bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA
Authors: Higgins D E , DiRita V J
Published: November 2016

Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein. These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin. ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade. ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB). The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites. Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V. cholerae. In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter. These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters.
The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli
Authors: Jourlin C , Simon G , Pommier J , Chippaux M , Méjean V
Published: November 2016

Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway.
The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair
Authors: Kogoma T , Cadwell G W , Barnard K G , Asai T
Published: November 2016

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.
Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli
Authors: Rao N N , Kornberg A
Published: November 2016

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.
Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA
Authors: La Farina M , Stira S , Mancuso R , Grisanti C
Published: November 2016

Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions.
An autolysin ring associated with cell separation of Staphylococcus aureus
Authors: Yamada S , Sugai M , Komatsuzawa H , Nakashima S , Oshida T , Matsumoto A , Suginaka H
Published: November 2016

atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 59-kDa endo-beta-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.
RNases involved in ribozyme degradation in Escherichia coli
Authors: Wang J Y , Qiu L , Wu E D , Drlica K
Published: November 2016

Hammerhead ribozymes are small catalytic RNA molecules that can be designed to specifically cleave other RNAs. These ribozymes have exhibited low efficiency when examined inside cells, perhaps in part because of their sensitivity to intracellular RNases. In an effort to better understand intracellular degradation of small, foreign RNAs and to develop more stable ribozymes, the ability of Escherichia coli RNase mutants to digest ribozymes was examined. In soluble extracts, most (80 to 90%) of the endonucleolytic activity was due to RNases I and I*, since degradative activity was inhibited by Mg2+ and by the rna-2 mutation. Degradation by exonucleolytic activities was temperature sensitive in extracts from an rna pnp rnb(Ts) triple mutant but not in extracts from an rna rnb(Ts) double mutant. Thus, the products of rnb and pnp, RNase II and polynucleotide phosphorylase, respectively, appear to be the major exonucleases that degrade hammerhead ribozymes. Examination of intracellular degradation revealed that RNases I and I* contributed to about half of the degradative activity as judged by comparison of the rate of ribozyme decay in wild-type and rna-2 mutant cells. Little additional effect was observed in rne(RNase E) and rnc (RNaseIII) mutants. Taken together, these data indicate that hammerhead ribozymes are digested largely by the degradative class of RNase (RNases I, I* and II and polynucleotide phosphorylase).
The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress
Authors: Lamark T , Røkenes T P , McDougall J , Strøm A R
Published: November 2016

The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline. It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase). The bet genes are regulated by oxygen, choline, and osmotic stress. Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts. The transcripts were initiated 61 bp apart. Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (beta-galactosidase) operon fusions. Mutation of betI on plasmid F'2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline. Both promoters remained inducible by osmotic stress in a betI mutant background. On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent. The bet promoters were repressed by ArcA under anaerobic growth conditions. An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8. Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters.
DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation
Authors: Rkenes T P , Lamark T , Strøm A R
Published: November 2016

The betT and betIBA genes govern glycine betaine synthesis from choline in Escherichia coli. In an accompanying paper we report that the betT and betI promoters are divergently organized and partially overlapping and that both are negatively regulated by BetI in response to choline. (T. Lamark, T.P. Rokenes, J. McDougall, and A.R. Strom, J. Bacteriol. 178:1655-1662, 1996). In this paper, we report that the in vivo synthesis rate of the BetI protein constituted only 10% of that of BetA and BetB dehydrogenase proteins, indicating the existence of a posttranscriptional control of the betIBA operon. A genetically modified BetI protein called BetI*, which carries 7 extra N-terminal amino acids, was purified as a glutathione S-transferase fusion protein. Gel mobility shift assays showed that BetI* formed a complex with a 41-bp DNA fragment containing the -10 and -35 regions of both promoters. Only one stable complex was detected with the 41-bp fragment and all larger promoter-containing fragments tested. In DNase I footprinting, BetI* protected a region of 21 nucleotides covering both the -35 boxes. Choline stimulated complex formation but did not change the binding site of BetI*. We conclude that in vivo BetI is bound to its operator in both repressed and induced cells and that BetI represents a new type of repressor.
Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer
Authors: Schoberth S M , Chapman B E , Kuchel P W , Wittig R M , Grotendorst J , Jansen P , DeGraff A A
Published: November 2016

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.
Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics
Authors: Bocchetta M , Huber R , Cammarano P
Published: November 2016

A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei, S. Altamura, R. Huber, K. O. Stetter, and P. Cammarano, J. offteriol. 170-4353-4360, 1988), Aquifex ribosomes are susceptible to all of the aminoglycosides tested (disubstituted 2-deoxystreptamines, monosubstituted 2-deoxystreptamines, sand streptidine compounds). The significance of this result in light of the Aquifex and Thermotoga placements in phylogenetic trees of molecular sequences is discussed.
Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis
Authors: Franke C M , Leenhouts K J , Haandrikman A J , Kok J , Venema G , Venema K
Published: November 2016

Four in-frame translational fusions to both the reporter proteins beta-galactosidase and alkaline phosphatase support a topological model of LcnD, a protein implicated in the transport of several bacteriocins from Lactococcus lactis, in which the N-terminal part is located intracellularly and one transmembrane helix spans the cytoplasmic membrane.
Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element
Authors: Zückert W R , Meyer J
Published: November 2016

We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids.
Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm
Authors: Pilsl H , Killmann H , Hantke K , Braun V
Published: November 2016

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.
Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K-12
Authors: Man T K , Zhao G , Winkler M E
Published: November 2016

We isolated 26 suppressor mutations that allowed growth of a delta pdxH::omega null mutant in the absence of pyridoxal. Each suppressor mapped to pdxJ, and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide. This bypass suppression suggests that PdxJ may participate in formation of the pyridine ring of pyridoxine 5'-phosphate.
Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis
Authors: Szekeres E S , Woodgate R , Lawrence C W
Published: November 2016

We have examined the effect of replacing umuDC with mucAB or rumAB on the mutagenic properties of a T-T cyclobutane dimer in an attempt to determine the molecular basis for the differences in UV-induced mutagenesis that are associated with these structurally and functionally related genes. A single-stranded vector carrying a site-specific T-T cis-syn cyclobutane dimer was transfected into a set of isogenic Escherichia coli delta umuDC strains harboring low-copy-number plasmids expressing UmuDC, MucAB, RumAB, or their genetically engineered and mutagenically active counterparts UmuD'C, MucA'B, and RumA'B, respectively. Although the overall mutation frequency was similar for all strains, the relative frequencies of the two classes of mutation induced by the T-T dimer varied according to the mutagenesis operon expressed. In umuDC strains, 3' T-->A mutations outnumbered 3' T-->C mutations, but the reverse was true for the mucAB and rumAB strains. We also found that the T-T dimer was bypassed with differing efficiencies in unirradiated cells expressing wild-type UmuDC, MucAB, and RumAB proteins. These differences can probably be attributed to the relative efficiency of the normal cellular posttranslational activation of UmuD, MucA, and RumA, respectively, since recombinant constructs expressing the mutagenically active UmuD'C, MucA'B, and RumA'B proteins all promoted similarly high levels of bypass in UV-irradiated cells. These results suggest that the UmuD'/UmuC complex and its homologs may differ in their relative abilities to promote elongation from T - T and T - G mismatched termini. Alternatively, they may differentially influence the efficiency with which these mismatches are edited or influence nucleotide insertion by the catalytic subunit of the DNA polymerase III.
Role of transmembrane pH gradient and membrane binding in nisin pore formation
Authors: Moll G N , Clark J , Chan W C , Bycroft B W , Roberts G C , Konings W N , Driessen A J
Published: November 2016

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.
Identification, sequencing, and enzymatic activity of the erythrose-4-phosphate dehydrogenase gene of Vibrio cholerae
Authors: Carroll P A , Zhao G , Boyko S A , Winkler M E , Calderwood S B
Published: November 2016

We have identified a gene in Vibrio cholerae (epd) which encodes an erythrose-4-phosphate dehydrogenase activity and is located immediately downstream of an iron-regulated virulence gene, irgA, and immediately upstream of a gene encoding phosphoglycerate kinase (pgk). Expression of epd in V. cholerae is not regulated by iron, nor is it required for virulence in an infant mouse model.
Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism
Authors: Leeds J A , Welch R A
Published: November 2016

Escherichia coli hlyCABD operons encode the polypeptide component (HlyA) of an extracellular cytolytic toxin as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD). The E. coli protein RfaH is required for wild-type hemolysin expression at the level of hlyCABD transcript elongation (J. A. Leeds and R. A. Welch, J. Bacteriol. 178:1850-1857, 1996). RfaH is also required for the transcription of wild-type levels of mRNA from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, supporting the role for RfaH in transcriptional elongation. All or portions of a common 39-bp sequence termed JUMPStart are present in the untranslated regions of RfaH-enhanced operons. In this study, we tested the model that the JUMPStart sequence and RfaH are part of the same functional pathway. We examined the effect of JUMPStart deletion mutations within the untranslated leader of a chromosomally derived hlyCABD operon on hly RNA and HlyA protein levels in either wild-type or rfaH null mutant E. coli. We also provide in vivo physical evidence that is consistent with RNA polymerase pausing at the wild-type JUMPStart sequences.
Stimulatory effect of trehalose on formation and activity of Escherichia coli RNA polymerase E sigma38 holoenzyme
Authors: Kusano S , Ishihama A
Published: November 2016

The intracellular concentration of trehalose increases in the stationary-phase cells of Escherichia coli. The effects of trehalose on transcription in vitro by E. coli RNA polymerase were compared for two holoenzymes, E sigma70 and E sigma38, which were reconstituted from purified core enzyme and either sigma70 (the major sigma at the exponential growth phase) or sigma38 (the essential sigma at the stationary growth phase), respectively. The optimum trehalose concentration giving maximum transcription by E sigma38 was higher than that by E sigma70. Transcription activation by trehalose was attributed to both increased formation of E sigma38 holoenzyme and increased transcription initiation by E sigma38 from sigma38-dependent promoters. The activation of E sigma38 by trehalose was additive with the transcription enhancement by decreased superhelicity of template DNA prepared from stationary-phase cells. We thus propose that the selective activation of transcription by E sigma38 holoenzyme takes place in the presence of specific conditions and factors present under stress conditions.
Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN)
Authors: Mathee K , McPherson C J , Ohman D E
Published: November 2016

Pseudomonas aeruginosa strains associated with cystic fibrosis are often mucoid due to the copious production of alginate, an exopolysaccharide and virulence factor. Alginate gene expression is transcriptionally controlled by a gene cluster at 68 min on the chromosome: algT (algU)-mucA-mucB (algN)-mucC (algM)-mucD (algY). The algT gene encodes a 22-kDa alternative sigma factor (sigma22) that autoregulates its own promoter (PalgT) as well as the promoters of algR, algB, and algD. The other genes in the algT cluster appear to regulate the expression or activity of sigma22. The goal of this study was to better understand the functional interactions between sigma22 and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing sigma22 in the cell by introducing a plasmid clone containing algT from mucA22(Def) strain FRD1. However, the bacterial cells remained nonmucoid if the transcriptionally coupled mucB on the clone remained intact. This suggested that a stoichiometric relationship between sigma22 and MucB may be required to control sigma factor activity. When the transcription and translational initiation of algT were measured with lacZ fusions, alginate production correlated with only about a 1.2- to 1.7-fold increase in algT-lacZ activity, respectively. An algR-lacZ transcriptional fusion showed a 2.8-fold increase in transcription with alginate production under the same conditions. A Western blot analysis of total cell extracts showed that sigma22 was approximately 10-fold higher in strains that overproduced alginate, even though algT expression increased less than 2-fold. This suggested that a post-transcriptional mechanism may exist to destabilize sigma22 in order to control certain sigma22-dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of sigma22 was found to localize to the periplasm of the cell. Similar experiments suggest that MucA localizes to the inner membrane via one transmembrane domain with amino- and carboxy-terminal domains in the cytoplasm and periplasm, respectively. These data were used to propose a model in which MucB-MucA-sigma22 interact via an inner membrane complex that controls the stability of sigma22 protein in order to control alginate biosynthesis.
Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution
Authors: Zarrouk H , Karibian D , Bodie S , Perry M B , Richards J C , Caroff M
Published: November 2016

The structures of lipids A isolated from the lipopolysaccharides (LPSs; endotoxins) of three different pathogenic Bordetella bronchiseptica strains were investigated by chemical composition and methylation analysis, gas chromatography-mass spectrometry, nuclear magnetic resonance, and plasma desorption mass spectrometry (PDMS). The analyses revealed that the LPSs contain the classical lipid A bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Their structures differ from that of the lipid A of Bordetella pertussis endotoxin by the replacement of hydroxydecanoic acid on the C-3 position with hydroxydodecanoic acid or dodecanoic acid and the presence of variable amounts of hexadecanoic acid. The dodecanoic acid is the first nonhydroxylated fatty acid to be found directly linked to a lipid A glucosamine. The lipids A were heterogeneous and composed of one to three major and several minor molecular species. The fatty acids in ester linkage were localized by PDMS of chemically modified lipids A. B. pertussis lipids A are usually hypoacylated with respect to those of enterobacterial lipids A. However, one of the three B. bronchiseptica strains had a major hexaacylated molecular species. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site of the polysaccharide. The structural variability seen in these three lipids A was unusual for a single species and may have consequences for the pathogenicity of this Bordetella species.
Characterization of DNA binding sites for the BvgA protein of Bordetella pertussis
Authors: Karimova G , Ullmann A
Published: November 2016

Expression of virulence-associated genes in Bordetella pertussis is under the control of the pleiotropic regulator BvgA. Although previous studies have identified recognition sequences for BvgA in several promoter regions, their structures have not been clearly characterized. We show that the BvgA binding sites within the bvgp(1) and cyaA promoters consist of inverted repeats and suggest that inverted-repeat motifs may represent the recognition elements for DNA-BvgA interaction.
Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene
Authors: Li L A , Tabita F R
Published: November 2016

Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Plasmid constructions containing the genes from Anabaena sp. strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins. The purified recombinant Anabaena sp. strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.
Integration host factor is required for 1,2-propanediol-dependent transcription of the cob/pdu regulon in Salmonella typhimurium LT2
Authors: Rondon M R , Escalante-Semerena J C
Published: November 2016

We show that integration host factor (IHF) is required for the activation of transcription of the cobalamin biosynthetic (cob) and 1,2-propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium LT2. A lack of IHF affected transcription of the cob/pdu regulon in at least two ways. First, the level of the regulatory protein PocR was decreased in ihfB (formerly himD) mutants, as judged by Western blot analysis with polyclonal antiserum raised against PocR. Second, even when PocR was available, in the absence of IHF, PocR was unable to activate transcription of cob/pdu in response to 1,2-PDL. This result suggested an additional role for IHF in PocR-dependent transcription activation. Consistent with these findings, ihfB mutants of this bacterium were unable to use 1,2-PDL as a carbon or energy source.
Interactions of the RepA1 protein with its replicon targets: two opposing roles in control of plasmid replication
Authors: Maas R , Wang C , Maas W K
Published: November 2016

By studying the interaction of derivatives of RepFIC miniplasmids, we were able to demonstrate that under certain conditions the RepA1 initiator protein inhibits plasmid replication. An analysis of cloned derivatives whose replication is inhibited by the RepA1 protein revealed the existence of two areas of the RepFIC genome that interact with RepA1 in the inhibition reaction. One of these areas, which occurs in the origin region, was explored by in vivo methylation protection footprinting studies. The protected area was 200 bp long and showed a definite periodicity of protected and hypersensitive sites, suggesting that RepA1 promotes a topological change in the RepFIC genome. The significance of our results is discussed in the context of plasmid replication control.
Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer
Authors: Ries W , Hotzy C , Schocher I , Sleytr U B , Sára M
Published: November 2016

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.
New classes of mutants in complementary chromatic adaptation provide evidence for a novel four-step phosphorelay system
Authors: Kehoe D M , Grossman A R
Published: November 2016

Complementary chromatic adaptation appears to be controlled by a complex regulatory system with similarity to four-step phosphorelays. Such pathways utilize two histidine and two aspartate residues for signal transduction. Previous studies of the signaling system controlling complementary chromatic adaptation have uncovered two elements of this pathway, a putative sensor, RcaE, and a response regulator, RcaC. In this work, we describe a second response regulator controlling complementary chromatic adaptation, RcaF, and identify putative DNA binding and histidine phosphoacceptor domains within RcaC. RcaF is a small response regulator with similarity to SpoOF of Bacillus subtilis; the latter functions in the four-step phosphorelay system controlling sporulation. We have also determined that within this phosphorelay pathway, RcaE precedes RcaF, and RcaC is probably downstream of RcaE and RcaF. This signal transduction pathway is novel because it appears to use at least five, instead of four, phosphoacceptor domains in the phosphorelay circuit.
Isolation and expression of the catA gene encoding the major vegetative catalase in Streptomyces coelicolor Müller
Authors: Cho Y H , Roe J H
Published: November 2016

We isolated the catA gene for the major vegetative catalase from Streptomyces coelicolor Müller. It encodes a polypeptide of 488 residues (55,440 Da) that is highly homologous to typical monofunctional catalases. We investigated catA expression by analyzing both catA mRNA and catalase activity. catA expression was increased by H2O2 treatment but did not increase during stationary phase. A putative catalase (CatB) cross-reactive with anti-CatA antibody appeared during stationary phase and in the aerial mycelium.
Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis
Authors: Trinh S , Reysset G
Published: November 2016

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.
The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain
Authors: Ziegelin G , Calendar R , Lurz R , Lanka E
Published: November 2016

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.
RNA polymerase beta mutations have reduced sigma70 synthesis leading to a hyper-temperature-sensitive phenotype of a sigma70 mutant
Authors: Zhou Y N , Jin D J
Published: November 2016

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.
The tgl gene: social motility and stimulation in Myxococcus xanthus
Authors: Rodriguez-Soto J P , Kaiser D
Published: November 2016

Mutations in the tgl locus inactivate social gliding motility in Myxococcus xanthus and block production of pili. The tgl locus is distinctive among the genes for social motility because social gliding and pili can be restored transiently to tgl mutant cells by mixing them with tgl+ cells, a process known as stimulation. The tgl locus was cloned with a linked insertion of transposon Tn5 by using the kanamycin resistance encoded by that transposon. A 16-kb segment of chromosomal DNA complemented the social motility defect when introduced into tgl mutant cells to form a tandem duplication tgl+/tgl heterozygote. To delimit the autonomous tgl transcription unit, subfragments of this 16-kb piece were integrated at the ectopic Mx8 prophage attachment site. A 1.7-kb DNA fragment was identified which, when integrated at the Mx8 site, simultaneously rescued social motility and pilus production. The ability to stimulate tgl mutants was also rescued by the 1.7-kb fragment. Because rescue of stimulation from an mgl-deficient donor strain which cannot swarm was observed, this demonstrates that a stimulation donor requires a tgl+ allele but does not require the capacity to swarm actively. The nucleotide sequence of the 1.7-kb fragment revealed two protein coding regions, open reading frame A and open reading frame B (ORFB). ORFB is the tgl gene, because a 613-bp DNA fragment which includes 75% of ORFB rescues tgl-1, -2, and -3 mutants and because disruption of ORFB by deletion or insertion of transposon Tn5lac constitutes a tgl mutation.
An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer
Authors: Bhandari P , Gowrishankar J
Published: November 2016

Salt-induced overexpression of genes cloned downstream of the phage T7 phi10 promoter was demonstrated in an Escherichia coli strain (GJ1158) which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the cis-regulatory elements of the osmoresponsive proU operon. Plasmids that have been constructed to obtain overproduction of individual target gene products in strain BL21(DE3) (by addition of isopropyl-beta-D-thiogalactopyranoside as an inducer) can directly be transformed into GJ1158. The NaCl induction regimen was also shown to be associated with a decreased propensity for sequestration of overexpressed target proteins within insoluble inclusion bodies.
The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ
Authors: Padmalayam I , Anderson B , Kron M , Kelly T , Baumstark B
Published: November 2016

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.
Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449
Authors: Umelo E , Trust T J
Published: November 2016

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.