bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation and Physiological Role of the
DAS1
Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast
Candida boidinii
Authors: Sakai Yasuyoshi , Nakagawa Tomoyuki , Shimase Masayuki , Kato Nobuo 
Published: December 2019

ABSTRACT

The physiological role of dihydroxyacetone synthase (DHAS) in
Candida boidinii
was evaluated at the molecular level. The
DAS1
gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that
DAS1
expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The
DAS1
gene was disrupted in one step in the host genome (
das1
Δ strain), and the growth of the
das1
Δ strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The
das1
Δ strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480–4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the
das1
Δ strain on media containing methylamine or choline as a nitrogen source) suggested that
DAS1
is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.
In Vivo Assay of Protein-Protein Interactions in Hin-Mediated DNA Inversion
Authors: Lee Sun Young , Lee Hee Jung , Lee Heejin , Kim Shukho , Cho Eun Hee , Lim Heon Man 
Published: December 2019

ABSTRACT

In order to form the catalytic nucleoprotein complex called the invertasome in the Hin-mediated DNA inversion reaction, interactions of the DNA-binding proteins Hin and Fis are required. Assays for these protein-protein interactions have been exploited with protein cross-linkers in vitro. In this study, an in vivo assay system that probes protein-protein interactions was developed. The formation of a DNA loop generated by protein interactions resulted in transcriptional repression of an artificially designed operon, which in turn increased the chance of survival of
Escherichia coli
host cells in a streptomycin-containing medium. Using this system, we were able to assay the Hin-Hin interaction that results in the pairing of the two recombination sites and protein interactions that result in the formation of the invertasome. This assay system also led us to find that an individual Hin dimer bound on a recombination site can form a stable complex with Fis bound on the recombinational enhancer; this finding has never been observed in in vitro studies. Possible pathways toward the formation of the invertasome are discussed based on the assay results for a previously reported Hin mutant.
The
yiaE
Gene, Located at 80.1 Minutes on the
Escherichia coli
Chromosome, Encodes a 2-Ketoaldonate Reductase
Authors: Yum Do-Young , Lee Bong-Yong , Hahm Dae-Hyum , Pan Jae-Gu 
Published: December 2019

ABSTRACT

An open reading frame located in the
bisC-cspA
intergenic region, or at 80.1 min on the
Escherichia coli
chromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the
E. coli
Genome Sequencing Project. We report here that the product of the gene (
yiaE
) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in
E. coli
, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH
2
-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2,5-diketo-
d
-gluconate to 5-keto-
d
-gluconate, 2-keto-
d
-gluconate (2KDG) to
d
-gluconate, 2-keto-
l
-gulonate to
l
-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from
Erwinia herbicola
. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in
E. coli. E. coli
W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in
E. coli
and the diminishment of produced 2KDG from
d
-gluconate in the cultivation of
E. coli
harboring a cloned gluconate dehydrogenase gene.
Anaerobic Expression ofEscherichia coliSuccinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth
Authors: Maklashina Elena , Berthold Deborah A. , Cecchini Gary 
Published: December 2019

ABSTRACTSuccinate-ubiquinone oxidoreductase (SQR) fromEscherichia coliis expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration byE. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of thefrdABCDoperon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth ofE. colibecause expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the PFRDpromoter of thefrdABCDoperon fused to thesdhCDABgenes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth ofE. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex,E. coliaccommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.
Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers
Authors: Higgs Penelope I. , Myers Paul S. , Postle Kathleen 
Published: December 2019

ABSTRACTThe cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.
The
Escherichia coli cmlA
Gene Encodes the Multidrug Efflux Pump Cmr/MdfA and Is Responsible for Isopropyl-β-
d
-Thiogalactopyranoside Exclusion and Spectinomycin Sensitivity
Authors: Bohn Chantal , Bouloc Philippe 
Published: December 2019

ABSTRACT

Expression of cloned genes from isopropyl-β-
d
-thiogalactopyranoside (IPTG)-regulated promoters is lowered when the
Escherichia coli
CmlA/Cmr/MdfA efflux pump is overexpressed, probably due to IPTG exclusion from the cytoplasm. The previously reported
cmlA1
mutation confers a similar phenotype.
cmlA1
contains an IS
30
insertion upstream of
cmr/mdfA
, which creates a putative promoter. CmlA overproduction also causes spectinomycin hypersensitivity.
Cytochrome P460 Genes from the Methanotroph
Methylococcus capsulatus
Bath
Authors: Bergmann David J. , Zahn James A. , Hooper Alan B. , DiSpirito Alan A. 
Published: December 2019

ABSTRACT

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium
Nitrosomonas europaea
(R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium
Methylococcus capsulatus
Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from
M. capsulatus
Bath that contains
cyp
, the gene encoding cytochrome P460.
cyp
is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (
cyp
) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from
M. capsulatus
Bath and
N. europaea
was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal,
c
-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using
cyp
indicated that a cytochrome P460 similar to that from
M. capsulatus
Bath may be present in the type II methanotrophs
Methylosinus trichosporium
OB3b and
Methylocystis parvus
OBBP but not in the type I methanotrophs
Methylobacter marinus
A45,
Methylomicrobium albus
BG8, and
Methylomonas
sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from
M. capsulatus
Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.
Novel Organization of the Genes for Phthalate Degradation from
Burkholderia cepacia
DBO1
Authors: Chang Hung-Kuang , Zylstra Gerben J. 
Published: December 2019

ABSTRACT

Burkholderia cepacia
DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (
ophA1
) is present by itself, while the genes for an inactive transporter (
ophD
) and 4,5-dihydroxyphthalate decarboxylase (
ophC
) are linked and the genes for phthalate dioxygenase oxygenase (
ophA2
) and
cis
-phthalate dihydrodiol dehydrogenase (
ophB
) are linked.
ophA1
and
ophDC
are adjacent to each other but are transcribed in opposite directions, while
ophA2B
is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for
ophD
are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.
The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid
Authors: Moll Gert N. , Konings Wil N. , Driessen Arnold J. M. 
Published: December 2019

ABSTRACT

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-
sn
-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant
k
of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.
Cytochrome
bd
Biosynthesis in
Bacillus subtilis
: Characterization of the
cydABCD
Operon
Authors: Winstedt Lena , Yoshida Ken-Ichi , Fujita Yasutaro , von Wachenfeldt Claes 
Published: December 2019

ABSTRACT

Under aerobic conditions
Bacillus subtilis
utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in
B. subtilis
: a
caa
3
-, an
aa
3
-, and a
bd
-type oxidase. We report here the cloning of the structural genes (
cydA
and
cydB
) encoding the cytochrome
bd
complex. Downstream of the structural genes,
cydC
and
cydD
are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of
cydA
or deletion of
cydABCD
resulted in the loss of spectral features associated with cytochrome
bd
. Gene disruption experiments and complementation analysis showed that the
cydC
and
cydD
gene products are required for the expression of a functional cytochrome
bd
complex. Disruption of the
cyd
genes had no apparent effect on the growth of cells in broth or defined media. The expression of the
cydABCD
operon was investigated by Northern blot analysis and by transcriptional and translational
cyd-lacZ
fusions. Northern blot analysis confirmed that
cydABCD
is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.
A Mutant of
Mycobacterium smegmatis
Defective in Dipeptide Transport
Authors: Bhatt Achal , Green Renee , Coles Roswell , Condon Michael , Connell Nancy D. 
Published: December 2019

ABSTRACT

A mutant of
Mycobacterium smegmatis
unable to use the dipeptide carnosine (β-alanyl-
l
-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on β-Ala and/or
l
-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium.
CcpB, a Novel Transcription Factor Implicated in Catabolite Repression in
Bacillus subtilis
Authors: Chauvaux Sylvie , Paulsen Ian T. , Saier Milton H. 
Published: December 2019

ABSTRACT

Recent work has shown that in
Bacillus subtilis
catabolite repression of several operons is mediated by a mechanism dependent on DNA-binding protein CcpA complexed to a seryl-phosphorylated derivative of HPr [HPr(Ser-P)], the small phosphocarrier protein of the phosphoenolpyruvate-sugar phosphotransferase system. In this study, it was found that a transposon insertional mutation resulted in the partial loss of gluconate (
gnt
) and xylose (
xyl
) operon catabolite repression by glucose, mannitol, and sucrose. The transposon insertion was localized to a gene, designated
ccpB
, encoding a protein 30% identical to CcpA, and relief from catabolite repression was shown to be due to the absence of CcpB rather than to the absence of a protein encoded by a downstream gene within the same operon. The relative intensities of CcpA- and CcpB-mediated catabolite repression depended on growth conditions. On solid media, and when cells were grown in liquid media with little agitation, CcpB and CcpA both proved to function in catabolite repression. However, when cells were grown in liquid media with much agitation, CcpA alone mediated catabolite repression. Like CcpA, CcpB appears to exert its catabolite-repressing effect by a mechanism dependent on the presence of HPr(Ser-P).
Bacillus subtilis
Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning
Authors: Sharpe Michaela E. , Hauser Philippe M. , Sharpe Robert G. , Errington Jeffery 
Published: December 2019

ABSTRACT

Fluorescence microscopic methods have been used to characterize the cell cycle of
Bacillus subtilis
at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of
Escherichia coli
, the period required by
B. subtilis
for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of
B. subtilis
differed from that of
E. coli
in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in
E. coli
, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in
B. subtilis
should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.
Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%
Authors: Teusink Bas , Diderich Jasper A. , Westerhoff Hans V. , van Dam Karel , Walsh Michael C. 
Published: December 2019

ABSTRACT

In
Saccharomyces cerevisiae
cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero
trans
-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.
Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in
Escherichia coli
Exposed to Hydrogen Peroxide
Authors: McCormick Michael L. , Buettner Garry R. , Britigan Bradley E. 
Published: December 2019

ABSTRACT

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O
2
·−
and H
2
O
2
. Previous work by others has shown that
Escherichia coli
mutants lacking SOD not only are more susceptible to DNA damage and killing by H
2
O
2
but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient
E. coli
cells are exposed to increased levels of hydroxyl radical (
·
OH) as a consequence of the reaction of H
2
O
2
with this increased iron pool. When the parental
E. coli
strain AB1157 was exposed to H
2
O
2
in the presence of an α-(4-pyridyl-1-oxide)-
N
-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–
·
CH(CH
3
)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating
·
OH production. When the isogenic
E. coli
mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H
2
O
2
in a similar manner, the magnitude of
·
OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of
·
OH spin trapped. Exogenous SOD failed to inhibit
·
OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in
E. coli
SOD mutants and (ii) support the hypothesis that a resulting increase in
·
OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H
2
O
2
-mediated killing seen in these mutants lacking SOD.
Purification of
Leuconostoc mesenteroides
Citrate Lyase and Cloning and Characterization of the
citCDEFG
Gene Cluster
Authors: Bekal Sadjia , Van Beeumen Jozef , Samyn Bart , Garmyn Dominique , Henini Samia , Diviès Charles , Prévost Hervé 
Published: December 2019

ABSTRACT

A citrate lyase (EC
4.1.3.6
) was purified 25-fold from
Leuconostoc mesenteroides
and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from
Leuconostoc mesenteroides
subsp.
cremoris
. This fragment was used as a probe for screening a
Leuconostoc
genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of
L. mesenteroides
is organized in three open reading frames,
citD
,
citE
, and
citF
, encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC
4.1.3.34
), and α (citrate:acetyl-ACP transferase; EC
2.8.3.10
). The gene (
citC
) encoding the citrate lyase ligase (EC
6.2.1.22
) was localized in the region upstream of
citD
. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of
Klebsiella pneumoniae
and
Haemophilus influenzae
. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the
citCDEFG
gene cluster in
Escherichia coli
led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in
E. coli
is not complete or not linked to the protein.