bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine
Authors: Smith L T , Smith G M , Madkour M A
Published: November 2016

We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed.
Regulation of photolyase in Escherichia coli K-12 during adenine deprivation
Authors: Alcorn J L , Rupert C S
Published: November 2016

DNA photolyase, a DNA repair enzyme encoded by the phr gene of Escherichia coli, is normally regulated at 10 to 20 active molecules per cell. In purA mutants deprived of adenine, this amount increased sixfold within 2 h. Operon fusions placing lacZ under transcriptional control of phr promoters indicated no change in transcription rate during adenine deprivation, and gene fusions of phr with lacZ showed a nearly constant level of translation as well. Immunoblot analysis indicated that the total amount of photolyase protein remained constant during enzyme amplification. On the other hand, treatment of cells with chloramphenicol during the adenine deprivation prevented any increase. DNA regions lying 1.3 to 4.2 kb upstream of the phr coding sequences were necessary for this amplification to occur and for this purpose would function in trans. These results suggest that adenine deprivation leads to a posttranslational change, involving synthesis of protein encoded by sequences lying upstream of phr, which increases photolyase activity. The amplification in activity was found to be reversible, for when adenine was restored, the photolyase activity declined before cell growth resumed.
The spoIIIA locus is not a major determinant of prespore-specific gene expression during sporulation in Bacillus subtilis
Authors: Illing N , Errington J
Published: November 2016

During sporulation in Bacillus subtilis, expression of several prespore-specific genes is strongly dependent on the spoIIIE and spoIIIG gene products. Previous reports have also indicated a requirement for the products of the spoIIIA locus. However, we have now systematically studied six different well-defined spoIIIA mutations and find that, relative to spoIIIE and spoIIIG mutations, they have only a minor effect on the expression of two different prespore-specific genes, spoVA and sspA. Moreover, we have shown that strain IS37, which has been used as a spoIIIA mutant in several previous studies, actually contains a lesion in the spo0A gene. We conclude that spoIIIA has a relatively minor or indirect role in the regulation of prespore-specific gene expression.
Molecular analysis of two genes of the Escherichia coli gab cluster: nucleotide sequence of the glutamate:succinic semialdehyde transaminase gene (gabT) and characterization of the succinic semialdehyde dehydrogenase gene (gabD)
Authors: Bartsch K , von Johnn-Marteville A , Schulz A
Published: November 2016

We have characterized two genes of the Escherichia coli K-12 gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway. The nucleotide sequence of gabT, coding for glutamate:succinic semialdehyde transaminase (EC 2.6.1.19), alternatively known as 4-aminobutyrate transaminase, was determined. The structural gene consists of 1,281 nucleotides specifying a protein of 426 amino acids with a molecular mass of 45.76 kDa. The protein shows significant homologies to the ornithine transaminases from Saccharomyces cerevisiae and from rat and human mitochondria. Three functionally and structurally important amino acid residues of the transaminase were identified by sequence comparison studies, and evolutionary relationships of the aminotransferases are discussed. The gabD gene, encoding succinic semialdehyde dehydrogenase (EC 1.2.1.16), was cloned and shown to be located adjacent to the 5' end of gabT. Expression studies with subfragments of the initially cloned DNA region revealed a maximal size of 1.7 kb for gabD. Both genes are cotranscribed from a promoter located upstream of gabD.
Genetic analysis of Escherichia coli urease genes: evidence for two distinct loci
Authors: Collins C M , Falkow S
Published: November 2016

Studies with two uropathogenic urease-producing Escherichia coli strains, 1021 and 1440, indicated that the urease genes of each are distinct. Recombinant plasmids encoding urease activity from E. coli 1021 and 1440 differed in their restriction endonuclease cleavage sites and showed minimal DNA hybridization under stringent conditions. The polypeptides encoded by the DNA fragments containing the 1021 and 1440 urease loci differed in electrophoretic mobility under reducing conditions. Regulation of urease gene expression differed in the two ureolytic E. coli. The E. coli 1021 locus is probably chromosomally encoded and has DNA homology to Klebsiella, Citrobacter, Enterobacter, and Serratia species and to about one-half of the urease-producing E. coli tested. The E. coli 1440 locus is plasmid encoded; plasmids with DNA homology to the 1440 locus probe were found in urease-producing Salmonella spp., Providencia stuartii, and two E. coli isolates. In addition, the 1440 urease probe was homologous to Proteus mirabilis DNA.
Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation
Authors: Spence J , Cegielska A , Georgopoulos C
Published: November 2016

Because of the highly conserved pattern of expression of the eucaryotic heat shock genes hsp70 and hsp84 or their cognates during sporulation in Saccharomyces cerevisiae and development in higher organisms, the role of the Escherichia coli homologs dnaK and htpG was examined during the response to starvation. The htpG deletion mutant was found to be similar to its wild-type parent in its ability to survive starvation for essential nutrients and to induce proteins specific to starvation conditions. The dnaK103 mutant, however, was highly susceptible to killing by starvation for carbon and, to a lesser extent, for nitrogen and phosphate. Analysis of proteins induced under starvation conditions on two-dimensional gels showed that the dnaK103 mutant was defective for the synthesis of some proteins induced in wild-type cells by carbon starvation and of some proteins induced under all starvation conditions, including the stationary phase in wild-type cells. In addition, unique proteins were synthesized in the dnaK103 mutant in response to starvation. Although the synthesis of some proteins under glucose starvation control was drastically affected by the dnaK103 mutation, the synthesis of proteins specifically induced by nitrogen starvation was essentially unaffected. Similarly, the dnaK103 mutant was able to grow, utilizing glutamine or arginine as a source of nitrogen, at a rate approximate to that of the wild-type parent, but it inefficiently utilized glycerol or maltose as carbon sources. Several differences between the protein synthetic pattern of the dnaK103 mutant and the wild type were observed after phosphate starvation, but these did not result in a decreased ability to survive phosphate starvation, compared with nitrogen starvation.
Role of purine biosynthetic intermediates in response to folate stress in Escherichia coli
Authors: Rohlman C E , Matthews R G
Published: November 2016

Folic acid plays a central role in anabolic metabolism by supplying single-carbon units at varied levels of oxidation for both nucleotide and amino acid biosyntheses. It has been proposed that 5-amino-4-imidazole carboxamide riboside 5'-triphosphate (ZTP), an intermediate in de novo purine biosynthesis, serves as a signal of cellular folate stress and mediates a physiologically beneficial response to folate stress in Salmonella typhimurium (B. R. Bochner, and B. N. Ames, Cell 29:929-937, 1982). We examined the physiological response of Escherichia coli to folate stress induced by the drugs psicofuranine, trimethoprim, and sodium sulfathiazole or by p-aminobenzoic acid (pABA) starvation. Analysis of nucleotide pools showed that psicofuranine or trimethoprim treatment of a prototrophic strain or growth of a pABA auxotroph on limiting pABA induced the production of the nucleotide ZTP, as previously observed in S. typhimurium by Bochner and Ames. Accumulation of ZTP and its precursor 5-amino-4-imidazole carboxamide riboside 5'-monophosphate (ZMP) did not correlate well with folate stress in E. coli, as measured by determination of the folate/protein ratios of extracts of treated cells. Treatment of cells with psicofuranine caused a marked accumulation of 5-amino-4-imidazole carboxamide ribonucleotides (Z-ribonucleotides) but a statistically insignificant drop in the folate/protein ratio of cell extracts. Sodium sulfathiazole treatment at a drug concentration that led to a threefold drop in the growth rate and in the folate/protein ratio of treated cells led to little accumulation of Z-ribonucleotides in E. coli A purF his+ strain which produces ZTP and ZMP when treated with trimethoprim was constructed. In this strain, histidine represses the synthesis of both ZMP and ZTP. Treatment of cells of this strain with trimethoprim resulted in a decrease in the folate/protein ratio of cell extracts, but a blockade of Z-ribonucleotide accumulation did not affect the extent of folate depletion seen in treated cells and had only a small effect on the resistance of this strain to growth inhibition by trimethoprim. The patterns of protein expression induced by treatment of this strain with trimethoprim or psicofuranine were examined by two-dimensional electrophoretic resolution of the total cellular proteins. No differences in protein expression were seen when the treatment were performed in media containing or lacking histidine. These studies failed to provide evidence in E. coli for a folate stress regulon controlled by ZTP.
Lysine uptake and exchange in Corynebacterium glutamicum
Authors: Bröer S , Krämer R
Published: November 2016

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.
Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus
Authors: Jensen S E , Wong A , Rollins M J , Westlake D W
Published: November 2016

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) was purified from Streptomyces clavuligerus by a combination of salt precipitation, ultrafiltration, and anion-exchange chromatography. The final purified material gave two protein bands with molecular weights of 283,000 and 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis in nondenaturing gels gave a single protein band with an estimated molecular weight of 560,000. These results suggest that ACVS is a multimer composed of nonidentical subunits.
Mapping of conformational epitopes of monoclonal antibodies against Escherichia coli penicillin-binding protein 1B (PBP 1B) by means of hybrid protein analysis: implications for the tertiary structure of PBP 1B
Authors: Den Blaauwen T , Pas E , Edelman A , Spratt B G , Nanninga N
Published: November 2016

We have analyzed the location of the epitope areas of the four monoclonal antibody groups against penicillin-binding protein 1B (PBP 1B; T. den Blaauwen, F. B. Wientjes, A. H. J. Kolk, B. G. Spratt, and N. Nanninga, J. Bacteriol. 171:1393-1401). They could be specified by studying monoclonal antibody binding patterns to amino- and carboxy-terminal truncated PBP 1B molecules. Monoclonal antibodies against conformational epitopes, with the exception of one epitope area, did not recognize PBP 1B molecules that had not been translocated across the membrane. Apparently, translocation is required for PBP 1B to fully obtain its native conformation.
Isolation and characterization of Bacillus stearothermophilus 30S and 50S ribosomal protein mutations
Authors: Schnier J , Gewitz H S , Behrens S E , Lee A , Ginther C , Leighton T
Published: November 2016

Bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated. Many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system. This collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.
Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila
Authors: Jablonski P E , DiMarco A A , Bobik T A , Cabell M C , Ferry J G
Published: November 2016

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.
Genes for phycocyanin subunits in Synechocystis sp. strain PCC 6701 and assembly mutant UV16
Authors: Anderson L K , Grossman A R
Published: November 2016

The cyanobacterial phycobilisome is a large protein complex located on the photosynthetic membrane. It harvests light energy and transfers it to chlorophyll for use in photosynthesis. Phycobilisome assembly mutants in the unicellular cyanobacterium Synechocystis sp. strain 6701 have been characterized. One such mutant, UV16, contains a defect in the assembly of the biliprotein phycocyanin. We report the cloning and sequencing of the phycocyanin genes from wild-type Synechocystis strain 6701 and demonstrate an alteration in the gene for the phycocyanin alpha subunit in UV16. Possible consequences of the lesion on phycobilisome assembly were assessed from its position in the phycocyanin tertiary and quaternary structures. The UV16 phenotype is complex and includes a reduced level of phycocyanin relative to that in the wild type. To determine whether the lower phycocyanin content results from lower transcript levels, a fragment of cpcBA was used as a probe for quantitating phycocyanin mRNA. Both the wild type and UV16 contained two phycocyanin transcripts of approximately 1.4 and 1.5 kilobases that were equal in abundance and that did not vary with light quality during cell growth. Equal levels of these transcripts in the wild type and UV16 suggest that the lower phycocyanin content in the mutant may be due to posttranscriptional events. The 5' ends of the two phycocyanin mRNAs were mapped at 100 and 223 base pairs upstream of the cpcB initiation codon. Homologous regions upstream of the putative transcription initiation sites may be important for maintaining high levels of transcription from the Synechocystis strain 6701 phycocyanin gene set.
Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains
Authors: Ebisu S , Tsuboi A , Takagi H , Naruse Y , Yamagata H , Tsukagoshi N , Udaka S
Published: November 2016

Bacillus brevis HPD31 contains a surface (S)-layer protein, termed the HWP, which forms a hexagonal array in the cell wall. The 5' region of the HWP gene was isolated from a DNA library constructed in bacteriophage vector EMBL3 from a partial BamHI digest of the chromosomal DNA. The 3' region contained in a 2.7-kilobase BglII fragment of the DNA was cloned into Escherichia coli, using pUC118 as a vector. On the basis of the chemically determined N-terminal amino acid sequence, the HWP gene was found to encode a polypeptide consisting of 1,087 amino acid residues with a signal peptide of 53 or 23 amino acid residues. The deduced amino acid composition was similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic relative to basic amino acids and in the very low content of sulfur-containing amino acids. The deduced amino acid sequence showed high homology (78%) with that of the middle wall protein of B. brevis 47. Furthermore, the multiple 5' ends of the HWP gene transcripts detected on S1 nuclease analysis closely resembled those of the middle wall protein gene transcripts. This complex structure was also conserved (greater than 85%) in the regulatory regions of two other cell wall protein genes isolated from B. brevis HPD52 and HP033, suggesting that the synthesis of the cell wall proteins is intricately regulated through a similar mechanism in protein-producing B. brevis.
Integration host factor plays a role in IS50 and Tn5 transposition
Authors: Makris J C , Nordmann P L , Reznikoff W S
Published: November 2016

In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.
Capsulation in distantly related strains of Haemophilus influenzae type b: genetic drift and gene transfer at the capsulation locus
Authors: Kroll J S , Moxon E R
Published: November 2016

Among natural populations of capsulate Haemophilus influenzae, clones of strains with type b capsular polysaccharide are found in each of two widely separated phylogenetic divisions. The chromosomal capsulation locus found in strains from either division has a three-segment organization, with serotype-specific DNA nested between elements common to all serotypes, but pairwise comparison of the segments between the divisions suggests that they have distinct phylogenetic histories. Genes clustered in one of the non-serotype-specific segments appear to have diverged from an ancestral element, reflected in 12% nucleotide sequence divergence in one homologous pair. In contrast, genes conferring the capacity to produce type-specific polysaccharide exhibit no such divergence, and we speculate that these have been subject more recently to horizontal transfer within the bacterial population. Clinically important capsulate gram-negative bacteria share a common organization of their capsulation loci, arguing convergence on a successful arrangement of genes. In H. influenzae this appears to have allowed the occasional exchange of serotype-specific capsulation genes between strains, a event of potential clinical importance in this major bacterial pathogen.
Transcription of the ftsZ gene and cell division in Escherichia coli
Authors: Robin A , Joseleau-Petit D , D'Ari R
Published: November 2016

The ftsZ gene of Escherichia coli, which lies in a cluster of cell division genes at 2 min on the genetic map, codes for a protein which is thought to play a key role in triggering cell division. Using an ftsZ::lacZ operon fusion, we have studied the transcription of the ftsZ gene under conditions in which cell division was either inhibited or synchronized in the bacterial population. In ftsZ, ftsA, ftsQ, and ftsI (or pbpB) mutants, there was no change in the differential rate of expression of the ftsZ gene in nonpermissive conditions, when cell division was completely blocked. Although the FtsZ protein is thought to be limiting for cell division, in synchronized cultures the ftsZ gene was expressed not only at the moment of septation initiation but throughout the cell cycle. Its expression, however, was not exponential but linear, with a rapid doubling in rate at a specific cell age; this age, about 20 min after division in a 60-min cycle, was different from the age at which the ftsZ::lacZ operon was duplicated. However, it was close to the age at which replication initiated and at which the rate of phospholipid synthesis doubled. During the transient division inhibition after a nutritional shift-up, ftsZ transcription again became linear, with two doublings in rate at intervals equal to the mass doubling time in the rich medium; it adopted the exponential rate typical of rich medium about 60 min after the shift-up, just before the bacterial population resumed cell division. The doubling in the rate of ftsZ transcription once per cycle in synchronized cultures and once per mass doubling time during the transition period after a nutritional shift-up reflects a new cell cycle event.
Hypoosmotic adaptation in Rhizobium meliloti requires beta-(1----2)-glucan
Authors: Dylan T , Helinski D R , Ditta G S
Published: November 2016

beta-(1----2)-Glucan, an unusual cyclic oligosaccharide, can be isolated from the periplasm of bacteria belonging to the family Rhizobiaceae. Data presented here suggest that the periplasmic beta-(1----2)-glucan of Rhizobium meliloti plays a major role in osmotic adaptation. First, growth of R. meliloti in a low-osmolarity medium causes a large accumulation of periplasmic beta-(1----2)-glucan. Second, mutations in the ndv genes, which prevent this accumulation of beta-(1----2)-glucan, reduce cell growth rates under low-osmolarity conditions and cause several other phenotypic changes indicative of an altered or stressed surface. Third, growth of the ndv mutants can be restored by raising the osmolarity of the medium with the addition of a variety of ionic or nonionic compounds. The phenotypic changes associated with the cell surface of the mutants can also be substantially suppressed by increasing the medium osmolarity. On the basis of these data and general considerations about the periplasmic space in gram-negative bacteria, we suggest a mechanism of hypoosmotic adaptation in R. meliloti in which beta-(1----2)-glucan plays an essential role.
Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase
Authors: Margolin W , Howe M M
Published: November 2016

Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70. This result suggests that C-dependent activation of Plys requires sigma 70. Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription.
Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas ruminantium
Authors: Michel T A , Macy J M
Published: November 2016

When Selenomonas ruminantium HD4 was grown in a chemostat, maximal succinate production and the highest molar growth yield values were both observed at a dilution rate of roughly 0.2 h-1. To determine the possible relationship between succinate efflux and high molar growth yields, the generation of a membrane potential by succinate efflux was studied in whole cells and vesicles (inside-out and right-side-out) prepared from S. ruminantium. Washed whole cells took up succinate in the absence of an exogenous energy supply; uptake was completely abolished by brief treatment with dinitrophenol or with nigericin and valinomycin. High levels of sodium ions (with respect to the intracellular sodium concentration in the assay buffer had a stimulatory effect on succinate uptake. When succinate was added to inside-out vesicles, a membrane potential (inside positive) was generated, as indicated by fluorescence quenching of the anionic lipophilic dye Oxonol V. Fluorescence quenching was sensitive to uncoupling by gramicidin D but only partially sensitive to the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In right-side-out vesicles, succinate uptake could be driven by an artificially imposed sodium gradient but not by a potassium diffusion potential; imposition of both a sodium gradient and potassium diffusion potential resulted in improved succinate uptake. The generation of a membrane potential (inside negative) upon succinate efflux was demonstrated directly in right-side-out vesicles when succinate-loaded vesicles were diluted into succinate-free buffer, and the lipophilic cationic probe tetraphenylphosphonium accumulated in the vesicles. Results indicate that an electrogenic succinate-sodium symporter is present in S. ruminantium. Transport of succinate out of the cell via the symporter might be responsible for the high molar growth yields obtained by this organism when it is grown at dilution rates where maximal succinate production occurs.
Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus
Authors: Ong C J , Wong M L , Smit J
Published: November 2016

Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond formations that occur not only at the time of holdfast production. Thus, we propose that the attachment of the holdfast to the cell is a true adhesion process and that the stalk tip and base of the flagellum must have compositions distinctly different from that of the remainder of the caulobacter cell surface.
Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins
Authors: Jennings M P , Beacham I R
Published: November 2016

Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme. A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported. We now present a molecular study of the gene, ansB, encoding L-asparaginase II. This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence. The nucleotide sequence of ansB, including 5'- and 3'-untranslated regions, was determined. The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence. The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues. A single region of sequence similarity is observed when ansA and ansB are compared. The transcriptional start site in ansB was determined, allowing the identification of the promoter region. The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter. Regulation by cyclic AMP receptor protein and anaerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated.
Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins
Authors: White D W , Jacobson G R
Published: November 2016

We have subcloned a portion of the Escherichia coli mtlA gene encoding the hydrophilic, C-terminal domain of the mannitol-specific enzyme II (mannitol permease; molecular mass, 68 kilodaltons [kDa]) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system. This mtlA fragment, encoding residues 379 to 637 (residue 637 = C terminus), was cloned in frame into the expression vector pCQV2 immediately downstream from the lambda pr promoter of the vector, which also encodes a temperature-sensitive lambda repressor. E. coli cells carrying a chromosomal deletion in mtlA (strain LGS322) and harboring this recombinant plasmid, pDW1, expressed a 28-kDa protein cross-reacting with antipermease antibody when grown at 42 degrees C but not when grown at 32 degrees C. This protein was relatively stable and could be phosphorylated in vitro by the general phospho-carrier protein of the phosphotransferase system, phospho-HPr. Thus, this fragment of the permease, when expressed in the absence of the hydrophobic, membrane-bound N-terminal domain, can apparently fold into a conformation resembling that of the C-terminal domain of the intact permease. When transformed into LGS322 cells harboring plasmid pGJ9-delta 137, which encodes a C-terminally truncated and inactive permease (residues 1 to ca. 480; molecular mass, 51 kDa), pDW1 conferred a mannitol-positive phenotype to this strain when grown at 42 degrees C but not when grown at 32 degrees C. This strain also exhibited phosphoenolpyruvate-dependent mannitol phosphorylation activity only when grown at the higher temperature. In contrast, pDW1 could not complement a plasmid encoding the complementary N-terminal part of the permease (residues 1 to 377). The pathway of phosphorylation of mannitol by the combined protein products of pGJ9-delta 137 and pDPW1 was also investigated by using N-ethylmaleimide to inactivate the second phosphorylation sites of these permease fragments (proposed to be Cys-384). These results are discussed with respect to the domain structure of the permease and its mechanism of transport and phosphorylation.
Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon
Authors: Downing W L , Sullivan S L , Gottesman M E , Dennis P P
Published: November 2016

Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized. The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P. J. Schatz, P. D. Riggs, A. Jacq, M. J. Fath, and J. Beckwith, Genes Dev. 3:1035-1044, 1989). The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination. The protein product of nusG is essential for bacterial viability. The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter. The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease. The role of transcript processing in the regulation of secE and nusG has not yet been established. The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.
Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism
Authors: Kuske C R , Kirkpatrick B C
Published: November 2016

Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome.
Membrane-associated chromate reductase activity from Enterobacter cloacae
Authors: Wang P C , Mori T , Toda K , Ohtake H
Published: November 2016

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.
Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles
Authors: Markiewicz Z , Tomasz A
Published: November 2016

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.
A new model for the penetration of prey cells by bdellovibrios
Authors: Tudor J J , McCann M P , Acrich I A
Published: November 2016

Bdellovibrio bacteriovorus 109J and most other bdellovibrios cause prey cells to round following penetration. Bdellovibrio sp. strain W does not cause rounding of the prey. Analysis of enzyme activities during the early stages of bdellovibrio attack indicated that strain W differs from most other bdellovibrios in that there is no glycanase activity produced during penetration. Likewise, heat-killed prey were penetrated normally by strain 109J, but the resulting bdelloplast did not become round and no glycanase was detected, indicating that glycanase is not essential for penetration. Peptidoglycan from prey cells penetrated by strain W was sensitive to lysozyme, but these cells were not susceptible to attack and penetration by strain 109J, indicating that peptidoglycan deacetylation is not the primary exclusion mechanism. We propose a model in which it is the peptidase activity of the bdellovibrios which allows them to breach the peptidoglycan of their prey and in which the glycanase activity exhibited by strain 109J and other bdellovibrios is responsible for the rounding of the bdelloplast.
A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis
Authors: Engel J N , Ganem D
Published: November 2016

Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.
DNA sequence and expression analysis of algP and algQ, components of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa: algP contains multiple direct repeats
Authors: Konyecsni W M , Deretic V
Published: November 2016

The complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.
Highly repetitive DNA sequences in cyanobacterial genomes
Authors: Mazel D , Houmard J , Castets A M , Tandeau de Marsac N
Published: November 2016

We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.
Bacterial transposon Tn7 utilizes two different classes of target sites
Authors: Kubo K M , Craig N L
Published: November 2016

Sites of transposon Tn7 insertion in the Escherichia coli chromosome were examined, and two distinct classes of target sites differing in nucleotide sequence were identified. The target site choice was found to be determined by Tn7-encoded transposition genes.
Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris
Authors: Hötte B , Rath-Arnold I , Pühler A , Simon R
Published: November 2016

Cosmid clones able to restore exopolysaccharide production in possibly insertion sequence element-induced surface mutants of Xanthomonas campestris pv. campestris were isolated. By fragment-specific Tn5-lac mutagenesis of one of the cosmids, pXCB1002, a new DNA region which is involved in exopolysaccharide biosynthesis and which is organized into at least 12 complementation groups was identified.
soxR, a locus governing a superoxide response regulon in Escherichia coli K-12
Authors: Tsaneva I R , Weiss B
Published: November 2016

The nfo (endonuclease IV) gene of Escherichia coli is induced by superoxide generators such as paraquat (methyl viologen). An nfo'-lacZ operon fusion was used to isolate extragenic mutations affecting its expression. The mutations also affected the expression of glucose 6-phosphate dehydrogenase, Mn2(+)-superoxide dismutase (sodA), and three lacZ fusions to soi (superoxide-inducible) genes of unknown function. The mutations were located 2 kilobases clockwise of ssb at 92 min on the current linkage map. One set of mutations, in a new gene designated soxR, caused constitutive overexpression of nfo and the other genes. It included insertions or deletions affecting the carboxyl end of a 17-kilodalton polypeptide. In a soxR mutant, the expression of sodA, unlike that of nfo, was also regulated independently by oxygen tension. Two other mutants were isolated in which the target genes were noninducible; they had an increased sensitivity to killing by superoxide-generating compounds. One had a Tn10 insertion in or near soxR; the other had a multigene deletion encompassing soxR. Therefore, the region functions as a positive regulator because it encodes one or more products needed for the induction of nfo. Regulation is likely to be at the level of transcription because the mutations were able to affect the expression of an nfo'-lac operon fusion that contained the ribosome-binding site for lacZ. Some mutant plasmids that failed to suppress (or complement) constitutivity in trans had insertion mutations several hundred nucleotides upstream of soxR in the general region of a gene for a 13-kilodalton protein encoded by the opposite strand, raising the possibility of a second regulatory gene in this region. The result define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E. coli.