bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of
Saccharomyces cerevisiae
Authors: Small W. Curtis , McAlister-Henn Lee 
Published: December 2019

ABSTRACT

The reoxidation of NADH generated in reactions within the mitochondrial matrix of
Saccharomyces cerevisiae
is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated
NDH1
results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated
NDH2
has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of
NDH1
and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.
Insertion Mutation of the Form I
cbbL
Gene Encoding Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in
Thiobacillus neapolitanus
Results in Expression of Form II RuBisCO, Loss of Carboxysomes, and an Increased CO
2
Requirement for Growth
Authors: Baker Stefanie H. , Jin Songmu , Aldrich Henry C. , Howard Gary T. , Shively Jessup M. 
Published: December 2019

ABSTRACT

It has been previously established that
Thiobacillus neapolitanus
fixes CO
2
by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme,
cbbL
and
cbbS
, are part of a putative carboxysome operon. In the present study,
cbbL
and
cbbS
were cloned and sequenced. Analysis of RNA showed that
cbbL
and
cbbS
are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into
cbbL
resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO
2
, but requires a CO
2
supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO
2
by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.
Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from
Sphingomonas
sp. Strain RW5
Authors: Werwath Jörn , Arfmann Hans-Adolf , Pieper Dietmar H. , Timmis Kenneth N. , Wittich Rolf-Michael 
Published: December 2019

ABSTRACT

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC
1.13.11.4
),
gtdA
, from
Sphingomonas
sp. strain RW5 was cloned and sequenced. The
gtdA
gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the
gtdA
gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (
phdI
) of the phenanthrene degradation in
Nocardioides
sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in
E. coli
. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (

K
m

) and specificity constants (
K
cat
/
K
m
) of the enzyme were determined to be 15 μM and 511 s
−1
M
−1
× 10
4
for gentisate and 754 μM and 20 s
−1
M
−1
× 10
4
for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of
gtdA
. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.
Localization and Function of Early Cell Division Proteins in Filamentous
Escherichia coli
Cells Lacking Phosphatidylethanolamine
Authors: Mileykovskaya Eugenia , Sun Qin , Margolin William , Dowhan William 
Published: December 2019

ABSTRACT

Escherichia coli
cells that contain the
pss-93
null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in
pss-93
filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.
The
Escherichia coli
Starvation Gene
cstC
Is Involved in Amino Acid Catabolism
Authors: Fraley C. D. , Kim J. H. , McCann M. P. , Matin A. 
Published: December 2019

ABSTRACT

Escherichia coli
strains mutant in the starvation gene
cstC
grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources. They are also compromised in starvation survival, where amino acid catabolism is important. The
cstC
gene encodes a 406-amino-acid protein that closely resembles the
E. coli
ArgD protein, which is involved in arginine biosynthesis. We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway. The
cstC
upstream region contains several regulatory consensus sequences. Both ς
S
and ς
54
promoters are probably involved in
cstC
transcription and appear to compete with each other, presumably to match
cstC
expression to the cellular amino acid catabolic needs.
Cloning and DNA Sequencing of the Genes Encoding
Clostridium josui
Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome
Authors: Kakiuchi Motohide , Isui Ayako , Suzuki Katsuhisa , Fujino Tsuchiyoshi , Fujino Emi , Kimura Tetsuya , Karita Shuichi , Sakka Kazuo , Ohmiya Kunio 
Published: December 2019

ABSTRACT

The
Clostridium josui cipA
and
celD
genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of
C. josui
CipA is similar to those of the scaffolding proteins reported so far, such as
Clostridium thermocellum
CipA,
Clostridium cellulovorans
CbpA, and
Clostridium cellulolyticum
CipC, but
C. josui
CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the
C. josui
cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.
Mechanism for Biotransformation of Nonylphenol Polyethoxylates to Xenoestrogens in
Pseudomonas putida
Authors: John Dominic M. , White Graham F. 
Published: December 2019

ABSTRACT

A strain of
Pseudomonas putida
isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEO

x

, where
x
is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEO
av6
, NPEO
av9
, and NPEO
av20
(mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO
2
). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEO

x

. Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEO

x

mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEO

x

-degrading activity was inducible by substrate, and cell extracts of NPEO
av9
-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO
8
), producing sequentially, under either aerobic or anaerobic conditions, AEO
7
, AEO
6
, AEO
5
, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEO
av9
or AEO
8
under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEO

x

and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEO

x
−1

, which then undergoes further cycles of the same reaction until
x
= 2.
Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in
Lactobacillus johnsonii
100-100
Authors: Elkins Christopher A. , Savage Dwayne C. 
Published: December 2019

ABSTRACT

Cytosolic extracts of
Lactobacillus johnsonii
100-100 (previously reported as
Lactobacillus
sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in
Escherichia coli
. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily.
E. coli
DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [
14
C]taurocholic acid that were increased as much as threefold over control levels. [
14
C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.
Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from
Pseudomonas
sp. Strain HR199 and Molecular Characterization of the Gene
Authors: Achterholt Sandra , Priefert Horst , Steinbüchel Alexander 
Published: December 2019

ABSTRACT

The coniferyl aldehyde dehydrogenase (CALDH) of
Pseudomonas
sp. strain HR199 (DSM7063), which catalyzes the NAD
+
-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α
2
structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The

K
m

values for coniferyl aldehyde and NAD
+
were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH
2
-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (
calB
). The corresponding nucleotide sequence was localized on a 9.4-kbp
Eco
RI fragment (E94), which was subcloned from a
Pseudomonas
sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD
+
-dependent aldehyde dehydrogenases from different sources.
Farnesol-Induced Generation of Reactive Oxygen Species via Indirect Inhibition of the Mitochondrial Electron Transport Chain in the Yeast
Saccharomyces cerevisiae
Authors: Machida Kiyotaka , Tanaka Toshio , Fujita Ken-ichi , Taniguchi Makoto 
Published: December 2019

ABSTRACT

The mechanism of farnesol (FOH)-induced growth inhibition of
Saccharomyces cerevisiae
was studied in terms of its promotive effect on generation of reactive oxygen species (ROS). The level of ROS generation in FOH-treated cells increased five- to eightfold upon the initial 30-min incubation, while cells treated with other isoprenoid compounds, like geraniol, geranylgeraniol, and squalene, showed no ROS-generating response. The dependence of FOH-induced growth inhibition on such an oxidative stress was confirmed by the protection against such growth inhibition in the presence of an antioxidant such as α-tocopherol, probucol, or
N
-acetylcysteine. FOH could accelerate ROS generation only in cells of the wild-type grande strain, not in those of the respiration-deficient petite mutant ([
rho
0
]), which illustrates the role of the mitochondrial electron transport chain as its origin. Among the respiratory chain inhibitors, ROS generation could be effectively eliminated with myxothiazol, which inhibits oxidation of ubiquinol to the ubisemiquinone radical by the Rieske iron-sulfur center of complex III, but not with antimycin A, an inhibitor of electron transport that is functional in further oxidation of the ubisemiquinone radical to ubiquinone in the Q cycle of complex III. Cellular oxygen consumption was inhibited immediately upon extracellular addition of FOH, whereas FOH and its possible metabolites failed to directly inhibit any oxidase activities detected with the isolated mitochondrial preparation. A protein kinase C (PKC)-dependent mechanism was suggested to exist in the inhibition of mitochondrial electron transport since FOH-induced ROS generation could be effectively eliminated with a membrane-permeable diacylglycerol analog which can activate PKC. The present study supports the idea that FOH inhibits the ability of the electron transport chain to accelerate ROS production via interference with a phosphatidylinositol type of signal.
Transcription Analysis of the Prolate-Headed Lactococcal Bacteriophage c2
Authors: Lubbers Mark W. , Schofield Karin , Waterfield Nick R. , Polzin Kayla M. 
Published: December 2019

ABSTRACT
A detailed transcription map of the prolate-headed lactococcal phage c2 has been constructed. Transcription of about one-third of the genome, encoding 22 open reading frames, began within the first 2 min of infection and produced at least 12 overlapping transcripts that persisted until lysis occurred at 30 min after initiation of infection. The remaining two-thirds of the genome, encoding 17 open reading frames, was divergently transcribed, beginning between 4 and 6 min after initiation of infection, and resulted in at least 18 overlapping transcripts that persisted until lysis. Five very strong, simultaneously active, and probably unregulated early promoters and a single positively regulated late promoter were identified. The late promoter had an extended −10 sequence, had a significant basal level of activity in the uninduced state, and was induced to high activity by a phage gene product. The complex overlapping pattern of transcripts resulted from the action of the multiple early promoters, inefficient termination of transcription, and (possibly) processing of a late precursor transcript(s). Phage proteins were not required for these processes, and the host RNA polymerase was probably used for both early and late transcription.
Identification of the Gene Encoding the Alternative Sigma Factor ς
B
from
Listeria monocytogenes
and Its Role in Osmotolerance
Authors: Becker Lynne A. , Çetin Mehmet Sevket , Hutkins Robert W. , Benson Andrew K. 
Published: December 2019

ABSTRACT

Listeria monocytogenes
is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by
L. monocytogenes
to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the
sigB
gene and several
rsb
genes from
L. monocytogenes
, encoding homologs of the alternative sigma factor ς
B
and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium
Bacillus subtilis
. The
L. monocytogenes
and
B. subtilis sigB
and
rsb
genes are similar in sequence and physical organization; however, we observed that the activity of ς
B
in
L. monocytogenes
was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ς
B
in coordinating osmotic responses in
L. monocytogenes
. A null mutation in the
sigB
gene led to substantial defects in the ability of
L. monocytogenes
to use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ς
B
reduced the ability of the cells to accumulate betaine. Thus, ς
B
coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of
L. monocytogenes
under conditions of high osmotic strength.
Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in
Escherichia coli
Authors: Subrahmanyam Satyanarayana , Cronan John E. 
Published: December 2019

ABSTRACT

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three
Escherichia coli
isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to
E. coli
, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.
Physiological Response of
Lactobacillus plantarum
to Salt and Nonelectrolyte Stress
Authors: Glaasker Erwin , Tjan Frans S. B. , Ter Steeg Pieter F. , Konings Wil N. , Poolman Bert 
Published: December 2019

ABSTRACT

In this report, we compared the effects on the growth of
Lactobacillus plantarum
of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K
+
(or Na
+
) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.
Salmonella typhimurium nit
Is
nadE
: Defective Nitrogen Utilization and Ammonia-Dependent NAD Synthetase
Authors: Schneider Barbara L. , Reitzer Lawrence J. 
Published: December 2019

ABSTRACT

S. typhimurium nit
mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes. Complementation, enzyme assays, and genetic mapping show that
nit
is
nadE
. We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype.