bluuebunny/crossref_metadata_2025 huggingface dataset:
nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein
Authors: de Maagd R A , Wijfjes A H , Spaink H P , Ruiz-Sainz J E , Wijffelman C A , Okker R J , Lugtenberg B J
Published: November 2016

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined. A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined. Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size. This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor. A conventional N-terminal signal sequence was not detected in the NodO protein. The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli. Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.
Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant
Authors: Garcia-Dominguez M , Martin J F , Liras P
Published: November 2016

Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.
Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus
Authors: Zulty J J , Speedie M K
Published: November 2016

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.
Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli
Authors: Guzman P , Rivera Chavira B E , Court D L , Gottesman M E , Guarneros G
Published: November 2016

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.
Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria
Authors: Ahmad S , Weisburg W G , Jensen R A
Published: November 2016

A comprehensive phylogenetic tree for virtually the entire assemblage of enteric bacteria is presented. Character states of aromatic amino acid biosynthesis are used as criteria, and the results are compared with partial trees based upon sequencing of 16S rRNA, 5S rRNA, and tryptophan leader peptide. Three major clusters are apparent. Enterocluster 1 possesses a gene fusion (trpG-trpD) encoding anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase of tryptophan biosynthesis. This cluster includes the genera Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, and Enterobacter. The remaining two clusters lack the trpG-trpD gene fusion, but differ in the presence (enterocluster 2) or absence (enterocluster 3) of the three-step overflow pathway to L-phenylalanine. Enterocluster 2 consists of the genera Serratia and Erwinia. Enterocluster 3 includes the genera Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia, and Morganella. Within these three major clusters, a tentative hierarchy of subcluster ordering is formulated on the basis of all data available. This hierarchical framework is proposed as a general working basis for continued refinement of the phylogenetic relationships of enteric bacteria.
The cisA cistron of Bacillus subtilis sporulation gene spoIVC encodes a protein homologous to a site-specific recombinase
Authors: Sato T , Samori Y , Kobayashi Y
Published: November 2016

The nucleotide sequence of the sporulation gene spoIVC cisA in Bacillus subtilis was determined and found to encode a protein of 500 amino acid residues with a calculated molecular weight of 57,481, which is in good agreement with the size of the gene product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminal region of this protein is homologous to the site-specific DNA recombinases. Hybridization of a 3.6-kilobase EcoRI fragment carrying the spoIVC cisA gene with the EcoRI-restricted chromosomal DNA prepared from cells of various stages showed that DNA rearrangement occurs only in the mother cell in the region adjacent to spoIVC cisA 3 h after the initiation of sporulation. This result coincides with that of Stragier et al. (P. Stragier, B. Kunkel, L. Kroos, and R. Losick, Science 243:507-512, 1989). The timing of the DNA rearrangement coincides very well with the timing of spoIVC cisA gene expression. The DNA rearrangement was not observed in spoIVC cisA mutants. These results strongly suggest that the spoIVC cisA gene encodes a site-specific DNA recombinase having a very important role in sporulation.
Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria
Authors: Hayase N , Taira K , Furukawa K
Published: November 2016

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.
A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression
Authors: Schultz D W , Yarus M
Published: November 2016

We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.
Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium
Authors: Smith N H , Selander R K
Published: November 2016

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.
Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa
Authors: Wong S C , Abdelal A T
Published: November 2016

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.
Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Vega-Palas M A , Madueño F , Herrero A , Flores E
Published: November 2016

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.
Colicin cleavage by OmpT protease during both entry into and release from Escherichia coli cells
Authors: Cavard D , Lazdunski C
Published: November 2016

Proteolysis of colicins A, E1, E2, and E3 was observed after they were added to whole cells carrying a functional ompT gene. Recombinant plasmid pML19 containing the ompT gene enabled two mutant strains to cleave the added colicins. On the other hand, two colicin A recombinants were split after release from the wild-type bacteria that produced them but not from ompT mutant cells.
Effects of pH, glucose, and chelating agents on lethality of paraquat to Escherichia coli
Authors: Minakami H , Kitzler J W , Fridovich I
Published: November 2016

Retention of paraquat by Escherichia coli B was greatest after exposure at pH 9.0 and was progressively less after exposure at pH 7.0 and 5.0, respectively. This retained paraquat was capable of persistent growth inhibition. Uptake and retention of paraquat by E. coli B was dependent upon a carbon source, such as glucose. Under comparable conditions E. coli K-12 did not retain paraquat. The lethality of paraquat was seen in TSY medium but not in VB medium. The addition of Soytone, tryptone, or yeast extract, to the VB medium allowed the lethality of paraquat to be seen. A variety of chelating agents, including EDTA, 8-hydroxyquinoline, and o-phenanthroline, prevented the lethal effect of paraquat in TSY medium. Although EDTA protected against the lethality of paraquat, it did not protect against its bacteriostatic effect.
A plasmid vector with a selectable marker for halophilic archaebacteria
Authors: Holmes M L , Dyall-Smith M L
Published: November 2016

A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type cells to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (greater than 10(6)/micrograms), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria.
Role of the promoter in activation of transcription by nitrogen regulator I phosphate in Escherichia coli
Authors: Ray L , Claverie-Martin F , Weglenski P , Magasanik B
Published: November 2016

The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.
A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis
Authors: Fouet A , Sonenshein A L
Published: November 2016

Expression of the aconitase (citB) gene of Bacillus subtilis is subject to catabolite repression in cells grown in minimal media. In nutrient broth medium, citB expression is low in growing cells but is induced when cells enter sporulation. A 600-base-pair DNA fragment that extends from positions -400 through +200, relative to the transcription start site, was shown to include all of the cis-acting sequences necessary for catabolite repression and sporulation-associated regulation. This was demonstrated by fusing this DNA fragment to the Escherichia coli lacZ gene, integrating the fusion in the amyE locus of the B. subtilis chromosome, and measuring the regulation of expression of beta-galactosidase. By creating a series of deletions from either end of the 600-base-pair fragment, it was possible to define a target for catabolite repression; at least part of this target lies within the sequence between positions -84 and -68. DNA fragments that included positions -84 through +36, when carried on high-copy plasmids, caused derepression of aconitase synthesis, as if a negative regulator were being titrated. The same plasmids caused derepression of citrate synthase activity as well. Deletion of the sequence between positions -84 and -67 abolished this titration effect for both enzymes. Mutations that altered the target for catabolite repression also affected the inducibility of citB at the onset of sporulation, at least when sporulation was induced by the addition of decoyinine, an inhibitor of guanine nucleotide synthesis. When sporulation was induced by exhaustion of nutrient broth, there was no detectable difference in expression of citB-lacZ fusions whether or not they had the citB sequence from positions -84 to -67, suggesting that the mechanisms of regulation of citB in minimal medium and nutrient broth are different.
Natural transformation in Campylobacter species
Authors: Wang Y , Taylor D E
Published: November 2016

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.
Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype
Authors: Farchaus J W , Gruenberg H , Oesterhelt D
Published: November 2016

The puf operon in Rhodobacter sphaeroides is composed of the genes for the photosynthetic reaction center L and M subunits, light-harvesting antenna complex I, and one other open reading frame termed pufX. Complementation of a reaction center-deficient, photosynthetically incompetent pufLMX deletion strain in trans with a fragment containing the entire puf operon, including pufX and an additional 1,100 base pairs of DNA downstream of pufX, restored the reaction center and the photosynthesis-positive phenotype. Complementation of the same strain with pufBALM restores the reaction center to the level seen with the entire puf operon but not the photosynthesis-positive phenotype. Northern (RNA) blot analysis revealed that oxygen regulated transcription was not blocked in the absence of pufX and the downstream region. Spectroscopic and protein analyses indicated that the pigment-binding protein complexes, including the reaction center, were expressed and showed normal absorption characteristics. A 20% reduction in the amount of light-harvesting antenna complex II and a corresponding increase in the amount of light-harvesting antenna complex I were observed in the deletion strain harboring the plasmid with the puf insert lacking the pufX gene and the downstream region compared with those complemented with the entire puf operon and an additional downstream 1,100 base pairs.
Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions
Authors: Lloyd A D , Kadner R J
Published: November 2016

The Escherichia coli uhpT protein catalyzes the active transport of sugar-phosphates by an obligatory exchange mechanism. To examine its transmembrane topology, we isolated a collection of uhpT-phoA fusions encoding hybrid proteins of different lengths from the N terminus of UhpT fused to alkaline phosphatase by using transposon TnphoA. These fusions displayed different levels of alkaline phosphatase activity, although comparable levels of full-length UhpT-PhoA proteins were produced in maxicells of both high- and low-activity fusions. The full-length protein species were unstable and were degraded to the size of the alkaline phosphatase moiety in the case of a high-activity fusion or to small fragments in the case of a low-activity fusion. The enzyme activity present in low-activity fusions appeared to result from export of a small proportion of the fusion proteins to the periplasmic space. Although fusions were not obtained in all predicted extramembranous loops, the deduced topology of UhpT was consistent with a model of 12 membrane-spanning regions oriented with the amino and carboxyl termini in the cytoplasm.
Binding-protein-dependent lactose transport in Agrobacterium radiobacter
Authors: Greenwood J A , Cornish A , Jones C W
Published: November 2016

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.
Regulation of the operon encoding ribonucleotide reductase: role of the negative sites in nrd repression
Authors: Tuggle C K , Fuchs J A
Published: November 2016

Expression of the nrd genes was previously shown to be controlled by both positive and negative regulation (C. K. Tuggle and J. A. Fuchs, EMBO J. 5:1077-1085, 1986). Two regions, one located 5' and one located 3' of the nrd promoter (nrdP), were identified as negative regulatory sites since deletion of these sequences increased nrd expression. These regions of DNA have sequence similarities, and a looping mechanism was proposed to explain the requirement for two distinct sites in nrd repression. To investigate the role of these sequences in regulating nrd, a gel electrophoresis assay was used to detect the proteins that bind to the nrd regulatory sites. A protein that bound to restriction fragments containing the negative regulatory sites but not to other DNA fragments was identified in cell extracts and was partially purified. DNase I footprinting experiments showed that the binding protein protects the 5' negative site previously identified in vivo. The 3' negative site also identified in vivo was not required in vitro for high-affinity protein binding to the 5' site, but lower-affinity binding to this site could be detected. Specific binding to the 5' site was found to be elevated approximately 10-fold in crude extracts from thymine-starved cells as compared with that in extracts from unstarved cells. This higher activity was also evident in purified preparations, suggesting that thymine starvation increases the expression of the negative regulatory protein. The finding that a purified protein preparation binds both negative regulatory sites indicates that this preparation contains the nrd repressor protein or proteins. Insertion of 37 base pairs (3.5 helix turns) of DNA at a HpaII site or 35 base pairs (3.3 turns) at a MnlI site between the 5' regulatory sites and nrdP abolished the increase in nrd expression resulting from thymine starvation in vivo, but negative regulation appeared to be less affected than when either negative site was deleted. Insertion of DNA in these constructs was shown not to affect repressor binding in vitro, indicating either that a simple model of DNA looping to bring equivalent operator sites into physical proximity does not explain repression at nrd or that the distance between sites is sufficient that helical turns are of little importance.
Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum
Authors: Carrion M , Bhat U R , Reuhs B , Carlson R W
Published: November 2016

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.
Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides
Authors: Hallenbeck P L , Lerchen R , Hessler P , Kaplan S
Published: November 2016

The Rhodobacter sphaeroides genome contains two unlinked genetic regions each encoding a series of proteins involved in CO2 fixation which include phosphoribulokinase (prkA and prkB) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcLS and rbcR) (P. L. Hallenbeck and S. Kaplan, Photosynth. Res. 19:63-71, 1988; F. R. Tabita, Microbiol. Rev. 52:155-189, 1988). We examined the effect of CO2 in the presence and absence of an alternate electron acceptor, dimethyl sulfoxide, on the expression of rbcR and rbcLS in photoheterotrophically grown R. sphaeroides. The expression of both rbcR and rbcLS was shown to depend on the CO2 concentration when succinate was used as the carbon source. It was also demonstrated that CO2 fixation is critical for photoheterotrophic growth but could be replaced by the alternative reduction of dimethyl sulfoxide to dimethyl sulfide. Dimethyl sulfoxide severely depressed both rbcR and rbcLS expression in cells grown photoheterotrophically at CO2 concentrations of 0.05% or greater. However, cells grown photoheterotrophically in the absence of exogenous CO2 but in the presence of dimethyl sulfoxide had intermediate levels of expression of rbcL and rbcR, suggesting partially independent control by limiting CO2 tension. We also present evidence for the existence of two gene products, namely, CfxA and CfxB, which are encoded by genes immediately upstream of rbcLS and rbcR, respectively. Strains were constructed which contained null mutations in cfxA and/or cfxB. Each mutation eliminated expression of the linked downstream rbc operon. Further, studies utilizing these strains demonstrated that each form of ribulose 1,5-bisphosphate carboxylase/oxygenase plays an essential role in maintaining the cellular redox balance during photoheterotrophic growth at differing CO2 concentrations.
Phosphoribulokinase activity and regulation of CO2 fixation critical for photosynthetic growth of Rhodobacter sphaeroides
Authors: Hallenbeck P L , Lerchen R , Hessler P , Kaplan S
Published: November 2016

The Rhodobacter sphaeroides genome contains two unlinked genetic regions each encoding numerous proteins involved in CO2 fixation which include phosphoribulokinases (prkA and prkB), ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcLS and rbcR) (P. L. Hallenbeck and S. Kaplan, Photosynth. Res. 19:63-71, 1988; F. R. Tabita, Microbiol. Rev. 52:155-189, 1988), and two open reading frames linked to rbcLS and rbcR, namely, cfxA and cfxB, respectively (P. L. Hallenbeck, R. Lerchen, P. Hessler, and S. Kaplan, J. Bacteriol. 172:1736-1748). In this study, we examined the unique role(s) of each phosphoribulokinase activity in the regulation of CO2 fixation. Strains were constructed which contain null mutations in prkA and/or prkB. Studies utilizing these strains suggested that CO2 fixation plays an essential role in attaining the cellular redox balance necessary for photoheterotrophic growth. The presence of an external electron acceptor can negate the requirement for CO2 for photoheterotrophic growth. Each form of phosphoribulokinase and ribulose 1,5-bisphosphate carboxylase/oxygenase was shown to have distinct roles in CO2 metabolism when cells were exposed to extremes in CO2 levels. Evidence is also presented which unequivocally demonstrated that regulation of the expression of the enzymes involved in CO2 metabolism is effective at the transcriptional level. Although the two regions of the DNA involved in CO2 fixation are physically unlinked, each region of the DNA can have a profound effect on the expression of the other region of the DNA.
Transcriptional organization of a cloned chemotaxis locus of Bacillus subtilis
Authors: Zuberi A R , Ying C W , Weinreich M R , Ordal G W
Published: November 2016

A cloned chemotaxis operon has been characterized. Thirteen representative che mutations from different complementation groups were localized on the physical map by recombination experiments. The use of integration plasmids established that at least 10 of these complementation groups within this locus are cotranscribed. An additional three complementation groups may form part of the same transcript. The direction of transcription and the time of expression were determined from chromosomal che-lacZ gene fusions. The promoter was cloned and localized to a 3-kilobase fragment. Expression of beta-galactosidase from this promoter was observed primarily during the logarithmic phase of growth. Three-factor PBS1 cotransduction experiments were performed to order the che locus with respect to adjacent markers. The cheF141 mutation is 70 to 80% linked to pyrD1. This linkage is different from that reported previously (G. W. Ordal, D. O. Nettleton, and J. A. Hoch, J. Bacteriol. 154:1088-1097, 1983). The cheM127 mutation is 57% linked by transformation to spcB3. The gene order determined from all crosses is pyrD-cheF-cheM-spcB.
Possible insertion sequences in a mosaic genome organization upstream of the exotoxin A gene in Pseudomonas aeruginosa
Authors: Pritchard A E , Vasil M L
Published: November 2016

Nucleotide sequence and Southern hybridization data revealed a mosaic genome organization in a region that extends several thousand base pairs upstream of the exotoxin A (toxA) gene in Pseudomonas aeruginosa. An interstrain comparison of DNA in this region showed a pattern of alternating segments of homologous and nonhomologous sequences. Two nonhomologous elements, approximately 1 kilobase pair upstream of the gene in strains PA103 and Ps388, were characterized in more detail. The sequence elements, denoted IS-PA-1 and IS-PA-2 for the different strains, are about 1,000 and 785 base pairs long, respectively, and have 5-base-pair direct repeats at their boundaries, consistent with their being DNA insertion sequences. The distribution of these elements in 34 different strains was determined. IS-PA-1 was found in a single copy upstream of toxA in half of the strains and was found in two copies in four of the strains. Some strains contained neither element, and one strain carried both. The genome of another strain, WR5, which lacks toxA, was shown to contain a 350-base-pair region that was highly homologous to DNA sequences located just upstream of toxA in other strains. The WR5 genome lacked several kilobase pairs of DNA that was found both upstream and downstream of this homologous region in the other strains.
SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells
Authors: Banerjee S K , Borden A , Christensen R B , LeClerc J E , Lawrence C W
Published: November 2016

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.
A mutation in P23, the first gene in the RNA polymerase sigma A (sigma 43) operon, affects sporulation in Bacillus subtilis
Authors: Zuberi A R , Doi R H
Published: November 2016

Mutations within P23, the first gene of the Bacillus subtilis sigma A operon, were not detrimental to vegetative growth or sporulation. One deletion of P23 resulted in a strain that sporulated earlier than the wild type. This aberrant phenotype may be due to the simultaneous deletion of a sigma H promoter from the sigma A operon.
Alkaline induction of a novel gene locus, alx, in Escherichia coli
Authors: Bingham R J , Hall K S , Slonczewski J L
Published: November 2016

A novel pH-regulated locus inducible over 100-fold in alkaline media was identified in Escherichia coli through screening of 93,000 Mu dI1734 (lacZ Kmr) operon fusions at pH 6.5 and pH 8.5. Four lacZ fusions that showed expression only at the higher pH were mapped at 67.5 min by P1 transduction crosses. The locus was designated alx.
DNA sequence of folate biosynthesis gene sulD, encoding hydroxymethyldihydropterin pyrophosphokinase in Streptococcus pneumoniae, and characterization of the enzyme
Authors: Lopez P , Greenberg B , Lacks S A
Published: November 2016

A cloned segment of the chromosome of Streptococcus pneumoniae, in which mutations to sulfonamide resistance occur, contains several genes encoding enzymes for folate biosynthesis. Determination of the DNA sequence of parts of this segment and identification of a putative promoter and terminator of transcription indicate an operon composed of four genes. The first, sulA, encodes the enzyme dihydropteroate synthase. The functions of the second and third possible genes, sulB and sulC, are not known. The last gene, sulD, encodes a 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase. The product of this enzyme is the substrate for dihydropteroate synthetase. The enzyme protein was partially purified and shown to consist of a single subunit of 31 kilodaltons, encoded by sulD. On the basis of gel filtration behavior, the native protein appears to be a trimer or tetramer. Subcloning of the sulD gene in an Escherichia coli expression vector increased expression of the pyrophosphokinase 1,000-fold over the level produced by a single copy of the chromosomal gene.
Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes: clarification of actinorhodin gene functions
Authors: Bartel P L , Zhu C B , Lampel J S , Dosch D C , Connors N C , Strohl W R , Beale J M , Floss H G
Published: November 2016

Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIII, actIV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-pair XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2]acetate feeding experiments. S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product. Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actIII, and actVII loci, the recombinant strain produced both aloesaponarin II and aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate 2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIII gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actVII, and actIV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-O-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.
Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin
Authors: Webb R , Reddy K J , Sherman L A
Published: November 2016

The molecular chaperonins such as GroEL are now widely regarded as essential components for the stabilization of integral membrane or secretory proteins before membrane insertion or translocation, as well as for the assembly of macromolecular complexes such as ribulose bisphosphate carboxylase-oxygenase. The groESL operon of Synechococcus sp. strain PCC 7942 was cloned as two independent lacZ-groEL translational fusions by immunoscreening a lambda ZAP genomic expression library and then sequenced. The derived amino acid sequences of the GroES and GroEL proteins demonstrated very high levels of amino acid identity with cognate chaperonins from bacteria and chloroplasts. The bicistronic 2.4-kilobase transcript from this operon, barely detectable in RNA preparations from cells grown at 30 degrees C, accumulated approximately 120-fold in preparations from cells grown for 20 min at 45 degrees C. Under these conditions, GroEL protein accumulated to 10-fold-higher levels. Primer extension analysis was used to identify a cyanobacterial heat shock promoter located at -81 base pairs from the groES initiation codon. The transcriptional -10 and -35 sequences differ slightly from Escherichia coli consensus heat shock promoter sequences.
Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen
Authors: Schmoll T , Ott M , Oudega B , Hacker J
Published: November 2016

S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the influence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme beta-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-lac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain 536WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain 536WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate had a strong influence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.
Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa
Authors: Kelly-Wintenberg K , Anderson T , Montie T C
Published: November 2016

Purified flagella from two strains of 32P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.
Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli
Authors: Young C C , Alvarez J D , Bernlohr R W
Published: November 2016

Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis (R. W. Bernlohr, A. L. Saha, C. C. Young, B. R. Toth, and K. J. Golden, J. Bacteriol. 170:4113-4118, 1988; K. J. Golden and R. W. Bernlohr, Mol. Gen. Genet. 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing.