bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the Multidrug Resistance Transporter NorA from
Staphylococcus aureus
Is Modified by a Two-Component Regulatory System
Authors: Fournier Bénédicte , Aras Rahul , Hooper David C. 
Published: July 2002

ABSTRACT

To dissect genetically the regulation of NorA, a multidrug transporter of
Staphylococcus aureus
, we analyzed the differential expression of the
norA
promoter using a transcriptional fusion with a β-lactamase reporter gene. Expression studies with an
arlS
mutant revealed that the
norA
promoter is ArlS dependent. The
arlR-arlS
locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of
norA
by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein. From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of
norA
expression in the presence of the
arlS
mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the
arlS
mutation, and the
arlS
mutation altered the growth-phase regulation of NorA. These results indicate that expression of
norA
is modified by a two-component regulatory system.
Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from
Rhodococcus erythropolis
Authors: Lichtinger Thomas , Reiss Gila , Benz Roland 
Published: July 2002

ABSTRACT

Organic solvent extracts of whole cells of the gram-positive bacterium
Rhodococcus erythropolis
contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of
R. erythropolis
was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of
R. erythropolis
were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.
PpdD Type IV Pilin of
Escherichia coli
K-12 Can Be Assembled into Pili in
Pseudomonas aeruginosa
Authors: Sauvonnet Nathalie , Gounon Pierre , Pugsley Anthony P. 
Published: July 2002

ABSTRACT

Escherichia coli
K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However,
E. coli
K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putative
E. coli
piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (
ppdD
) and the linked assembly genes
hofB
and
hofC
(homologues of the
Pseudomonas aeruginosa
type IV pilus assembly genes
pilB
and
pilC
) did not lead to pilus production. However, expression of the
ppdD
gene in
P. aeruginosa
led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in
P. aeruginosa
was dependent on the expression of the
pilB
and
pilC
genes and independent of expression of the
P. aeruginosa
pilin structural gene
pilA.
Evolution of Arginine Biosynthesis in the Bacterial Domain: Novel Gene-Enzyme Relationships from Psychrophilic
Moritella
Strains (
Vibrionaceae
) and Evolutionary Significance of
N
-α-Acetyl Ornithinase
Authors: Xu Ying , Liang Ziyuan , Legrain Christianne , Rüger Hans J. , Glansdorff Nicolas 
Published: July 2002

ABSTRACT

In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of
N
-α-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (
argJ
encoded). Only two exceptions had been reported—the
Enterobacteriaceae
and
Myxococcus xanthus
(members of the γ and δ groups of the class
Proteobacteria
, respectively)—in which ornithine is produced from
N
-α-acetylornithine by a deacylase, acetylornithinase (AOase) (
argE
encoded). We have investigated the gene-enzyme relationship in the arginine regulons of two psychrophilic
Moritella
strains belonging to the
Vibrionaceae
, a family phylogenetically related to the
Enterobacteriaceae
. Most of the
arg
genes were found to be clustered in one continuous sequence divergently transcribed in two wings,
argE
and
argCBFGH(A)
[“
H(A)
” indicates that the argininosuccinase gene consists of a part homologous to known
argH
sequences and of a 3′ extension able to complement an
Escherichia coli
mutant deficient in the
argA
gene, encoding
N
-α-acetylglutamate synthetase, the first enzyme committed to the pathway]. Phylogenetic evidence suggests that this new clustering pattern arose in an ancestor common to
Vibrionaceae
and
Enterobacteriaceae
, where OATase was lost and replaced by a deacylase. The AOase and ornithine carbamoyltransferase of these psychrophilic strains both display distinctly cold-adapted activity profiles, providing the first cold-active examples of such enzymes.
Biochemical and Physical Properties of theMethanococcus jannaschii20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome
Authors: Wilson Heather L. , Ou Mark S. , Aldrich Henry C. , Maupin-Furlow Julie 
Published: July 2002

ABSTRACTThe 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophileMethanococcus jannaschii. In addition, theM. jannaschii20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited byN-ethylmaleimide andp-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively highVmaxfor ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Piper min per mg of protein as well as a relatively low affinity for CTP and ATP withKmvalues of 307 and 497 μM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Δ1–73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that theM. jannaschiias well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.
Membrane Topology of the NixA Nickel Transporter of
Helicobacter pylori
: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III
Authors: Fulkerson John F. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

NixA, the high-affinity cytoplasmic membrane nickel transport protein of
Helicobacter pylori
, imports Ni
2+
into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with β-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni
2+
transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni
2+
transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.
Two ResD-Controlled Promoters Regulate
ctaA
Expression in
Bacillus subtilis
Authors: Paul Salbi , Zhang Xiaohui , Hulett F. Marion 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration.
ctaA
is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The
ctaAB
-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full
ctaA
promoter activity. In this study, we demonstrate the role of ResD∼P in the activation of the
ctaA
promoter using an in vitro transcription system. The results indicate that the
ctaA
promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by Eς
A
, transcription which was enhanced approximately 1.5-fold by ResD and 5-fold by ResD∼P. The downstream promoter (Ps) required both binding sites A2 and A3 and was not transcribed by Eς
A
with or without ResD∼P. RNA polymerase (RNAP) isolated from
B. subtilis
when cells were at the end of exponential growth (T
0
) or 3, 4, or 5 h into the stationary phase (T
3
, T
4
, or T
5
, respectively) was used in in vitro transcription assays. Maximal transcription from Ps required T
4
RNAP plus ResD∼P. RNAP isolated from a
spo0A
or a
sigE
mutant strain was not capable of Ps transcription. Comparison of the Ps promoter sequence with the SigE binding consensus suggests that the
ctaA
Ps promoter may be a SigE promoter. The collective data from ResD footprinting, in vivo promoter deletion analysis, and in vitro transcription assays suggest that
ctaA
is transcribed during late exponential to early stationary phases of growth from the Pv promoter, which requires ResD binding site A2, Eς
A
, and ResD∼P, and during later stationary phase from Ps, which requires binding sites A2 and A3, ResD∼P, and Eς
E
or a sigma factor whose transcription is dependent on SigE.
Autoregulation of the
dnaA-dnaN
Operon and Effects of DnaA Protein Levels on Replication Initiation in
Bacillus subtilis
Authors: Ogura Yoshitoshi , Imai Yukiho , Ogasawara Naotake , Moriya Shigeki 
Published: July 2002

ABSTRACT

In
Escherichia coli,
the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in
B. subtilis,
we constructed a strain in which a copy of the
dnaA
gene was integrated at the
purA
locus on the chromosome under the control of an isopropyl-β
-d-
thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native
dnaA-dnaN
operon was greatly reduced at high DnaA levels, but it was increased in a
dnaA
-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the
dnaN
gene was added downstream of the additional
dnaA
gene at
purA
, the cells grew at high DnaA levels, suggesting that depletion of DnaN (β subunit of DNA polymerase III) within the cell by repression of the native
dnaA-dnaN
operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in
B. subtilis
.
Succinyl-CoA:(
R
)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria
Authors: Leutwein Christina , Heider Johann 
Published: July 2002

ABSTRACT

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (
R
)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(
R
)-benzylsuccinate CoA-transferase activating (
R
)-benzylsuccinate to the CoA-thioester was purified and characterized from
Thauera aromatica
. The enzyme is fully reversible and forms exclusively the 2-(
R
)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (
R
)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α
2
β
2
conformation. Identity of the subunits with the products of the previously identified toluene-induced
bbsEF
genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (
E
)-cinnamoyl-CoA:(
R
)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates.
Dehalogenation of Dichloromethane by Dichloromethane Dehalogenase/Glutathione
S
-Transferase Leads to Formation of DNA Adducts
Authors: Kayser Martin F. , Vuilleumier Stéphane 
Published: July 2002

ABSTRACT

Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione
S
-transferase was demonstrated. Adducts included dichloromethane carbon and glutathione sulfur atoms. A reaction with DNA occurred preferentially at guanine bases. Increased DNA degradation in a
polA
mutant of
Methylobacterium dichloromethanicum
DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.
Gene Cluster on pAO1 of
Arthrobacter nicotinovorans
Involved in Degradation of the Plant Alkaloid Nicotine: Cloning, Purification, and Characterization of 2,6-Dihydroxypyridine 3-Hydroxylase
Authors: Baitsch Daniel , Sandu Cristinel , Brandsch Roderich , Igloi Gabor L. 
Published: July 2002

ABSTRACT

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of
Arthrobacter nicotinovorans
. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in
Escherichia coli
. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.
Noncatalytic Docking Domains of Cellulosomes of Anaerobic Fungi
Authors: Steenbakkers Peter J. M. , Li Xin-Liang , Ximenes Eduardo A. , Arts Jorik G. , Chen Huizhong , Ljungdahl Lars G. , Op den Camp Huub J. M. 
Published: July 2002

ABSTRACT

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus
Piromyces
sp. strain E2 and the polycentric fungus
Orpinomyces
sp. strain PC-2. Through PCR using cDNA from
Orpinomyces
sp. and genomic DNA from
Piromyces
sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an
Orpinomyces
sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus
Orpinomyces
sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.
LuxArray, a High-Density, Genomewide Transcription Analysis of
Escherichia coli
Using Bioluminescent Reporter Strains
Authors: Van Dyk Tina K. , DeRose Ellen J. , Gonye Gregory E. 
Published: July 2002

ABSTRACT

A sequenced collection of plasmid-borne random fusions of
Escherichia coli
DNA to a
Photorhabdus luminescens luxCDABE
reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions. This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in
E. coli
. High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression. The cellular response to nalidixic acid perturbation was analyzed using this format. As expected, fusions to promoters of LexA-controlled SOS-responsive genes
dinG
,
dinB
,
uvrA
, and
ydjM
were found to be upregulated in the presence of nalidixic acid. In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated. The responses of two of these, fusions to
oraA
and
yigN
, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon. The responses of the other four were neither induced by mitomycin C nor dependent on
lexA
function. Thus, the promoters of
ycgH, intG
,
rihC
, and a putative operon consisting of
lpxA, lpxB, rnhB,
and
dnaE
were not generally DNA damage responsive and represent a more specific response to nalidixic acid. These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses.
Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of
Haemophilus ducreyi
: Restoration of Full-Length LOS Restores Pyocin Sensitivity
Authors: Filiatrault Melanie J. , Munson Robert S. , Campagnari Anthony A. 
Published: July 2002

ABSTRACT

DNA sequence and Southern blot analyses were used to determine the genetic defect of a
Haemophilus ducreyi
pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire
waaQ
gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of
H. influenzae
, the gene encoding an argininosuccinate synthase homolog, and a change in the 3′ sequence of the
lgtF
gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.