bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
Saccharomyces cerevisiae
Weak-Acid-Inducible ABC Transporter Pdr12 Transports Fluorescein and Preservative Anions from the Cytosol by an Energy-Dependent Mechanism
Authors: Holyoak Caroline D. , Bracey Danielle , Piper Peter W. , Kuchler Karl , Coote Peter J. 
Published: December 2019

ABSTRACT

Growth of
Saccharomyces cerevisiae
in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257–4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C
1
to C
7
. Exposure to acids with aliphatic chain lengths greater than C
7
resulted in no observable sensitivity of
Δpdr12
mutant cells compared to the parent. Parent and
Δpdr12
mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the
Δpdr12
mutant were unable to efflux any of the dye. When both parent and
Δpdr12
mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.
Obg, an Essential GTP Binding Protein of
Bacillus subtilis
, Is Necessary for Stress Activation of Transcription Factor ς
B
Authors: Scott Janelle M. , Haldenwang W. G. 
Published: December 2019

ABSTRACT

ς
B
, the general stress response ς factor of
Bacillus subtilis
, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates ς
B
by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of ς
B
from an anti-ς factor inhibitor. By using the yeast dihybrid selection system to identify
B. subtilis
proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX).
B. subtilis
depleted of Obg no longer activated ς
B
in response to environmental stress, but it retained the ability to activate ς
B
by the ATP responsive pathway. Stress pathway components activated ς
B
in the absence of Obg if the pathway’s most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of ς
B
and suggest that Obg may directly communicate with one or more ς
B
regulators.
Complementation of Conjugation Functions of
Streptomyces lividans
Plasmid pIJ101 by the Related
Streptomyces
Plasmid pSB24.2
Authors: Pettis Gregg S. , Prakash Shubha 
Published: December 2019

ABSTRACT

A database search revealed extensive sequence similarity between
Streptomyces lividans
plasmid pIJ101 and
Streptomyces
plasmid pSB24.2, which is a deletion derivative of
Streptomyces cyanogenus
plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the
cis
-acting locus for transfer (
clt
) and the transfer-associated
korB
gene, were shown to be capable of complementing the pIJ101
clt
and
korB
functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.
Involvement of the RNA Polymerase α-Subunit C-Terminal Domain in LuxR-Dependent Activation of the
Vibrio fischeri
Luminescence Genes
Authors: Stevens Ann M. , Fujita Nobuyuki , Ishihama Akira , Greenberg E. P. 
Published: December 2019

ABSTRACT

LuxR is a ς
70
RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the
Vibrio fischeri lux
operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the α-subunit C-terminal domain (αCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRΔN) was used to study the activation of transcription from the
luxI
promoter in vitro. Initiation of
lux
operon transcription was observed in the presence of LuxRΔN and wild-type RNAP but not in the presence of LuxRΔN and RNAPs with truncated αCTDs. We also studied the in vivo role of the RNAP αCTD in activation of
lux
transcription in
Escherichia coli
. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRΔN. These in vivo studies indicated that both LuxR and LuxRΔN require the RNAP αCTD for activity. The results of DNase I protection studies showed that LuxRΔN-RNAP complexes can bind and protect the
luxI
promoter, but with less efficacy when the αCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the
lux
operon involves the RNAP αCTD and suggest that αCTD-LuxR interactions may play a role in recruitment of RNAP to the
luxI
promoter.
Serotype 1a O-Antigen Modification: Molecular Characterization of the Genes Involved and Their Novel Organization in the
Shigella flexneri
Chromosome
Authors: Adhikari Pradip , Allison Gwen , Whittle Belinda , Verma Naresh K. 
Published: December 2019

ABSTRACT

The factors responsible for serotype 1a O-antigen modification in
Shigella flexneri
were localized to a 5.8-kb chromosomal
Hin
dIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the
dsdC
and the
thrW proA
genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.
RpoS Synthesis Is Growth Rate Regulated in Salmonella typhimurium, but Its Turnover Is Not Dependent on Acetyl Phosphate Synthesis or PTS Function
Authors: Cunning Christofer , Elliott Thomas 
Published: December 2019

ABSTRACTThe RpoS sigma factor of enteric bacteria is either required for or augments the expression of a number of genes that are induced during nutrient limitation, growth into stationary phase, or in response to stresses, including high osmolarity. RpoS is regulated at multiple levels, including posttranscriptional control of its synthesis, protein turnover, and mechanisms that affect its activity directly. Here, the control of RpoS stability was investigated inSalmonella typhimuriumby the isolation of a number of mutants specifically defective in RpoS turnover. These included 20 mutants defective inmviA, the ortholog ofEscherichia coli rssB/sprE, and 13 mutants defective in eitherclpPorclpXwhich encode the protease active on RpoS. Anhnsmutant was also defective in RpoS turnover, thus confirming thatS. typhimuriumandE. colihave identical genetic requirements for this process. Some current models predict the existence of a kinase to phosphorylate the response regulator MviA, but no mutants affecting a kinase were recovered. AnmviAmutant carrying the D58N substitution altering the predicted phosphorylation site is substantially defective, suggesting that phosphorylation of MviA on D58 is important for its function. No evidence was obtained to support models in which acetyl phosphate or the PTS system contributes to MviA phosphorylation. However, we did find a significant (fivefold) elevation of RpoS during exponential growth on acetate as the carbon and energy source. This behavior is due to growth rate-dependent regulation which increases RpoS synthesis at slower growth rates. Growth rate regulation operates at the level of RpoS synthesis and is mainly posttranscriptional but, surprisingly, is independent ofhfqfunction.
Identification of a Gene in Staphylococcus xylosus Encoding a Novel Glucose Uptake Protein
Authors: Fiegler Heike , Bassias Joannis , Jankovic Ivana , Brückner Reinhold 
Published: December 2019

ABSTRACTBy transposon Tn917mutagenesis, two mutants ofStaphylococcus xylosuswere isolated that showed higher levels of β-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designatedglcU, encoding a protein involved in glucose uptake inS. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of β-galactosidase, α-glucosidase, and β-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons intoglcUreduced glucose dehydrogenase activity, suggesting cotranscription ofglcUandgdh. Insertional inactivation of thegdhgene and deletion of theglcUgene without affectinggdhexpression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in theglcUmutant strains. With clonedglcU, anEscherichia colimutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized byglcUin nonphosphorylated form. A gene fromBacillus subtilis,ycxE, that is homologous toglcU, could substitute forglcUin theE. coliglucose growth experiments and restored glucose repression in theS. xylosus glcUmutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.
Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica
Authors: Perkins-Balding Donna , Duval-Valentin Guy , Glasgow Anna C. 
Published: December 2019

ABSTRACTThe gram-negative marine bacteriumPseudoalteromonas atlanticaproduces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production inP. atlantica. Examination of IS492transposition inP. atlanticaby using a PCR-based assay revealed a circular form of IS492that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492insertion sites on theP. atlanticachromosome that we identified by using inverse PCR. IS492circle formation correlated with precise excision of IS492from theP. atlantica epstarget sequence when introduced intoEscherichia colion a plasmid. Deletion analyses of the flanking host sequences at theepsinsertion site for IS492demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492and circle formation inE. coli. Excision of IS492inE. colialso depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492is suggested by the creation of a new strong promoter for expression ofmooVby the joining of the ends of the insertion sequence element at the circle junction.
Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation
Authors: Ju Jingliang , Mitchell Theresa , Peters Howard , Haldenwang W. G. 
Published: December 2019

ABSTRACTAsBacillus subtilisproceeds through sporulation, the principal vegetative cell ς subunit (ςA) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific ς factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with ςAand two mother cell ς factors, ςEand ςK, which successively replace ςAon RNAP. Although the relative abundance of ςAwith respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable ςAwhich cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T0) to 2 to 8% by 3 h into the process (T3). In a strain that failed to synthesize ςE, the first of the mother cell-specific ς factors, approximately 40% of the ςAremained associated with RNAP atT3. The level of ςA-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ςEvariant (ςECR119) that could bind to RNAP but was unable to direct ςE-dependent transcription. The E-ςE-to-E-ςKchangeover was characterized by both the displacement of ςEfrom RNAP and the disappearance of ςEfrom the cell. Analyses of extracts from wild-type and mutantB. subtilisshowed that the ςKprotein is required for the displacement of ςEfrom RNAP and also confirmed that ςKis needed for the loss of the ςEprotein. The results indicate that the successive appearance of mother cell ς factors, but not necessarily their activities, is an important element in the displacement of preexisting ς factors from RNAP. It suggests that competition for RNAP by consecutive sporulation ς factors may be an important feature of the holoenzyme changeovers that occur during sporulation.
Transcriptional Analysis of the hmw Gene Cluster of Mycoplasma pneumoniae
Authors: Waldo Robert H. , Popham Phillip L. , Romero-Arroyo Cynthia E. , Mothershed Elizabeth A. , Lee Kyungok K. , Krause Duncan C. 
Published: December 2019

ABSTRACT

Mycoplasma pneumoniae adherence to host cells is a multifactorial process that requires the cytadhesin P1 and additional accessory proteins. The hmw gene cluster consists of the genes p30, hmw3, and hmw1, the products of which are known to be essential for cytadherence, therpsD gene, and six open reading frames of unknown function. Putative transcriptional terminators flank this locus, raising the possibility that these genes are expressed as a single transcriptional unit. However, S1 nuclease protection and primer extension experiments identified probable transcriptional start sites upstream of thep32, p21, p50, and rpsDgenes. Each was preceded at the appropriate spacing by the −10-like sequence TTAAAATT, but the −35 regions were not conserved. Analysis of the M. pneumoniae genome sequence indicated that this promoter-like sequence is found upstream of only a limited number of open reading frames, including the genes for P65 and P200, which are structurally related to HMW1 and HMW3. Promoter deletion studies demonstrated that the promoter-like region upstream ofp21 was necessary for the expression of p30 and an hmw3-cat fusion in M. pneumoniae, while deletion of the promoter-like region upstream of p32 had no apparent effect. Analysis by reverse transcription-PCR confirmed transcriptional linkage of all the open reading frames in thehmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts.
Identification of a New Regulator inStreptococcus pneumoniae Linking Quorum Sensing to Competence for Genetic Transformation
Authors: Lee Myeong S. , Morrison Donald A. 
Published: December 2019

ABSTRACT
Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB,cilC, cilD, cilE,cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at −10 and −25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE andcomAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA,cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comXexpression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes
Authors: Kinoshita Hiroshi , Tsuji Tomohiro , Ipposhi Hiroomi , Nihira Takuya , Yamada Yasuhiro 
Published: December 2019

ABSTRACT
BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barBupstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barBtranslational start codon. The BARE located on the barAupstream region (BARE-3) was found 101 to 76 bp upstream of thebarA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barBtranscription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.
Disruption of narG, the Gene Encoding the Catalytic Subunit of Respiratory Nitrate Reductase, Also Affects Nitrite Respiration in Pseudomonas fluorescens YT101
Authors: Ghiglione Jean-François , Philippot Laurent , Normand Philippe , Lensi Robert , Potier Patrick 
Published: December 2019

ABSTRACT
The Pseudomonas fluorescens YT101 genenarG, which encodes the catalytic α subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar− mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N2O respiration was not affected, anaerobic growth with NO2 as the sole electron acceptor was delayed for all of the Nar− mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.
Functional Interaction between PomA and PomB, the Na+-Driven Flagellar Motor Components of Vibrio alginolyticus
Authors: Yorimitsu Tomohiro , Sato Ken , Asai Yukako , Kawagishi Ikuro , Homma Michio 
Published: December 2019

ABSTRACT
Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to form a sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB. As a result, we could demonstrate that PomA and PomB functionally interact with each other.
DsbA Is Required for Stable Expression of Outer Membrane Protein YscC and for Efficient Yop Secretion inYersinia pestis
Authors: Jackson Michael W. , Plano Gregory V. 
Published: December 2019

ABSTRACT
The role of the periplasmic disulfide oxidoreductase DsbA in Yop secretion was investigated in Yersinia pestis. A Y. pestis dsbA mutant secreted reduced amounts of the V antigen and Yops and expressed reduced amounts of the full-sized YscC protein. Site-directed mutagenesis of the four cysteine residues present in the YscC protein resulted in defects similar to those found in thedsbA mutant. These results suggest that YscC contains at least one disulfide bond that is essential for the function of this protein in Yop secretion.