bluuebunny/crossref_metadata_2025_split huggingface dataset:
Balance between Endogenous Superoxide Stress and Antioxidant Defenses
Authors: Gort Amy Strohmeier , Imlay James A. 
Published: December 2019

ABSTRACT

Cells devoid of cytosolic superoxide dismutase (SOD) suffer enzyme inactivation, growth deficiencies, and DNA damage. It has been proposed that the scant superoxide (O
2
−
) generated by aerobic metabolism harms even cells that contain abundant SOD. However, this idea has been difficult to test. To determine the amount of O
2
−
that is needed to cause these defects, we modulated the O
2
−
concentration inside
Escherichia coli
by controlling the expression of SOD. An increase in O
2
−
of more than twofold above wild-type levels substantially diminished the activity of labile dehydratases, an increase in O
2
−
of any more than fourfold measurably impaired growth, and a fivefold increase in O
2
−
sensitized cells to DNA damage. These results indicate that
E. coli
constitutively synthesizes just enough SOD to defend biomolecules against endogenous O
2
−
so that modest increases in O
2
−
concentration diminish cell fitness. This conclusion is in excellent agreement with quantitative predictions based upon previously determined rates of intracellular O
2
−
production, O
2
−
dismutation, dehydratase inactivation, and enzyme repair. The vulnerability of bacteria to increased intracellular O
2
−
explains the widespread use of superoxide-producing drugs as bactericidal weapons in nature.
E. coli
responds to such drugs by inducing the SoxRS regulon, which positively regulates synthesis of SOD and other defensive proteins. However, even toxic amounts of endogenous O
2
−
did not activate SoxR, and SoxR activation by paraquat was not at all inhibited by excess SOD. Therefore, in responding to redox-cycling drugs, SoxR senses some signal other than O
2
−
.
Unusual Organization of the Genes Coding for HydSL, the Stable [NiFe]Hydrogenase in the Photosynthetic Bacterium
Thiocapsa roseopersicina
BBS
Authors: Rakhely Gabor , Colbeau Annette , Garin Jerome , Vignais Paulette M. , Kovacs Kornel L. 
Published: December 2019

ABSTRACT

The characterization of a
hyd
gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in
Thiocapsa roseopersicina
BBS, a purple sulfur photosynthetic bacterium belonging to the family
Chromatiaceae
, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947–951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567–3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes
hydS
and
hydL
, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the
hmc
operon, coding for a transmembrane electron transfer complex in
Desulfovibrio vulgaris
. Other accessory proteins are not found immediately downstream or upstream of
hydSL
. A
hup
gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in
T. roseopersicina
was reported earlier (A. Colbeau et al. Gene 140:25–31, 1994). The deduced amino acid sequences of the two small (
hupS
and
hydS
) and large subunit (
hupL
and
hydL
) sequences share 46 and 58% identity, respectively. The
hup
and
hyd
genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by
hydSL
, and the inducible and unstable second hydrogenase may be the product of the
hup
gene cluster.
In Vivo Protein Interactions within the
Escherichia coli
DNA Polymerase III Core
Authors: Jonczyk Piotr , Nowicka Adrianna , Fijałkowska Iwona J. , Schaaper Roel M. , Cieśla Zygmunt 
Published: December 2019

ABSTRACT

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of
Escherichia coli
could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the
Saccharomyces cerevisiae
two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the
mutD5
and
dnaQ926
mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, the
dnaQ49
mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of
dnaQ49
may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.
Molecular Cloning, Expression, and Characterization of the Genes Encoding the Two Essential Protein Components of
Micrococcus luteus
B-P 26 Hexaprenyl Diphosphate Synthase
Authors: Shimizu Naoto , Koyama Tanetoshi , Ogura Kyozo 
Published: December 2019

ABSTRACT

The structural genes encoding the two essential components A and B of hexaprenyl diphosphate synthase, which produce the precursor of the prenyl side chain of menaquinone-6, were cloned from
Micrococcus luteus
B-P 26.
Isolation and Purification of Two Novel Streptomycete RNase Inhibitors, SaI14 and SaI20, and Cloning, Sequencing, and Expression inEscherichia coliof the Gene Coding for SaI14
Authors: Krajcikova Daniela , Hartley Robert W. , Sevcik Jozef 
Published: December 2019

ABSTRACTTwo new RNase inhibitors, SaI14 (Mr, ∼14,000) and SaI20 (Mr, ∼20,000), were isolated and purified from aStreptomyces aureofaciensstrain. The genesai14, coding for SaI14 protein, was cloned and expressed inEscherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor fromBacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.
The Folate Branch of the Methionine Biosynthesis Pathway in
Streptomyces lividans
: Disruption of the 5,10-Methylenetetrahydrofolate Reductase Gene Leads to Methionine Auxotrophy
Authors: Blanco Jorge , Coque Juan Jose R. , Martin Juan F. 
Published: December 2019

ABSTRACT

In enterobacteria, the methyl group of methionine is donated by 5-methyltetrahydrofolate that is synthesized from
N
5,10
-methylenetetrahydrofolate by the 5,10-methylenetetrahydrofolate reductase. The
Streptomyces lividans metF
gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids with a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of the transcription initiation site showed that the
metF
gene is expressed, forming a leaderless mRNA. A 13-bp tandem repeat located immediately upstream of the promoter region shows homology with the consensus MetR-binding sequence of
Salmonella typhimurium
. Expression of
metF
in multicopy plasmids in
S. lividans
resulted in accumulation of a 32-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Disruption of the
metF
gene led to methionine auxotrophy. Integration of the disrupting plasmid at the
metF
locus was confirmed by Southern hybridization in three randomly isolated transformants. The methionine auxotrophy was complemented by transformation of the auxotrophs with an undisrupted
metF
gene. These results indicate that the folate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.
Cloning and Sequencing of a Form II Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from the Bacterial Symbiont of the Hydrothermal Vent Tubeworm
Riftia pachyptila
Authors: Robinson Jonathan J. , Stein Jeffrey L. , Cavanaugh Colleen M. 
Published: December 2019

ABSTRACT

The bacterial symbiont of the hydrothermal vent tubeworm fixes carbon via the Calvin-Benson cycle and has been shown previously to express a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene
cbbM
, which encodes this enzyme, has been cloned and sequenced. The gene has the highest identity with the
cbbM
gene from
Rhodospirillum rubrum
, and analysis of the inferred amino acid sequence reveals that all active-site residues are conserved. This is the first form II RubisCO cloned and sequenced from a chemoautotrophic symbiont and from a deep-sea organism.
The
ars
Operon in the
skin
Element of
Bacillus subtilis
Confers Resistance to Arsenate and Arsenite
Authors: Sato Tsutomu , Kobayashi Yasuo 
Published: December 2019

ABSTRACT

The
Bacillus subtilis skin
element confers resistance to arsenate and arsenite. The
ars
operon in the
skin
element contains four genes in the order
arsR
,
ORF2
,
arsB
, and
arsC
. Three of these genes are homologous to the
arsR
,
arsB
, and
arsC
genes from the staphylococcal plasmid pI258, while no homologs of
ORF2
have been found. Inactivation of
arsR
,
arsB
, or
arsC
results in either constitutive expression of
ars
, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the
ars
operon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length
ars
transcript of about 2.4 kb was induced by arsenate and that the
ars
promoter contains sequences that resemble the −10 and −35 regions of promoters that are recognized by Eς
A
.
Involvement of
recF
,
recO
, and
recR
Genes in UV-Radiation Mutagenesis of
Escherichia coli
Authors: Liu Ying-Hsiu , Cheng Ann-Joy , Wang Tzu-chien V. 
Published: December 2019

ABSTRACT

The
recF
,
recO
, and
recR
genes were originally identified as those affecting the RecF pathway of recombination in
Escherichia coli
cells. Several lines of evidence suggest that the
recF
,
recO
, and
recR
genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in
recF
,
recO
, or
recR
greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp
−
(
trpE65
) to a Trp
+
phenotypes. Introduction of the defective
lexA51
mutation [
lexA51
(Def)] and/or UmuD′ into
recF
,
recO
, and
recR
mutants failed to restore normal UVM in the mutants. On the other hand, the presence of
recA2020
, a suppressor mutation for
recF
,
recO
, and
recR
mutations, restored normal UVM in
recF
,
recO
, and
recR
mutants. These results indicate an involvement of the
recF
,
recO
, and
recR
genes and their products in UVM, possibly by affecting the third role of RecA in UVM.
Probing the Yeast Phase-Specific Expression of the
CBP1
Gene in
Histoplasma capsulatum
Authors: Patel Jean Baldus , Batanghari Janet West , Goldman William E. 
Published: December 2019

ABSTRACT

Histoplasma capsulatum
is a pathogenic fungus that exists in two distinct forms. The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes. Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism. In this study, we show that the gene encoding CBP (
CBP1
) is transcriptionally regulated. To identify promoter sequences that are important for yeast phase-specific activity, we created a series of fusions between successively truncated
CBP1
5′ untranslated regulatory sequences and the
Eschericha coli lacZ
gene. The fusions were constructed on a telomeric shuttle plasmid that can replicate autonomously in the fungus. By assaying for β-galactosidase activity from
H. capsulatum
transformants, we identified a 102-bp region that mediates promoter activation and yeast phase promoter activity. Base pair substitution analysis suggests that the sequences between 839 and 877 bp upstream of the start codon are the most important for this positive regulation.
The Methyl Group of the
N
6
-Methyl-
N
6
-Threonylcarbamoyladenosine in tRNA of
Escherichia coli
Modestly Improves the Efficiency of the tRNA
Authors: Qian Qiang , Curran James F. , Björk Glenn R. 
Published: December 2019

ABSTRACT

tRNA species that read codons starting with adenosine (A) contain
N
6
-threonylcarbamoyladenosine (t
6
A) derivatives adjacent to and 3′ of the anticodons from all organisms. In
Escherichia coli
there are 12 such tRNA species of which two (tRNA
GGU
Thr1
and tRNA
GGU
Thr3
) have the t
6
A derivative
N
6
-methyl-
N
6
-threonylcarbamoyladenosine (m
6
t
6
A37). We have isolated a mutant of
E. coli
that lacks the m
6
t
6
A37 in these two tRNA
GGU
Thr
species. These tRNA species in the mutant are likely to have t
6
A37 instead of m
6
t
6
A37. We show that the methyl group of m
6
t
6
A37 originates from
S
-adenosyl-
l
-methionine and that the gene (
tsaA
) which most likely encodes tRNA(m
6
t
6
A37)methyltransferase is located at min 4.6 on the
E. coli
chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA
GGU
Thr
to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the
tsaA1
mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m
6
t
6
A37-containing tRNA
GGU
Thr
species. We show that the
tsaA1
mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m
6
t
6
A37 in tRNA
GGU
Thr
slightly reduces the efficiency of this tRNA to read cognate codon ACC.
The
Escherichia coli mrsC
Gene Is Required for Cell Growth and mRNA Decay
Authors: Granger Laurie L. , O’Hara Eileen B. , Wang Rong-Fu , Meffen Frances V. , Armstrong Katherine , Yancey Stephanie D. , Babitzke Paul , Kushner Sidney R. 
Published: December 2019

ABSTRACT

We have identified a gene in
Escherichia coli
that is required for both the normal decay of mRNA and RNA synthesis. Originally designated
mrsC
(mRNA stability), the
mrsC505
mutation described here is, in fact, an allele of the
hflB/ftsH
locus (R.-F. Wang et al., J. Bacteriol. 180:1929–1938, 1998). Strains carrying the thermosensitive
mrsC505
allele stopped growing soon after the temperature was shifted to 44°C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44°C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the
mrsC505
single mutant. In an
rne-1 mrsC505
double mutant, the average half-life was 19.8 min. Inactivating
mrsC
significantly increased the half-lives of the
trxA
,
cat
,
secG
, and
kan
mRNAs, particularly in an
mrsC505 pnp-7 rnb-500 rne-1
multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of
mrsC505
multiple mutants at 44°C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.
Levels of the Vsr Endonuclease Do Not Regulate Stationary-Phase Reversion of a Lac
−
Frameshift Allele in
Escherichia coli
Authors: Foster Patricia L. , Rosche William A. 
Published: December 2019

ABSTRACT

Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac
−
frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.
Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in
Escherichia coli
Authors: Murphy Kenan C. 
Published: December 2019

ABSTRACT

Replacement of
Escherichia coli
’s RecBCD function with phage λ’s Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a
recBC sbcBC
or
recD
strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The Δ
recBCD
::P

lac

-red kan
replacement allele can be P1 transduced to other
E. coli
strains, making the hyper-Rec phenotype easily transferable.
Isolation and Characterization of
EPD1
, an Essential Gene for Pseudohyphal Growth of a Dimorphic Yeast,
Candida maltosa
Authors: Nakazawa Takanobu , Horiuchi Hiroyuki , Ohta Akinori , Takagi Masamichi 
Published: December 2019

ABSTRACT

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast,
Candida maltosa
(T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030–5036, 1997). To understand the mechanism of this transition, we screened the gene library of
C. maltosa
for sequences which could suppress this morphological change. As a result, we isolated the 5′ end of a new gene,
EPD1
(for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epd1p was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of
Saccharomyces cerevisiae
, and Phr1p and Phr2p of
Candida albicans
. The expression of
EPD1
was moderately regulated by environmental pH. A homozygous
EPD1
null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble β-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by
n
-hexadecane at pH 4 or pH 7, suggesting that
EPD1
is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the
GAL
promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length
EPD1
did not. This result and the initial isolation of the 5′ end of
EPD1
as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.
IS
6110
Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related
Mycobacterium tuberculosis
Strains
Authors: Fang Z. , Morrison N. , Watt B. , Doig C. , Forbes K. J. 
Published: December 2019

ABSTRACT

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the
Mycobacterium tuberculosis
genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS
6110
, and IS
1081
. These, especially IS
6110
and the DR locus, have been widely used as genetic markers to differentiate
M. tuberculosis
isolates and will continue to be so used, due to the conserved nature of the genome of
M. tuberculosis
. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of
M. tuberculosis
. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS
6110
transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among
M. tuberculosis
strains. Our results suggest that IS
6110
restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS
6110
, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS
6110
copy in the DR locus was noted, and the mechanisms of this loss are discussed.
Transformation of
Rickettsia prowazekii
to Rifampin Resistance
Authors: Rachek Lyudmila I. , Tucker Aimee M. , Winkler Herbert H. , Wood David O. 
Published: December 2019

ABSTRACT

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial
rpoB
gene that confers resistance to rifampin and used it to demonstrate allelic exchange in
R. prowazekii
. Comparison of the
rpoB
sequences from the rifampin-sensitive (Rif
s
) Madrid E strain and a rifampin-resistant (Rif
r
) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the
R. prowazekii
RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rif
s
Madrid E strain of
R. prowazekii
by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rif
r
region of rickettsial
rpoB
. This is the first successful demonstration of genetic transformation of
Rickettsia prowazekii
and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.