bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Expression of
cadD
, a New Cadmium Resistance Gene of
Staphylococcus aureus
Authors: Crupper Scott S. , Worrell Veronica , Stewart George C. , Iandolo John J. 
Published: December 2019

ABSTRACT

A cadmium resistance gene, designated
cadD
, has been identified in and cloned from the
Staphylococcus aureus
plasmid pRW001. The gene is part of a two-component operon which contains the resistance gene
cadD
and an inactive regulatory gene,
cadX*
. A high degree of sequence similarity was observed between
cadD
and the
cadB
-like gene from
S. lugdunensis
, but no significant similarity was found with either
cadA
or
cadB
from the
S. aureus
plasmids pI258 and pII147. The positive regulatory gene
cadX*
is identical to
cadX
from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS
257
. The expression of
cadD
in
S. aureus
and
Bacillus subtilis
resulted in low-level resistance to cadmium; in contrast,
cadA
and
cadB
from
S. aureus
induced higher level resistance. However, when the truncated version of
cadX
contained in pRW001 is complemented in
trans
with
cadX
from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version of
cadX
contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.
ScoC Regulates Peptide Transport and Sporulation Initiation in
Bacillus subtilis
Authors: Koide Akiko , Perego Marta , Hoch James A. 
Published: December 2019

ABSTRACT

Oligopeptides are transported into
Bacillus subtilis
by two ABC transport systems, App and Opp. Transcription of the operon encoding the Opp system was found to occur during exponential growth, whereas the
app
operon was induced at the onset of stationary phase. Transcription of both operons was completely curtailed by overproduction of the ScoC regulator from a multicopy plasmid and was enhanced in strains with the
scoC
locus deleted. ScoC, a member of the MarR family of transcription regulators, is known from previous studies to be a negative regulator of sporulation and of protease production that acts by binding directly to the promoters of the genes it regulates. Since peptide transport is essential for inactivation of the negative regulation of sporulation by Rap phosphatases, the control of ScoC transcription repression activity plays a crucial role in the initiation of sporulation.
Molecular Cloning and Characterization of a Locus Responsible for O Acetylation of the O Polysaccharide of
Legionella pneumophila
Serogroup 1 Lipopolysaccharide
Authors: Zou Chang Hua , Knirel Yuriy A. , Helbig Jürgen H. , Zähringer Ulrich , Mintz Clifford S. 
Published: December 2019

ABSTRACT

Complementation experiments, Tn
5
mutagenesis, and DNA sequencing were used to identify a locus (
lag-1
) that participates in acetylation of
Legionella pneumophila
serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of lipopolysaccharides from mutant and complemented strains suggest that
lag-1
is responsible for O acetylation of serogroup 1 O polysaccharide.
The Genes Encoding Formamidopyrimidine and MutY DNA Glycosylases in
Escherichia coli
Are Transcribed as Part of Complex Operons
Authors: Gifford Christine M. , Wallace Susan S. 
Published: December 2019

ABSTRACT

Escherichia coli
formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine. The genes encoding these proteins,
fpg
and
mutY
, are both cotranscribed as part of complex operons.
fpg
is the terminal gene in an operon with the gene order
radC
,
rpmB
,
rpmG
, and
fpg
. This operon has transcription initiation sites upstream of
radC
, in the
radC
coding region, and immediately upstream of
fpg
. There is a strong attenuator in the
rpmG-fpg
intergenic region and three transcription termination sites downstream of
fpg
. There is an additional site, in the
radC-rpmB
intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site.
mutY
is the first gene in an operon with the gene order
mutY
,
yggX
,
mltC
, and
nupG
. This operon has transcription initiation sites upstream of
mutY
, in the
mutY
coding region, and immediately upstream of
nupG
. There also appear to be attenuators in the
yggX-mltC
and
mltC-nupG
intergenic regions. The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms.
A
Vibrio cholerae
LysR Homolog, AphB, Cooperates with AphA at the
tcpPH
Promoter To Activate Expression of the ToxR Virulence Cascade
Authors: Kovacikova Gabriela , Skorupski Karen 
Published: December 2019

ABSTRACT

We describe here a new member of the LysR family of transcriptional regulators, AphB, which is required for activation of the
Vibrio cholerae
ToxR virulence cascade. AphB activates the transcription of the
tcpPH
operon in response to environmental stimuli, and this process requires cooperation with a second protein, AphA. The expression of neither
aphA
or
aphB
is strongly regulated by environmental stimuli, raising the possibility that the activities of the proteins themselves may be influenced under various conditions. Strains of the El Tor biotype of
V. cholerae
typically exhibit lower expression of ToxR-regulated virulence genes in vitro than classical strains and require specialized culture conditions (AKI medium) to induce high-level expression. We show here that expression of
aphB
from the
tac
promoter in El Tor biotype strains dramatically increases virulence gene expression to levels similar to those observed in classical strains under all growth conditions examined. These results suggest that AphB plays a role in the differential regulation of virulence genes between the two disease-causing biotypes.
Genetic Organization of the
citCDEF
Locus and Identification of
mae
and
clyR
Genes from
Leuconostoc mesenteroides
Authors: Bekal-Si Ali Sadjia , Diviès Charles , Prévost Hervé 
Published: December 2019

ABSTRACT

In this paper, we describe two open reading frames coding for a NAD-dependent malic enzyme (
mae
) and a putative regulatory protein (
clyR
) found in the upstream region of
citCDEFG
of
Leuconostoc mesenteroides
subsp.
cremoris
195. The transcriptional analysis of the citrate lyase locus revealed one polycistronic mRNA covering the
mae
and
citCDEF
genes. This transcript was detected only on RNA prepared from cells grown in the presence of citrate. Primer extension experiments suggest that
clyR
and the citrate lyase operon are expressed from a bidirectional A-T-rich promoter region located between
mae
and
clyR.
Physicochemical Evidence that
Treponema pallidum
TroA Is a Zinc-Containing Metalloprotein That Lacks Porin-Like Structure
Authors: Deka Ranjit K. , Lee Yong-Hwan , Hagman Kayla E. , Shevchenko Dmitriy , Lingwood Clifford A. , Hasemann Charles A. , Norgard Michael V. , Radolf Justin D. 
Published: December 2019

ABSTRACT

Although TroA (Tromp1) was initially reported to be a
Treponema pallidum
outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein’s amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive β-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.
Methanobacterium thermoautotrophicum
RNA Polymerase and Transcription In Vitro
Authors: Darcy Trevor J. , Hausner Winfried , Awery Donald E. , Edwards Aled M. , Thomm Michael , Reeve John N. 
Published: December 2019

ABSTRACT

RNA polymerase (RNAP) purified from
Methanobacterium thermoautotrophicum
ΔH has been shown to initiate transcription accurately in vitro from the
hmtB
archaeal histone promoter with either native or recombinant forms of the
M. thermoautotrophicum
TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the
M. thermoautotrophicum
RNAP and TFB has been demonstrated.
Isolation of Additional Bacteriophages with Genomes of Segmented Double-Stranded RNA
Authors: Mindich Leonard , Qiao Xueying , Qiao Jian , Onodera Shiroh , Romantschuk Martin , Hoogstraten Deborah 
Published: December 2019

ABSTRACT

Eight different bacteriophages were isolated from leaves of
Pisum sativum
,
Phaseolus vulgaris
,
Lycopersicon esculentum
,
Daucus carota sativum
,
Raphanus sativum
, and
Ocimum basilicum
. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage φ6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of φ6. Three of the viruses have little or no base sequence identity to φ6. Two of the viruses, φ8 and φ12, contain proteins with a size distribution very different from that of φ6 and do not package genomic segments of φ6. Whereas φ6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in
Escherichia coli
or
Salmonella typhimurium
. One of the isolates, φ8, can form plaques on heptoseless strains of
S. typhimurium
.
Citromicrobium bathyomarinum
, a Novel Aerobic Bacterium Isolated from Deep-Sea Hydrothermal Vent Plume Waters That Contains Photosynthetic Pigment-Protein Complexes
Authors: Yurkov Vladimir V. , Krieger Steven , Stackebrandt Erko , Beatty J. Thomas 
Published: December 2019

ABSTRACT

We have taxonomically and phylogenetically characterized a new aerobic bacterial strain (JF-1) that contains photosynthetic pigment-protein complexes and which was recently isolated from black smoker plume waters of the Juan de Fuca Ridge. Strain JF-1 is a gram-negative, yellow-pigmented, motile bacterium that is salt-, pH-, and thermotolerant. These properties are consistent with an oligotrophic adaptation to varied environmental conditions thought to exist around deep-sea hydrothermal vents. The analysis of 16S rDNA sequences revealed that strain JF-1 forms a separate phylogenetic branch between the genus
Erythromonas
and the
Erythromicrobium-Porphyrobacter-Erythrobacter
cluster within the α subclass of the
Proteobacteria
. The taxonomic name
Citromicrobium bathyomarinum
(gen. nov., sp. nov.) is proposed for strain JF-1.
Molecular Cloning, Sequence Analysis, and Characterization of a Penicillin-Resistant
dd
-Carboxypeptidase of
Myxococcus xanthus
Authors: Kimura Yoshio , Takashima Yukie , Tokumasu Yushi , Sato Masayuki 
Published: December 2019

ABSTRACT

We have cloned a gene,
pdcA
, from the genomic library of
Myxococcus xanthus
with an oligonucleotide probe representing conserved regions of penicillin-resistant
dd
-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted
pdcA
gene product showed significant sequence similarity to
N
-acetylmuramoyl-
l
-alanine amidase and penicillin-resistant
dd
-carboxypeptidase, respectively. The
pdcA
gene was expressed in
Escherichia coli
, and the characteristics of the gene product were similar to those of
dd
-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in
pdcA
deletion mutants.
Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents
Authors: Moukadiri Ismaïl , Jaafar Lahcen , Zueco Jesús 
Published: December 2019

ABSTRACT
In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of aSaccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with β-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, andPIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.
Regulation and Adaptation of Glucose Metabolism of the Parasitic Protist Leishmania donovani at the Enzyme and mRNA Levels
Authors: ter Kuile Benno H. 
Published: December 2019

ABSTRACT
Adaptation of the glucose metabolism of Leishmania donovani promastigotes (insect stage) was investigated by simultaneously measuring metabolic rates, enzyme activities, message levels, and cellular parameters under various conditions. Chemostats were used to adapt cells to different growth rates with growth rate-limiting or excess glucose concentrations. L. donovanicatabolized glucose to CO2, succinate, acetate, and pyruvate in ratios that depended on growth rate and glucose availability. Rates of glucose consumption were a linear function of growth rate and were twice as high in excess glucose-grown cells as in glucose-limited organisms. The major end product was CO2, but organic end products were also formed in ratios that varied strongly with growth conditions. The specific activities of the 14 metabolic enzymes measured varied by factors of 3 to 17. Two groups of enzymes adapted specific activities in parallel, but there was no correlation between the groups. The activities of only one group correlated with specific rates of glucose metabolism. Total RNA content per cellular protein varied by a factor of 6 and showed a linear relationship with the rate of glucose consumption. There was no correlation between steady-state message levels and activities of the corresponding enzymes, suggesting regulation at the posttranscriptional level. A comparison of the adaptation of energy metabolism in L. donovani and other species suggests that the energy metabolism ofL. donovani is inefficient but is well suited to the environmental challenges that it encounters during residence in the sandfly, its insect vector.
The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii
Authors: Hales Laura M. , Shuman Howard A. 
Published: December 2019

ABSTRACTTo investigate regulatory networks inLegionella pneumophila, the gene encoding the homolog of theEscherichia colistress and stationary-phase sigma factor RpoS was identified by complementation of anE. coli rpoSmutation. An open reading frame that is approximately 60% identical to theE. coli rpoSgene was identified. Western blot analysis showed that the level ofL. pneumophilaRpoS increased in stationary phase. An insertion mutation was constructed in therpoSgene on the chromosome ofL. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates thatL. pneumophilaRpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host,Acanthamoeba castellanii. These data suggest thatL. pneumophilapossesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role ofrpoSinL. pneumophilais very different from what has previously been reported forE. coli rpoS.
The Pseudomonas aeruginosa Exotoxin A Regulatory Gene, ptxS: Evidence for Negative Autoregulation
Authors: Swanson Britta L. , Colmer Jane A. , Hamood Abdul N. 
Published: December 2019

ABSTRACTWe have previously described aPseudomonas aeruginosagene,ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene,ptxS, which is transcribed divergently fromptxRand interferes with the enhancement of exotoxin A synthesis byptxR. However, the mechanisms through whichptxRand/orptxSare regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine ifP. aeruginosacontains a potential regulatory protein that binds specifically to theptxRorptxSupstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing theptxR-ptxSintergenic region was incubated with the lysate ofP. aeruginosaPAO1. The strongest binding activity was detected with a smaller fragment that represents theptxSupstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream ofptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of theptxSisogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carryingptxSintact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate ofEscherichia coliin whichptxSwas overexpressed from the T7 promoter. The effect of PtxS onptxSexpression was examined by using aptxS-lacZfusion plasmid. The level of β-galactosidase activity produced by PAO1::ptxScarrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within theptxSupstream region.
A Megaplasmid-Borne Anaerobic Ribonucleotide Reductase in Alcaligenes eutrophus H16
Authors: Siedow Anja , Cramm Rainer , Siddiqui Roman A. , Friedrich Bärbel 
Published: December 2019

ABSTRACTThe conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth ofAlcaligenes eutrophusH16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdDandnrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion ofnrdDandnrdGinA. eutrophusabolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant ofA. eutrophuswith a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate thatnrdDandnrdGare cotranscribed and that the translation efficiency ofnrdDis 40-fold higher than that ofnrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.
Proton-Nuclear Magnetic Resonance Analyses of the Substrate Specificity of a β-Ketolase from Pseudomonas putida, Acetopyruvate Hydrolase
Authors: Pokorny Diana , Brecker Lothar , Pogorevc Mateja , Steiner Walter , Griengl Herfried , Kappe Thomas , Ribbons Douglas W. 
Published: December 2019

ABSTRACTA revised purification of acetopyruvate hydrolase from orcinol-grownPseudomonas putidaORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of ∼38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by1H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three1H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A1H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized π-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg2+bound to the enzyme was made.1H–2H exchange reactions showed the complete, fast equilibration of2H into the C-3 of acetopyruvate chemically; this accounts for the appearance of2H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional1H-NMR in normal1H2O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.