terminal end of maltose-binding protein was purified from Escherichia coli. Specific binding to the promoter regions of the scpA and emm alleles of the type M6 GAS strain JRS4 was demonstrated by electrophoresis of the DNA-protein complex. Competition studies showed that the region upstream of scpA bound MBP-Mga with a slightly higher affinity than did the region upstream of emm. DNase I protection experiments identified a single 45-bp binding site immediately upstream of and overlapping the -35 region of both promoters. Sequences homologous to the protected regions were found in the promoters of many emm, scp, and emm-like genes from strains of different serotypes of GAS, and a consensus Mga binding site was deduced.
A functional origin of transfer (oriT) on the conjugative transposon Tn916
Authors: Jaworski D D , Clewell D B
Published: November 2016

The origin of transfer (oriT) of the 18-kb conjugative transposon Tn916 has been localized to a 466-bp region which spans nucleotides 15215 to 15681 on the transposon map. The oriT lies within an intercistronic region between open reading frames ORF20 and ORF21 that contains six sets of inverted repeats ranging from 10 to 20 bp in size. The segment contains three sequences showing identity in 9 of 12 bp to the consensus nicking site (nic) of the IncP family of conjugative plasmids found in gram-negative bacteria. Overlapping one of these sequences is a region similar to the nic site of the F plasmid. Functionality was based on the ability of the oriT-containing sequence to provide a cis-acting mobilization of chimeras involving the shuttle vector pWM401 in response to activation in trans by an intact chromosome-borne transposon Tn916 delta E. Cloned segments of 466 or 376 nucleotides resulted in unselected cotransfer of the plasmid at levels of about 40% when selection was for Tn916 delta E, whereas a 110-bp segment resulted in cotransfer at a frequency of about 7%. Mobilization was specific in that gram-positive plasmids, such as pAD1 and pAM beta 1, and the gram-negative plasmids pOX38 (a derivative of F) and RP1 did not mobilize oriT-containing chimeras.
Regulation of malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability
Authors: Park S J , Cotter P A , Gunsalus R P
Published: November 2016

Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. It participates as a member of the tricarboxylic acid cycle and the branched noncyclic pathways under aerobic and anaerobic cell growth conditions, respectively. To investigate how the mdh gene is expressed under these different conditions, an mdh-lacZ operon fusion was constructed and analyzed in vivo. The mdh-lacZ fusion was expressed about twofold higher under aerobic conditions than under anaerobic cell growth conditions on most media tested. This anaerobic response is modulated by the ArcA protein, which functions as a repressor of mdh gene expression under both aerobic and anaerobic conditions. In contrast, mutations in the fnr gene did not affect mdh gene expression. Interestingly, cells grown anaerobically with glycerol and trimethylamine N-oxide or fumarate showed higher levels of mdh expression than did cells that were grown aerobically. Depending on the type of carbon compound used for cell growth, mdh expression varied by 11-fold and 5-fold under aerobic and anaerobic conditions, respectively. While mdh transcription was shown to be inversely proportional to the cell growth rate, cellular heme limitation stimulated a fivefold increase in mdh gene expression. The mdh gene appears to be highly regulated to adapt to changing conditions of aerobic and anaerobic cell growth with various types of carbon substrates.
Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene
Authors: Vrijbloed J W , van Hylckama Vlieg J , van der Put N M , Hessels G I , Dijkhuizen L
Published: November 2016

An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized.
Expression of kinA and accumulation of sigma H at the onset of sporulation in Bacillus subtilis
Authors: Asai K , Kawamura F , Yoshikawa H , Takahashi H
Published: November 2016

Induction of the Bacillus subtilis kinA gene, which codes for a major kinase of the phosphorelay pathway, required the spo0H gene, coding for the sigma H protein, but not the genes spo0A, spo0B, and spo0F at the onset of sporulation. Also, the levels of sigma H in spo0A, spo0B, and spo0F mutants were increased at the onset of sporulation, though induction of spo0H transcription in all of these mutants was appreciably inhibited. In addition, kinA expression was almost completely eliminated in a medium supplemented with excess glucose and glutamine, even though the usual stationary-phase-associated increase in sigma H was observed under these conditions.
Gene expression enhancement due to plasmid maintenance
Authors: Diaz-Ricci J C , Bode J , Rhee J I , Schügerl K
Published: November 2016

Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.
Growth of Escherichia coli K88 in piglet ileal mucus: protein expression as an indicator of type of metabolism
Authors: Blomberg L , Gustafsson L , Cohen P S , Conway P L , Blomberg A
Published: November 2016

The physiological and molecular responses of enterotoxigenic Escherichia coli K88 strain Bd 1107/7508 during growth in piglet ileal mucus and lipids extracted from mucus were studied in terms of growth rate, protein expression, and rate of heat production. E. coli K88 multiplied at maximum speed in mucus and in lipids extracted from mucus. By two-dimensional gel electrophoresis of [35S]methionine-labelled cells, it was demonstrated that the synthesis of a subclass of 13 proteins was changed at least fourfold during exponential growth in mucus compared with growth in M9 minimal medium. Ten of these proteins were repressed, while three were induced, and one of the induced proteins was identified as heat shock protein GroEL. Furthermore, two-dimensional analysis of E. coli K88 cells grown on lipids extracted from mucus revealed a set of lipid utilization-associated proteins. None of these was induced fourfold during exponential growth in mucus. Microcalorimetric measurements (monitoring the rate of heat production) of E. coli K88 grown in mucus indicated metabolic shifts in the stationary phase, in which five of the lipid utilization-associated proteins were expressed at a higher level. An isogenic E. coli K88 fadAB mutant deficient in fatty acid degradation genes grew as well as the wild type on mucus and mucus lipids. The heat production rate curve of the mutant grown in mucus differed from that of the wild type only during the stationary phase. From these results it was concluded that protein expression is influenced when E. coli K88 is grown in piglet ileal mucus rather than in M9 minimal medium. Lipids extracted from ileal mucus can serve as a substrate for E. coli K88 but appear not to be utilized during exponential growth in mucus. Stationary-phase cells metabolize fatty acids; however, the functional purpose of this is unclear.
Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)
Authors: Masoud H , Sadovskaya I , de Kievit T , Altman E , Richards J C , Lam J S
Published: November 2016

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one- and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.
Activation of the Ras/cyclic AMP pathway in the yeast Saccharomyces cerevisiae does not prevent G1 arrest in response to nitrogen starvation
Authors: Markwardt D D , Garrett J M , Eberhardy S , Heideman W
Published: November 2016

Cells carrying mutations that activate the Ras/cyclic AMP (Ras/cAMP) pathway fail to accumulate in G1 as unbudded cells and lose viability in response to nitrogen starvation. This observation has led to the idea that cells carrying this type of mutation are sensitive to nitrogen starvation because they are unable to appropriately arrest in G1. In this study, we tested predictions made by this model. We found that cells with activating Ras/cAMP pathway mutations do not continue to divide after nitrogen starvation, show a normal decrease in steady state levels of START-specific transcripts, and are not rescued by removal of cAMP during nitrogen starvation. These findings are inconsistent with the idea that activation of the Ras/cAMP pathway prevents growth arrest in cells starved for nitrogen. Our finding that cells with an active Ras/cAMP pathway have dramatically reduced amino acid stores suggests an alternative model. We propose that cells at high cAMP levels are unable to store sufficient nutrients to allow return to the G1 phase of the cell cycle when they are suddenly deprived of nitrogen. It is this inability to return to G1, rather than a failure to arrest, which leaves cells at different points in the cell cycle following nitrogen starvation.
Superoxide and the production of oxidative DNA damage
Authors: Keyer K , Gort A S , Imlay J A
Published: November 2016

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.
Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane
Authors: Lathrop J T , Wei B Y , Touchie G A , Kadner R J
Published: November 2016

The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.
Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38
Authors: Jishage M , Ishihama A
Published: November 2016

The intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma s, the rpoS gene product), in Escherichia coli MC4100 were determined by a quantitative Western immunoblot analysis. Results indicate that the level of sigma 70 is maintained at 50 to 80 fmol per micrograms of total proteins throughout the transition from the exponential growth phase to the stationary phase, while the level of sigma 38 protein is below the detection level at the exponential growth phase but increases to 30% of the level of sigma 70 when cell growth stops to enter into the stationary phase. Beside the stationary phase, the increase in sigma 38 level was observed in two cases: exposure to heat shock at the exponential phase and osmotic shock at the stationary phase.