bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation and Organization of the
dcuA
and
dcuB
Genes, Encoding Homologous Anaerobic C
4
-Dicarboxylate Transporters in
Escherichia coli
Authors: Golby Paul , Kelly David J. , Guest John R. , Andrews Simon C. 
Published: December 2019

ABSTRACT

The
dcuA
and
dcuB
genes of
Escherichia coli
encode homologous proteins that appear to function as independent and mutually redundant C
4
-dicarboxylate transporters during anaerobiosis. The
dcuA
gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (
aspA
), while
dcuB
is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (
fumB
). To learn more about the respective roles of the
dcu
genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy
dcuA
- and
dcuB-lacZ
transcriptional fusions. The results show that
dcuA
is constitutively expressed whereas
dcuB
expression is highly regulated. The
dcuB
gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition,
dcuB
is strongly induced by C
4
-dicarboxylates, suggesting that
dcuB
is under the control of an uncharacterized C
4
-dicarboxylate-responsive gene regulator. Northern blotting confirmed that
dcuA
(and
aspA
) is expressed under both aerobic and anaerobic conditions and that
dcuB
(and
fumB
) is induced anaerobically. Major monocistronic transcripts were identified for
aspA
and
dcuA
, as well as a minor species possibly corresponding to an
aspA-dcuA
cotranscript. Five major transcripts were observed for
dcuB
and
fumB
: monocistronic transcripts for both
fumB
and
dcuB
; a
dcuB-fumB
cotranscript; and two transcripts, possibly corresponding to
dcuB-fumB
and
fumB
mRNA degradation products. Primer extension analysis revealed independent promoters for
aspA
,
dcuA
, and
dcuB
, but surprisingly no primer extension product could be detected for
fumB
. The expression of
dcuB
is entirely consistent with a primary role for DcuB in mediating C
4
-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.
Disruption of
PMR1
, Encoding a Ca
2+
-ATPase Homolog in
Yarrowia lipolytica
, Affects Secretion and Processing of Homologous and Heterologous Proteins
Authors: Sohn Young-Sun , Park Cheon Seok , Lee Sun-Bok , Ryu Dewey D. Y. 
Published: December 2019

ABSTRACT

The
Yarrowia lipolytica PMR1
gene (
YlPMR1
) is a
Saccharomyces cerevisiae PMR1
homolog which encodes a putative secretory pathway Ca
2+
-ATPase. In this study, we investigated the effects of a
YlPMR1
disruption on the processing and secretion of native and foreign proteins in
Y. lipolytica
and found variable responses by the
YlPMR1
-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the
YlPMR1
-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the
YlPMR1
-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca
2+
-ATPase genes on the secretion of rice α-amylase was also observed in the
Y. lipolytica
and
S. cerevisiae PMR1
-disrupted mutants. Unlike rice α-amylase, the secretion of
Trichoderma reesei
endoglucanase I (EGI) was not influenced by the
YlPMR1
disruption. However, the secreted EGI from the
YlPMR1
-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the
YlPMR1
-disrupted mutant. Our results indicate that the effects of the
YlPMR1
disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.
Superimposition of TyrR Protein-Mediated Regulation on Osmoresponsive Transcription of
Escherichia coli proU
In Vivo
Authors: Gowrishankar J. , Pittard A. J. 
Published: December 2019

ABSTRACT

Osmotic regulation of
proU
expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the ς
70
-controlled promoter of
proU
in
Escherichia coli
, we were able to demonstrate a superposed TyrR-mediated activation by
l
-phenylalanine (Phe), as well as repression by
l
-tyrosine, of
proU
expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of
proU
at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in
E. coli.
Identification and Characterization of the
Erwinia amylovora rpoS
Gene: RpoS Is Not Involved in Induction of Fireblight Disease Symptoms
Authors: Anderson M. , Pollitt C. E. , Roberts I. S. , Eastgate J. A. 
Published: December 2019

ABSTRACT

The
Erwinia amylovora rpoS
gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, an
E. amylovora rpoS
mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.
Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe
Authors: Madison-Antenucci Susan , Steege Deborah A. 
Published: December 2019

ABSTRACT

Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it. Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from
Escherichia coli
. The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs. Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V. The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation. The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, ∼75-fold. As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred.
Toxicity of Linoleic Acid Hydroperoxide to
Saccharomyces cerevisiae
: Involvement of a Respiration-Related Process for Maximal Sensitivity and Adaptive Response
Authors: Evans Marguerite V. , Turton Hal E. , Grant Chris M. , Dawes Ian W. 
Published: December 2019

ABSTRACT

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type
Saccharomyces cerevisiae
at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (
gsh1
and
gsh2
), glutathione reductase (
glr1
), respiratory competence ([
rho
0
] petite), and yAP-1p-mediated expression (
yap1
) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [
rho
0
] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the
yap1
and
glr1
mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 μg ml
−1
). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and
tert
-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37°C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.
Immunolocalization of Hsp60 in
Legionella pneumophila
Authors: Garduño Rafael A. , Faulkner Gary , Trevors Mary A. , Vats Neeraj , Hoffman Paul S. 
Published: December 2019

ABSTRACT

One of the most abundant proteins synthesized by
Legionella pneumophila
, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including
L. pneumophila
, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in
L. pneumophila
grown in vitro, or in vivo inside of HeLa cells. For comparative purposes
Escherichia coli
, expressing recombinant
L. pneumophila
Hsp60, was employed. In contrast to
E. coli
, where Hsp60 was localized exclusively in the cytoplasm, in
L. pneumophila
Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked
L. pneumophila
organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by
L. pneumophila
was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in
L. pneumophila
and further suggest both the existence of a novel secretion mechanism (not present in
E. coli
) and a potential role in pathogenesis.
Demonstration of the Key Role Played by the PufX Protein in the Functional and Structural Organization of Native and Hybrid Bacterial Photosynthetic Core Complexes
Authors: Fulcher Timothy K. , Beatty J. Thomas , Jones Michael R. 
Published: December 2019

ABSTRACT

The role of a component of the bacterial photosystem, the PufX protein, was examined by heterologous expression of the
pufX
gene from
Rhodobacter capsulatus
in a strain of
R. sphaeroides
that lacks the native
pufX
gene. The strain of
R. sphaeroides
containing the
R. capsulatus
PufX protein was capable of efficient transduction of light energy despite a low degree of sequence conservation between the PufX proteins from the two species. The organization of the hybrid reaction center/LH1 photosystem in strains of
R. sphaeroides
containing the
R. capsulatus
LH1 antenna complex was affected differently by the
R. sphaeroides
and
R. capsulatus
PufX proteins. We discuss the implications of our findings for the role of the PufX protein in organizing the bacterial photosystem for efficient transduction of light energy.
Expression and Characterization of (
R
)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by
Aeromonas caviae
Authors: Fukui Toshiaki , Shiomi Naofumi , Doi Yoshiharu 
Published: December 2019

ABSTRACT

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of
Aeromonas caviae
proved that ORF3 in the
pha
locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as

phaJ
Ac
. Escherichia coli

BL21(DE3) carrying

phaJ
Ac

under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of

phaJ
Ac

except for the initial Met residue. The enoyl-CoA hydratase encoded by

phaJ
Ac

exhibited (
R
)-specific hydration activity toward
trans
-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (
R
)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of

phaJ
Ac

is a channeling pathway for supplying (
R
)-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate-
co
-3-hydroxyhexanoate) biosynthesis in
A. caviae.
A New Transformation-Deficient Mutant of
Haemophilus influenzae
Rd with Normal DNA Uptake
Authors: Gwinn M. L. , Ramanathan R. , Smith H. O. , Tomb J.-F. 
Published: December 2019

ABSTRACT

Haemophilus influenzae
Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type
H. influenzae
and whose phage recombination is 8 times lower was isolated. The affected gene,
comM
, is induced during competence development in wild-type
H. influenzae
but not in RJ248.
Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog
Authors: Cunningham Adam F. , Spreadbury Claire L. 
Published: December 2019

ABSTRACT

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How
Mycobacterium tuberculosis
survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy.
Mycobacterium bovis
BCG and
M. tuberculosis
H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured
Mycobacterium smegmatis
. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in
M. bovis
BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of
M. tuberculosis.
Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in
Saccharomyces cerevisiae
Authors: Wang Xinping , Mann Craig J. , Bai Yinlin , Ni Li , Weiner Henry 
Published: December 2019

ABSTRACT

The full-length DNAs for two
Saccharomyces cerevisiae
aldehyde dehydrogenase (ALDH) genes were cloned and expressed in
Escherichia coli
. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K
+
ions. In contrast, the activity of ALDH5 increased in the presence of K
+
ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δ
ald1
and Δ
ald2
strains on ethanol was marginally slower than that of the parent strain. The Δ
ald1 Δald2
double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.
Biochemical and Genetic Characterization of
trans
-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading
Nocardioides
Strain
Authors: Iwabuchi Tokuro , Harayama Shigeaki 
Published: December 2019

ABSTRACT

trans
-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium,
Nocardioides
sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The

K
m

and
k
cat values of this enzyme for
trans
-2′-carboxybenzalpyruvate were 50 μM and 13 s
−1
, respectively.
trans
-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of
trans
-2′-hydroxybenzalpyruvate hydratase-aldolases from
Pseudomonas putida
PpG7 and
Pseudomonas
sp. strain C18.
Mismatch Repair in
Escherichia coli
Cells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ
Authors: Harris Reuben S. , Ross Kimberly J. , Lombardo Mary-Jane , Rosenberg Susan M. 
Published: December 2019

ABSTRACT

In vitro, the methyl-directed mismatch repair system of
Escherichia coli
requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.
Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of
Alcaligenes eutrophus
Authors: Massanz Christian , Schmidt Silke , Friedrich Bärbel 
Published: December 2019

ABSTRACT

The cytoplasmic, NAD-reducing hydrogenase (SH) of
Alcaligenes eutrophus
H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most of
hoxF
,
hoxU
, and
hoxY
led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of
hoxF
and
hoxU
, displayed H
2
-dependent dye-reducing activity, the monomeric form did not mediate the activation of H
2
, although nickel was incorporated into HoxH. Deletion of
hoxH
and
hoxY
led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.
Importance of RpoS and Dps in Survival of Exposure of Both Exponential- and Stationary-Phase
Escherichia coli
Cells to the Electrophile
N
-Ethylmaleimide
Authors: Ferguson G. P. , Creighton R. I. , Nikolaev Y. , Booth I. R. 
Published: December 2019

ABSTRACT

The mechanisms by which
Escherichia coli
cells survive exposure to the toxic electrophile
N
-ethylmaleimide (NEM) have been investigated. Stationary-phase
E. coli
cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing
E. coli
strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting
E. coli
cells against NEM.