bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Hutchins Andrea M. , Holden James F. , Adams Michael W. W. 
Published: July 2002

ABSTRACT

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon
Pyrococcus furiosus
. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (∼1.6 MDa) complex that is not active. The apparent

K
m

values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 10
4
mM
−1
· s
−1
and 0.39 mM and 3.40 × 10
3
mM
−1
· s
−1
, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [
k
cat
/

K
m

= 32 (mM · s)
−1
]. In contrast to several other nucleotide-dependent enzymes from
P. furiosus
, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (∼5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.
Definition of the
Bacillus subtilis
PurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with
glyA
,
guaC
,
pbuG
,
xpt-pbuX
,
yqhZ-folD
, and
pbuO
Authors: Saxild Hans H. , Brunstedt Katja , Nielsen Karin I. , Jarmer Hanne , Nygaard Per 
Published: July 2002

ABSTRACT

The expression of the
pur
operon, which encodes enzymes of the purine biosynthetic pathway in
Bacillus subtilis
, is subject to control by the
purR
gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the
purA
and
purR
genes. A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900–3906, 1998). To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the
B. subtilis
genome sequence and found several candidate PurBoxes. By the use of transcriptional
lacZ
fusions, five selected genes or operons (
glyA
,
yumD
,
yebB
,
xpt-pbuX
, and
yqhZ-folD
), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence with the inverted repeats separated by 16 or 17 nucleotides. A computerized search revealed one additional PurR-regulated gene,
ytiP
. The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of
glyA
. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis showed that
yebB
encodes the previously characterized hypoxanthine-guanine permease PbuG and that
ytiP
encodes another guanine-hypoxanthine permease and is now named
pbuO. yumD
encodes a GMP reductase and is now named
guaC
.
Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria
Authors: Haldimann Andreas , Wanner Barry L. 
Published: July 2002

ABSTRACT

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of
Escherichia coli
and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision (“curing”), or retrieval of the CRIM plasmids.
Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the
pigA
Gene of
Pseudomonas aeruginosa
Authors: Ratliff Melanie , Zhu Wenming , Deshmukh Rahul , Wilks Angela , Stojiljkovic Igor 
Published: July 2002

ABSTRACT

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The
pigA
gene of
Pseudomonas aeruginosa
is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of
Neisseria meningitidis
. When the gene encoding the neisserial heme oxygenase,
hemO
, was replaced with
pigA
, we demonstrated that
pigA
could functionally replace
hemO
and allow for heme utilization by neisseriae. Furthermore, when
pigA
was disrupted by cassette mutagenesis in
P. aeruginosa
, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO
4
, indicating that the mutant did not have a defect in iron metabolism, and by in
trans
complementation with
pigA
from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader
Rhodococcus
sp. Strain RHA1
Authors: Kitagawa Wataru , Miyauchi Keisuke , Masai Eiji , Fukuda Masao 
Published: July 2002

ABSTRACT

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader,
Rhodococcus
sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes,
benABCDK
, exhibit 33 to 65% identity with those of
Acinetobacter
sp. strain ADP1. The gene organization of the RHA1
benABCDK
genes differs from that of ADP1. The RHA1
benABCDK
region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids.
Escherichia coli
cells containing RHA1
benABCD
transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1
benA
gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned
benABCD
genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1
benABC
. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1
benABC
homologs, suggesting that many
Rhodococcus
strains contain benzoate catabolic genes similar to RHA1
benABC
.
Characterization of the
cydAB
-Encoded Cytochrome
bd
Oxidase from
Mycobacterium smegmatis
Authors: Kana Bavesh D. , Weinstein Edward A. , Avarbock David , Dawes Stephanie S. , Rubin Harvey , Mizrahi Valerie 
Published: July 2002

ABSTRACT

The
cydAB
genes from
Mycobacterium smegmatis
have been cloned and characterized. The
cydA
and
cydB
genes encode the two subunits of a cytochrome
bd
oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The
cydD
and
cydC
genes located immediately downstream of
cydB
encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type
M. smegmatis
displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome
bd
oxidase. Inactivation of
cydA
or
cydB
by insertion of a kanamycin resistance marker resulted in loss of
d-
heme absorbance at 631 nm. The
d
-heme could be restored by transformation of the
M. smegmatis cyd
mutants with a replicating plasmid carrying the highly homologous
cydABDC
gene cluster from
Mycobacterium tuberculosis
. Inactivation of
cydA
had no effect on the ability of
M. smegmatis
to exit from stationary phase at 37 or 42°C. The growth rate of the
cydA
mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 10
2
Pa of pO
2
or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 10
2
Pa of pO
2
or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in
cydAB
gene expression observed upon reduction of the pO
2
of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in
d
-heme absorbance in spectra of membranes isolated from wild-type
M. smegmatis
cultured at 1% air saturation. Finally, the
cydA
mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome
bd
is an important terminal oxidase in
M. smegmatis.
Novel Genes Affecting Urease Activity inActinobacillus pleuropneumoniae
Authors: Bossé Janine T. , Gilmour Hali D. , MacInnes Janet I. 
Published: July 2002

ABSTRACTCharacterization of a series of urease-negative transposon mutations ofActinobacillus pleuropneumoniaerevealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF,apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. ThecbiMandcbiQgenes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and thecbiOgene encodes a cobalt transport ATP-binding protein homologue. The product of thecbiKgene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein.Escherichia coliclones containing this putative transport operon together with the urease genes ofA. pleuropneumoniaewere urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl2for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF,utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function inA. pleuropneumoniaeremains to be determined.
Recruitment of the
mecA
Gene Homologue of
Staphylococcus sciuri
into a Resistance Determinant and Expression of the Resistant Phenotype in
Staphylococcus aureus
Authors: Wu Shang Wei , de Lencastre Herminia , Tomasz Alexander 
Published: July 2002

ABSTRACT

Strains of methicillin-resistant
Staphylococcus aureus
(MRSA) have become the most important causative agents of hospital-acquired diseases worldwide. The genetic determinant of resistance,
mecA
, is not a gene native to
S. aureus
but was acquired from an extraspecies source by an unknown mechanism. We recently identified a close homologue of this gene in isolates of
Staphylococcus sciuri
, a taxonomically primitive staphylococcal species recovered most frequently from rodents and primitive mammals. In spite of the close sequence similarity between the
mecA
homologue of
S. sciuri
and the antibiotic resistance determinant
mecA
of
S. aureus
,
S. sciuri
strains were found to be uniformly susceptible to β-lactam antibiotics. In an attempt to activate the apparently “silent”
mecA
gene of
S. sciuri
, a methicillin-resistant derivative, K1M200 (for which the MIC of methicillin is 200 μg/ml), was obtained through stepwise exposure of the parental strain
S. sciuri
K1 (methicillin MIC of 4 μg/ml) to increasing concentrations of methicillin. DNA sequencing of the
mecA
homologue from K1M200 revealed the introduction of a point mutation into the −10 consensus of the promoter: the replacement of a thymine residue at nucleotide 1577 in the susceptible strain K1 by adenine in the resistant strain K1M200, which was accompanied by a drastic increase in transcription rate and the appearance of a new protein that reacted with monoclonal antibody prepared against the penicillin-binding protein 2A (PBP2A), i.e., the gene product of
S. aureus mecA
. Transduction of
mecA
from K1M200 (cloned into a plasmid vector) into a methicillin-susceptible
S. aureus
mutant resulted in a significant increase of methicillin resistance (from a methicillin MIC of 4 μg/ml to 12 and up to 50 μg/ml), the appearance of a low-affinity PBP detectable by the fluorographic assay, and the production of a protein that reacted in a Western blot with monoclonal antibody to PBP2A. Antibiotic resistance and the protein products disappeared upon removal of the plasmid-borne
mecA
homologue. The observations support the proposition that the
mecA
homologue ubiquitous in the antibiotic-susceptible animal species
S. sciuri
may be an evolutionary precursor of the methicillin resistance gene
mecA
of the pathogenic strains of MRSA.
The Evolving Genome of
Salmonella enterica
Serovar Pullorum
Authors: Liu Gui-Rong , Rahn Andrea , Liu Wei-Qiao , Sanderson Kenneth E. , Johnston Randal N. , Liu Shu-Lin 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make
S. enterica
serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of
S. enterica
serovar Pullorum RKS5078, using I-
Ceu
I,
Xba
I,
Avr
II, and
Spe
I and Tn
10
insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas
.
However, the genome of
S. enterica
serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most
S. enterica
serovar Pullorum strains examined, with different structures in a minority of
S. enterica
serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with
S. enterica
serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among
S. enterica
serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the
S. enterica
serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.
The
Escherichia coli
Cell Division Protein FtsW Is Required To Recruit Its Cognate Transpeptidase, FtsI (PBP3), to the Division Site
Authors: Mercer Keri L. N. , Weiss David S. 
Published: July 2002

ABSTRACT

The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting
Escherichia coli
cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues—which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation—is to recruit their cognate transpeptidases to the correct subcellular location.
Analysis of the
Bacillus subtilis spoIIIJ
Gene and Its Paralogue Gene,
yqjG
Authors: Murakami Takako , Haga Koki , Takeuchi Michio , Sato Tsutomu 
Published: July 2002

ABSTRACT

The
Bacillus subtilis spoIIIJ
gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that
spoIIIJ
and its paralogous gene,
yqjG
, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the
Escherichia coli
YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for
spoIIIJ
and
yqjG
during vegetative growth and sporulation. In rich media, the growth of
spoIIIJ
and
yqjG
single mutants were the same as that of the wild type, whereas
spoIIIJ yqjG
double inactivation was lethal, indicating that together these
B. subtilis
translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth.
spoIIIJ
mutations have already been established to block sporulation at stage III. In contrast, disruption of
yqjG
did not interfere with sporulation. We further show that high level expression of
spoIIIJ
during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of
spoIIIJ
is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of
spoIIIJ
during sporulation.
Critical Regions of
secM
That Control Its Translation and Secretion and Promote Secretion-Specific
secA
Regulation
Authors: Sarker Shameema , Oliver Donald 
Published: July 2002

ABSTRACT

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins.
secA
is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of
secA
,
secM
. The
secM
translational pause prevents formation of an RNA helix that normally blocks
secA
translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical
secM
signal peptide and found that mutations within the amino-terminal region specifically affect the
secM
translational pause and
secA
regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and
secA
regulation. In addition, mutational analysis of the 3′ end of
secM
allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the
secM
translational pause model of
secA
regulation, and they pinpoint key sequences within
secM
that promote this important regulatory system.