bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions
Authors: Boyd D , Traxler B , Beckwith J
Published: November 2016

An approach to analyzing the topology of membrane proteins with alkaline phosphatase fusions is described. Precise fusions were constructed by using polymerase chain reaction at the C terminus of each hydrophilic region of the membrane protein. The disruption of topogenic signals is thereby minimized, and predictable anomalous results are avoided. The Escherichia coli MalG protein has been analyzed.
Polarized cells, polar actions
Authors: Maddock J R , Alley M R , Shapiro L
Published: November 2016

The recognition of polar bacterial organization is just emerging. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. The significance of this observation remains unclear. Polarity in bacteria poses many problems, including the necessity for a mechanism for asymmetrically distributing proteins as well as a mechanism by which polar localization is maintained. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35). The constraining features for membrane components are not known. For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. In most cases of cellular asymmetry, bacteria are able to discriminate between the new pole and the old pole and to utilize this information for localization specificity. The maturation of new pole to old pole appears to be a common theme as well. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized.
Conditional synthesis and utilization of 1,5-anhydroglucitol in Escherichia coli
Authors: Shiga Y , Mizuno H , Akanuma H
Published: November 2016

A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is widely detected in most organisms, although little is known about its metabolism and physiological roles. The present study demonstrates the synthesis of AG in Escherichia coli C600. The major portion of the synthesized AG was indicated to be derived from glucose retaining all the six carbon atoms, and only 5% was attributed to AG synthesized from C3 compounds. AG synthesis is apparent in an early stage of the stationary phase, and accumulation is transient both in cells and in medium. Evidence is also presented for AG uptake and metabolism and for effects of cyclic AMP.
The Haemophilus influenzae adenylate cyclase gene: cloning, sequence, and essential role in competence
Authors: Dorocicz I R , Williams P M , Redfield R J
Published: November 2016

Competence for transformation in Haemophilus influenzae is stimulated by cyclic AMP (cAMP) and requires the cAMP-dependent catabolite regulatory protein CRP. Thus, understanding the control of competence will require understanding how cAMP levels are regulated. As a first step, we have cloned the H. influenzae adenylate cyclase gene (cya) by complementing the Lac- phenotype of delta cya Escherichia coli. Its sequence specifies an 843-amino-acid protein which has significant identity to other known bacterial adenylate cyclases (41 to 43% and 61% identical to the cya genes of enteric bacteria and of Pasteurella multocida, respectively). As seen in other bacterial cya genes, there is evidence for regulation similar to that demonstrated for E. coli: the presence of a strong consensus CRP binding site within the promoter of the gene may provide feedback control of cAMP levels by repressing cya transcription, and translation may be limited by the weak ribosome binding site and by initiation of protein synthesis with GUG rather than AUG or the UUG used in other bacterial cya genes. We confirmed the essential role of cAMP in competence by constructing and characterizing H. influenzae cya mutants. This strain failed to develop competence either spontaneously or after transfer to a competence-inducing medium. However, it became as competent as its wild-type parent in the presence of exogenous cAMP. This result suggests that the failure of exogenously added cAMP to induce optimum competence in wild-type cells is not due to a limitation to the entry of cAMP into the cells. Rather, it strongly favors models in which competence induction requires both an increase in intracellular cAMP and a second as yet unidentified regulatory event. H. influenzae strains mutant in cya or crp were unable to ferment xylose or ribose. This confirms that influenzae, like E. coli, uses cAMP and CRP to regulate nutrient uptake and utilization and lends increasing support to the hypothesis that DNA uptake is mechanism of nutrient acquisition.
Cloning and characterization of btr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator
Authors: Bannan J D , Moran M J , MacInnes J I , Soltes G A , Friedman R L
Published: November 2016

To determine whether hemolytic factors other than the bifunctional hemolysin-adenylate cyclase toxin (cyclolysin) are expressed by Bordetella pertussis, a gene library was constructed from a virulent strain of B. pertussis, BP504, transformed into nonhemolytic Escherichia coli, and screened on blood agar plates. A strongly hemolytic colony which contained the plasmid pHLY1A was isolated. Nucleotide sequencing of pHLY1A revealed an open reading frame that could encode a 27-kDa protein. No similarity was detected between the deduced amino acid sequence of this open reading frame and those of any known bacterial cytolysins. However, significant homology was detected with FNR of E. coli and several other transcriptional regulators including HylX from Actinobacillus pleuropneumoniae, which can also confer a hemolytic phenotype on E. coli. An fnr mutant of E. coli, JRG1728, could be complemented by pHLY1A. Thus, the B. pertussis transcriptional regulator-like gene and the protein which it encoded were named btr and BTR, respectively. A BTR-deficient B. pertussis strain, BJB1, was constructed. The btr::kan mutation had no effect on the expression of hemolytic activity or on phase variation. Northern (RNA) blotting revealed that btr expression was not regulated by the BvgAS two-component sensor-regulator. On the basis of sequence similarity to FNR-like transcriptional regulators and the ability to complement an anaerobically deficient E. coli strain (JRG1728) in growing anaerobically, BTR may regulate B. pertussis gene expression in response to changes in oxygen levels or to changes in the redox potential of the bacterial environment. Its role in virulence remains to be determined.
Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene
Authors: Zulty J J , Barcak G J
Published: November 2016

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.
dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product
Authors: Henrich B , Becker S , Schroeder U , Plapp R
Published: November 2016

Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties.
Multilevel regulation of the sporulation transcription factor sigma K in Bacillus subtilis
Authors: Oke V , Losick R
Published: November 2016

Gene expression in the mother-cell compartment of the Bacillus subtilis sporangium is governed in part by the sporulation transcription factor sigma K. The production of sigma K is controlled at three levels: by a chromosomal rearrangement that generates the sigma K-coding sequence (sigK), by compartment-specific transcription of sigK, and by conversion of the inactive pro-protein product of sigK (pro-sigma K) to sigma K. To investigate the function of these multiple levels of regulation, we constructed a set of strains that bypass the chromosomal rearrangement, pro-protein processing, or both levels of control. Here we show that one of the functions of the chromosomal rearrangement and pro-protein processing is to prevent inappropriate production of sigma K under nonsporulation conditions. In the absence of both of these levels of control, a low level of sigma K-directed gene expression is observed during stationary phase after growth in rich medium. The appearance of sigma K under these conditions is probably due to a low level of sigma K-directed transcription from the sigK promoter in a positive feedback loop. We also report the construction of a strain that produces high levels of sigma K during growth. Using this strain, we demonstrate that the production of sigma K during growth is sufficient to induce a cascade of gene expression that closely mimics late events in the mother-cell line of gene expression.
Functional domains of the penicillinase repressor of Bacillus licheniformis
Authors: Wittman V , Lin H C , Wong H C
Published: November 2016

The penicillinase repressor (PENI) negatively regulates expression of the penicillinase gene (penP) in Bacillus licheniformis by binding to its operators located within the promoter region of penP.penI codes for a protein with 128 amino acids. Filter-binding analyses suggest that the active form of the repressor is a dimer. Genetic analyses of PENI derivatives showed that the repressor carrying either a 6-amino-acid deletion near the N terminus or a 14-amino-acid deletion at the C terminus was functionally inactive in vivo. A repressor derivative carrying a 6-amino-acid deletion within its N-terminal region was extensively purified and used in DNA footprinting and subunit cross-linking analyses. The results of these studies showed that the repressor derivative had lost its ability to bind operator specifically even though it could dimerize effectively. In similar studies, we demonstrated that an N-terminal portion of PENI with a molecular mass of 10 kDa derived by digestion with papain was able to bind operator specifically but with reduced affinity and had completely lost its ability to dimerize. These data suggest that the repressor has two functional and separable domains. The amino-terminal domain of the repressor is responsible for operator recognition, and the carboxyl-terminal domain is involved in subunit dimerization.
Hemin uptake in Porphyromonas gingivalis: Omp26 is a hemin-binding surface protein
Authors: Bramanti T E , Holt S C
Published: November 2016

A 26-kDa outer membrane protein (Omp26) has been proposed to play a role in hemin acquisition by Porphyromonas gingivalis (T. E. Bramanti and S. C. Holt, J. Bacteriol. 174:5827-5839, 1992). We studied [55Fe]hemin uptake in P. gingivalis grown under conditions of hemin starvation (Omp26 expressed on the outer membrane surface) and hemin excess (Omp26 not expressed on surface). [55Fe]hemin uptake occurred rapidly in hemin-starved cells which incorporated up to 70% of total [55Fe]hemin within 3 min. P. gingivalis grown under hemin-starved conditions or treated with the iron chelator 2,2'-bipyridyl to induce an iron stress took up six times more [55Fe]hemin than hemin-excess-grown cells. Polyclonal monospecific anti-Omp26 antibody added to hemin-starved cells inhibited [55Fe]hemin uptake by more than 50%, whereas preimmune serum had no effect. [55Fe]hemin uptake in hemin-starved P. gingivalis was inhibited (36 to 67%) in the presence of equimolar amounts of unlabeled hemin, protoporphyrin IX, zinz protoporphyrin, and Congo red dye but was not inhibited in the presence of non-hemin-containing iron sources. Heat shock treatment (45 degrees C) of hemin-excess-grown P. gingivalis (which cases translocation of Omp26 to the surface) increased [55Fe]hemin uptake by threefold after 3 min in comparison with cells grown at 37 degrees C. However, no [55Fe] hemin uptake beyond 3 min was observed in either hemin-excess-grown or hemin-starved cells exposed to heat shock. In experiments using heterobifunctional cross-linker analysis, hemin and selected porphyrins were cross-linked to Omp26 in hemin-starved P. gingivalis, but no cross-linking was seen with hemin-excess-grown cells. However, cross-linking of hemin to Omp26 was observed after heat shock treatment of hemin-excess-grown cells. Finally, anti-Omp26 antibody inhibited cross-linked of hemin to Omp26. These findings indicate that hemin binding and transport into P.gingivalis cell mediated by Omp26.
Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants
Authors: Peterson K R , Mount D W
Published: November 2016

RecBCD protein, necessary for Escherichia coli dam mutant viability, is directly required for DNA repair. Recombination genes recF+, recN+, recO+, and recQ+ are not essential for dam mutant viability; they are required for recBC sbcBC dam mutant survival. mutH, mutL, or mutS mutations do not suppress subinduction of SOS genes in dam mutants.
Formation of the K30 (group I) capsule in Escherichia coli O9:K30 does not require attachment to lipopolysaccharide lipid A-core
Authors: MacLachlan P R , Keenleyside W J , Dodgson C , Whitfield C
Published: November 2016

Escherichia coli K antigens (capsular polysaccharides) are divided into two broad classes, designated groups I and II, on the basis of a number of chemical, physical, and genetic criteria. Group I K antigens can be further subdivided on the basis of the absence (group IA) or presence (group IB) of amino sugars in the repeating unit of the K antigen. One criterion proposed for inclusion in group I is covalent linkage of the capsular polysaccharide to the lipid A-core of lipopolysaccharide (LPS). E. coli O9:K30 is a strain with a representative group IA K antigen. This organism synthesizes an LPS-associated low-molecular-weight form of K30 antigen which is called K(LPS). To determine the involvement of LPS lipid A-core in expression of the K30 capsular polysaccharide, E. coli K30/K-12 hybrid strains were constructed with mutations in the E. coli K-12 rfa locus, responsible for the biosynthesis of the LPS core oligosaccharide. These strains lack K(LPS), indicating that a full-length core is required for K(LPS) expression. However, formation of a K30 capsule was unaffected by rfa defects, indicating that attachment to lipid A-core is not an obligatory step for either export of high-molecular-weight capsular polysaccharide or maintenance of the capsular structure on the cell surface. Silver-stained tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of lipopolysaccharides from other E. coli K serotypes showed that all strains with group IB K antigens expressed some K(LPS). In contrast, some strains with group IA K antigens appear to lack K(LPS). Consequently, although association of group 1 K antigens with lipid A-core is common, it is not a universal marker for inclusion in group I.
Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine
Authors: Grandoni J A , Fulmer S B , Brizzio V , Zahler S A , Calvo J M
Published: November 2016

The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.
Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides
Authors: Karls R K , Jin D J , Donohue T J
Published: November 2016

We have been characterizing RNA polymerase holoenzymes from Rhodobacter sphaeroides. RNA polymerase purified from R. sphaeroides transcribed from promoters recognized by Escherichia coli E sigma 32 or E sigma 70 holoenzyme. Antisera to E. coli sigma 32 or sigma 70 indicated that related polypeptides of approximately 37 kDa (sigma 37) and 93 kDa (sigma 93), respectively, are present in this preparation. Transcription of sigma 32-dependent promoters was observed in a further fractionated R. sphaeroides holoenzyme containing the sigma 37 polypeptide, while a preparation enriched in sigma 93 transcribed sigma 70-dependent promoters. To demonstrate further that the sigma 93 polypeptide functions like E. coli sigma 70, we obtained an R. sphaeroides E sigma 93 holoenzyme capable of transcription from sigma 70-dependent promoters by combining sigma 93 with (i) an E sigma 37 fraction with diminished sigma 93 polypeptide content or (ii) E. coli core RNA polymerase. The generation of analogous DNase I footprints on the lacUV5 promoter by R. sphaeroides E sigma 93 and by E. coli E sigma 70 suggests that the overall structures of these two holoenzymes are similar. However, some differences in promoter specificity between R. sphaeroides E sigma 93 and E. coli E sigma 70 exist because transcription of an R. sphaeroides rRNA promoter was detected only with E sigma 93.
Insertional inactivation of genes to isolate mutants of Synechococcus sp. strain PCC 7942: isolation of filamentous strains
Authors: Dolganov N , Grossman A R
Published: November 2016

We have developed a simple procedure for generating mutants of the cyanobacterium Synechococcus sp. strain PCC 7942 in which the site of the lesion can be readily identified. This procedure involves transforming Synechococcus sp. strain PCC 7942 with a library of its own DNA that was fully digested with Sau3A and ligated into the plasmid vector pUC8. The homologous integration of the recombinant plasmid into the genome will often result in the disruption of a gene and the loss of gene function. We have used this method to generate many mutants of Synechococcus sp. strain PCC 7942 which grow as multicellular filaments rather than as unicells. Since the gene harboring the lesion was tagged with pUC8, it was easily isolated. In this paper, we discuss the usefulness of this procedure for the generation of mutants, and we characterize one mutant in which the lesion may be in an operon involved in the assembly of lipopolysaccharides.
In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation
Authors: Lee H S , Narberhaus F , Kustu S
Published: November 2016

In the free-living diazotroph Klebsiella pneumoniae, the NifA protein is required for transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself. NifA activates transcription of nif operons by the alternative holoenzyme form of RNA polymerase, sigma 54 holoenzyme. In vivo, NifL is known to antagonize the action of NifA in the presence of molecular oxygen or combined nitrogen. We now demonstrate inhibition by NifL in vitro in both a coupled transcription-translation system and a purified transcription system. Crude cell extracts containing NifL inhibit NifA activity in the coupled system, as does NifL that has been solubilized with urea and allowed to refold. Inhibition is specific to NifA in that it does not affect activation by NtrC, a transcriptional activator homologous to NifA, or transcription by sigma 70 holoenzyme. Renatured NifL also inhibits transcriptional activation by a maltose-binding protein fusion to NifA in a purified transcription system, indicating that no protein factor other than NifL is required. Since inhibition in the purified system persists anaerobically, our NifL preparation does not sense molecular oxygen directly.
Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies
Authors: Wendell D L , Bisson L F
Published: November 2016

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.
Biosynthesis of biotin from dethiobiotin by the biotin auxotroph Lactobacillus plantarum
Authors: Bowman W C , DeMoll E
Published: November 2016

Lactobacillus plantarum requires biotin for growth. We show that in the presence of high levels of the biotin biosynthetic precursor, dethiobiotin, L. plantarum synthesizes biotin and grows in medium with dethiobiotin but without biotin. Lactobacillus casei also grew under similar conditions.
Azotobacter vinelandii mutS: nucleotide sequence and mutant analysis
Authors: Le O , Shen B , Iismaa S E , Burgess B K
Published: November 2016

An Azotobacter vinelandii homolog to the Salmonella typhimurium mutS gene was discovered upstream of the fdxA gene. The product of this gene is much more similar to S. typhimurium MutS than either is to the HexA protein of Streptococcus pneumoniae. An A. vinelandii delta mutS mutant strain was shown to have a spontaneous mutation frequency 65-fold greater than that of the wild type.
The pss and psd genes are required for motility and chemotaxis in Escherichia coli
Authors: Shi W , Bogdanov M , Dowhan W , Zusman D R
Published: November 2016

Mutants of Escherichia coli defective in phosphatidylserine synthase (encoded by pss) and phosphatidylserine decarboxylase (encoded by psd) make cell membranes deficient in phosphatidylethanolamine. In this report we show that wild-type pss and psd genes are required for motility and chemotaxis. Null mutants or strains with temperature-sensitive pss or psd mutations grown at high temperature (35 degrees C) were nonmotile. They lacked flagella and showed reduced rates of transcription of the flhD master operon (encoding FlhD and FlhC), the fliA operon (encoding sigma F), and the fliC operon (encoding flagellin). At low temperature (25 degrees C), the temperature-sensitive mutant cells showed motility and chemotaxis but at reduced levels. The extent of the motility and chemotaxis defects in the mutants was correlated with the amount of phosphatidylethanolamine in the membranes, suggesting a link between membrane phospholipid composition and expression of the flagellum chemotaxis regulon.
Altered-function mutations in the Agrobacterium tumefaciens OccR protein and in an OccR-regulated promoter
Authors: Cho K , Winans S C
Published: November 2016

OccR is a LysR-type transcriptional activator that controls the occQ and traR promoters of octopine-type Ti plasmids. The opine octopine converts OccR from a repressor to an activator of occQ, shortens the protein's DNase I footprint, and decreases the angle of an OccR-caused DNA bend at the occQ promoter. In this study we first localized the cis-acting DNA sequences required for regulated expression of occQ. To understand better the mechanism of activation of OccR, we isolated mutations both in the occQ promoter and in the occR gene which function differently from the wild type. An occQ promoter mutation that changes the putative -35 region of occQ from TTGACC to TTGACA increases the basal expression of occQ about 15-fold. Three mutations in occR were also identified, one of which activates occQ at fully constitutive levels in both the absence and presence of octopine. This mutation (E23G) is located in the first helix of a putative helix-turn-helix DNA-binding motif. The other two occR mutations cause the protein to detect much lower concentrations of octopine than wild-type OccR protein does. These mutations (F113L and G148D) are located in a region of the protein that is predicted to contain the ligand-binding site.
Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa
Authors: Huang H , Hancock R E
Published: November 2016

Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.
Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway
Authors: Cohen S P , Levy S B , Foulds J , Rosner J L
Published: November 2016

Since the growth of wild-type Escherichia coli in salicylate results in a multiple antibiotic resistance phenotype similar to that of constitutive mutants (Mar) of the chromosomal mar locus, the effect of salicylate on the expression of the marRAB operon was investigated. The amount of RNA hybridizing with a mar-specific DNA probe was 5 to 10 times higher in wild-type cells grown with sodium salicylate (5.0 mM) than in untreated controls. Untreated Mar mutants had three to five times more mar-specific RNA than wild-type cells did. When a Mar mutant was treated with salicylate, a 30- to 50-fold increase of mar-specific RNA was seen. In wild-type cells bearing a mar promoter-lacZ fusion on the chromosome, salicylate increased beta-galactosidase activity by sixfold. Thus, salicylate induces transcription of the marRAB operon. Other inducers of phenotypic multiple antibiotic resistance, e.g., benzoate, salicyl alcohol, and acetaminophen, but not acetate, also increased transcription from the mar promoter but to a lesser extent than did salicylate. Both in wild-type and mar-deficient strains, growth in salicylate resulted in increased antibiotic resistance, decreased permeation of the outer membrane to cephaloridine, increased micF transcription, and decreased amounts of OmpF. However, the magnitude of these changes was generally greater in wild-type (mar-containing) cells. Thus, salicylate and other compounds can induce transcription of the mar operon and, presumably, give rise to multiple antibiotic resistance via this pathway. However, salicylate can also activate an unidentified, mar-independent pathway(s) which engenders multiple antibiotic resistance.
The ancestry of insertion sequences common to Escherichia coli and Salmonella typhimurium
Authors: Bisercić M , Ochman H
Published: November 2016

Despite very restricted gene exchange between Escherichia coli and Salmonella typhimurium, both species harbor several of the same classes of insertion sequences. To determine whether the present-day distribution of these transposable elements is due to common ancestry or to horizontal transfer, we determined the sequences of IS1 and IS200 from natural isolates of S. typhimurium and E. coli. One strain of S. typhimurium harbored an IS1 element identical to that originally recovered from E. coli, suggesting that the element was recently transferred between these two species. The level of sequence divergence between copies of IS200 from E. coli and S. typhimurium ranged from 9.5 to 10.7%, indicating that IS200, unlike IS1, has not been repeatedly transferred between these enteric species since E. coli and S. typhimurium diverged from a common ancestor. Levels of variability in IS1 and IS200 for strains of E. coli and S. typhimurium show that each class of insertion sequence has a characteristic pattern of transposition within and among host genomes.
The chromosomal virulence gene, chvE, of Agrobacterium tumefaciens is regulated by a LysR family member
Authors: Doty S L , Chang M , Nester E W
Published: November 2016

Certain plant phenolic compounds and monosaccharides induce the transcription of virulence (vir) genes of Agrobacterium tumefaciens through the VirA-VirG two-component regulatory system. The product of the chromosomal virulence gene chvE is homologous to galactose-binding protein of Escherichia coli and is required for vir gene induction by sugars. Adjacent to, but divergent in transcription from, chvE is an open reading frame, now termed gbpR (galactose-binding protein regulator), that is homologous to the LysR family of transcriptional regulators. chvE::lacZ expression was induced by L-arabinose, D-galactose, and D-fucose when gbpR was present. In the absence of inducer, GbpR repressed chvE::lacZ expression. In addition, GbpR negatively regulated its own expression.
A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1
Authors: Wei P , Stewart C R
Published: November 2016

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.
A survey of the Mycoplasma genitalium genome by using random sequencing
Authors: Peterson S N , Hu P C , Bott K F , Hutchison C A
Published: November 2016

A total of 508 random clones from five Mycoplasma genitalium genomic libraries were partially sequenced and analyzed. This resulted in the identification of 291 unique contigs. Sequence information from these clones (100,993 nucleotides), representing approximately 17% of this pathogen's genome, was analyzed by comparison to the DNA and protein sequence data bases. The frequency with which clones could be identified, by virtue of possessing homology to another data base entry, was 46%. Sequence analysis indicated the following. (i) The M. genitalium genome contains many genes involved in various metabolic processes. (ii) Repetitive DNA may comprise as much as 4% of this genome. (iii) The MgPa adhesin gene may be the result of horizontal transfer from an unknown origin. (iv) Not all dinucleotide pairs are present in this genome at the expected frequency. (v) This genome potentially encodes approximately 390 proteins and makes very efficient use of its limited amount of DNA. In addition, this study allowed us to estimate the number of genes involved with various cellular functions.
Stress-induced activation of the sigma B transcription factor of Bacillus subtilis
Authors: Boylan S A , Redfield A R , Brody M S , Price C W
Published: November 2016

The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.
HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family
Authors: Wei Z M , Beer S V
Published: November 2016

HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv. vesicatoria. Cells of E. amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin. When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant. Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered. Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin. Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway.
Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA
Authors: Chu S , Gustafson C E , Feutrier J , Cavaignac S , Trust T J
Published: November 2016

The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer. Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences. Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A. salmonicida. Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A. salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures. Northern analysis of cells grown at 15 degrees C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25). At 15 degrees C, the half-life of the vapA mRNA was 22 min, while at 20 degrees C, the half-life was significantly shorter, 11 min. The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin. Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA. vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein.
Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp
Authors: Gentry D R , Hernandez V J , Nguyen L H , Jensen D B , Cashel M
Published: November 2016

Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.
A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae
Authors: Li Z , Dumas F , Dubreuil D , Jacques M
Published: November 2016

We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen.
Cloning, sequencing, expression, and complementation analysis of the Escherichia coli K1 kps region 1 gene, kpsE, and identification of an upstream open reading frame encoding a protein with homology to GutQ
Authors: Cieslewicz M J , Steenbergen S M , Vimr E R
Published: November 2016

The kps locus for polysialic acid capsule expression in Escherichia coli K1 is composed of a central group of biosynthetic neu genes, designated region 2, flanked on either side by region 1 or region 3 kps genes with poorly defined functions. Chromosomal mutagenesis with MudJ and subsequent complementation analysis, maxicell and in vitro protein expression studies, and nucleotide sequencing identified the region 1 gene, kpsE, which encodes a 39-kDa polypeptide. Polarity of the kpsE::lacZ mutation suggests an operonic structure for region 1. KpsE is homologous to putative polysaccharide-translocation components previously identified in Haemophilus influenzae type b and Neisseria meningitidis group B. An open reading frame upstream of kpsE encodes a 35-kDa polypeptide with homology to GutQ, a putative ATP-binding protein of unknown function encoded by gutQ of the glucitol utilization operon. Whether expression of the gutQ homolog as the potential first gene of region 1 is required for polysialic acid synthesis or localization is presently unknown.