bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of
Escherichia coli
FtsZ
Authors: Ma Xiaolan , Margolin William 
Published: December 2019

ABSTRACT

In
Escherichia coli
, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of
E. coli
FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal
ftsZ
mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an
ftsZ
chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal
ftsZ
mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.
Cloning and Characterization of the
Pseudomonas fluorescens
ATP-Binding Cassette Exporter, HasDEF, for the Heme Acquisition Protein HasA
Authors: Idei Akiko , Kawai Eri , Akatsuka Hiroyuki , Omori Kenji 
Published: December 2019

ABSTRACT

Two ATP-binding cassette (ABC) exporters are present in
Pseudomonas fluorescens
no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The
hasDEF
genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the
Serratia marcescens
ABC exporter Lip system.
P. fluorescens
HasA showed sequence identity of 40 to 49% with HasA proteins from
Pseudomonas aeruginosa
and
Serratia marcescens
. The
P. fluorescens
Has exporter secreted HasA proteins from
P. fluorescens
and
P. aeruginosa
but not
S. marcescens
HasA in
Escherichia coli
, whereas the Has exporter from
S. marcescens
allowed secretion of all three HasA proteins. The
P. fluorescens
HasDEF system also promoted the secretion of the lipase and alkaline protease of
P. fluorescens
. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both
P. fluorescens
and
S. marcescens
sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the
P. fluorescens
Has exporter.
Separate Roles of
Escherichia coli
Replication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA
Authors: Miller Christine , Cohen Stanley N. 
Published: December 2019

ABSTRACT

We report here that the
Escherichia coli
replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive
dnaB
mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of
dnaA
mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.
Conditional Sigma Factor Expression, Using the Inducible Acetamidase Promoter, Reveals that the
Mycobacterium tuberculosis sigF
Gene Modulates Expression of the 16-Kilodalton Alpha-Crystallin Homologue
Authors: Manabe Yukari C. , Chen Jong Min , Ko Chiew G. , Chen Ping , Bishai William R. 
Published: December 2019

ABSTRACT

A chemically inducible acetamidase promoter-
sigF
fusion gene was integrated into the chromosome of
Mycobacterium bovis
BCG. Two-dimensional protein gel analysis permitted the identification of a number of protein spots whose expression was SigF related. One spot upregulated by inappropriate induction of
sigF
expression corresponded to the 16-kDa antigen alpha-crystallin.
A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in
Hansenula polymorpha
DL-1
Authors: Sohn Jung-Hoon , Choi Eui-Sung , Kang Hyun Ah , Rhee Joon-Shick , Rhee Sang-Ki 
Published: December 2019

ABSTRACT

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of
Hansenula polymorpha
DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of
H. polymorpha
. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of
H. polymorpha
with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease
Bal
31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.
Two Roles for the Leucine-Responsive Regulatory Protein in Expression of the Alanine Catabolic Operon (
dadAB
) in
Klebsiella aerogenes
Authors: Janes Brian K. , Bender Robert A. 
Published: December 2019

ABSTRACT

The
lrp
gene, which codes for the leucine-responsive regulatory protein (Lrp), was cloned from
Klebsiella aerogenes
W70. The DNA sequence was determined, and the clone was used to create a disruption of the
lrp
gene. The lack of functional Lrp led to an increased expression of the alanine catabolic operon (
dad
) in the absence of the inducer
l
-alanine but also to a decreased expression of the operon in the presence of
l
-alanine. Thus, Lrp is both a repressor and activator of
dad
expression. Lrp is also necessary for glutamate synthase formation but not for the formation of two other enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate dehydrogenase and histidase.
The bZip Transcription Factor Cap1p Is Involved in Multidrug Resistance and Oxidative Stress Response in
Candida albicans
Authors: Alarco Anne-Marie , Raymond Martine 
Published: December 2019

ABSTRACT

Candida albicans
is an opportunistic pathogenic yeast which frequently develops resistance to the antifungal agent fluconazole (FCZ) in patients undergoing long-term therapy. FCZ-resistant strains often display a reduced intracellular FCZ accumulation which correlates with the overexpression of the ATP-binding cassette transporters
CDR1
and
CDR2
or the major facilitator (MF)
MDR1
. We have recently cloned a
C. albicans
gene, named
CAP1
, which codes for a bZip transcription factor of the AP-1 family homologous to the Yap1 protein involved in multidrug resistance and response to oxidative stress in
Saccharomyces cerevisiae. CAP1
was found to confer FCZ resistance in
S. cerevisiae
by transcriptionally activating
FLR1
, a gene coding for an MF homologous to the
C. albicans MDR1
gene product (A.-M. Alarco, I. Balan, D. Talibi, N. Mainville, and M. Raymond, J. Biol. Chem. 272:19304–19313, 1997). To study the role of
CAP1
in
C. albicans
, we constructed a CAI4-derived mutant strain carrying a homozygous deletion of the
CAP1
gene (CJD21). We found that deletion of
CAP1
did not affect the susceptibility of CJD21 cells to FCZ, cerulenin, brefeldin A, and diamide but caused hypersensitivity to cadmium, 4-nitroquinoline
N
-oxide, 1,10-phenanthroline, and hydrogen peroxide, an effect which was reverted by reintroduction of the
CAP1
gene in these cells. Introduction of a hyperactive truncated allele of
CAP1
(
CAP1-TR
) in CJD21 resulted in resistance of the cells to all of the above compounds except hydrogen peroxide. The hyperresistant phenotype displayed by the CJD21
CAP1-TR
transformants was found to correlate with the overexpression of a number of potential
CAP1
transcriptional targets such as
MDR1
,
CaYCF1
,
CaGLR1
, and
CaTRR1
. Taken together, our results demonstrate that
CAP1
is involved in multidrug resistance and oxidative stress response in
C. albicans
. Finally, disruption of
CAP1
in strain FR2, selected in vitro for FCZ resistance and constitutively overexpressing
MDR1
, did not suppress but rather increased the levels of
MDR1
expression, demonstrating that
CAP1
acts as a negative transcriptional regulator of the
MDR1
gene in FR2 and is not responsible for
MDR1
overexpression in this strain.
Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from
Escherichia coli
Authors: Sheppard Christal A. , Trimmer Elizabeth E. , Matthews Rowena G. 
Published: December 2019

ABSTRACT

A K-12 strain of
Escherichia coli
that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the

K
m

for NADH is 13 μM, the

K
m

for CH
2
-H
4
folate is 0.8 μM, and the turnover number under
V
max
conditions estimated for the reaction is 1,800 mol of NADH oxidized min
−1
(mol of enzyme-bound FAD)
−1
. NADPH also serves as a reductant, but exhibits a much higher

K
m

. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from
E. coli
differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen
Clostridium formicoaceticum
and more closely resemble those of the NADH-dependent enzyme from
Peptostreptococcus productus
and the NADPH-dependent enzymes from eukaryotes.
Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium
Pseudomonas syringae
pv. glycinea
Authors: Rohde Bettina H. , Schmid Roland , Ullrich Matthias S. 
Published: December 2019

ABSTRACT

The phytopathogenic bacterium
Pseudomonas syringae
pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of
Pseudomonas putida
M10. The respective
P. syringae
gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated
ncr
for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding
morB
gene of
P. putida
M10. The
ncr
gene was overexpressed in
Escherichia coli
, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the
ncr
gene in different
Pseudomonas
species and various strains of
P. syringae
was analyzed by PCR and Southern blot hybridization. The results indicated that the
ncr
gene is widespread among
P. syringae
pv. glycinea strains but not in other pathovars of
P. syringae
or in any of the other
Pseudomonas
strains tested.
Biochemical and Genetic Evidence for
meta
-Ring Cleavage of 2,4,5-Trihydroxytoluene in
Burkholderia
sp. Strain DNT
Authors: Haigler Billy E. , Johnson Glenn R. , Suen Wen-Chen , Spain Jim C. 
Published: December 2019

ABSTRACT

2,4,5-Trihydroxytoluene (THT) oxygenase from
Burkholderia
sp. strain DNT catalyzes the conversion of THT to an unstable ring fission product. Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction. The THT oxygenase gene (
dntD
) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831–1837, 1993). Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa. The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively. The results suggested that the native protein consists of two identical subunits. The colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site. The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (
meta
-cleavage dioxygenases). In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol. The chemical analysis of the THT ring cleavage product showed that the product was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.
Mycoplasma pneumoniae
Protein P30 Is Required for Cytadherence and Associated with Proper Cell Development
Authors: Romero-Arroyo Cynthia E. , Jordan Jarrat , Peacock Susan J. , Willby Melisa J. , Farmer Mark A. , Krause Duncan C. 
Published: December 2019

ABSTRACT

The attachment organelle of
Mycoplasma pneumoniae
is a polar, tapered cell extension containing an intracytoplasmic, electron-dense core. This terminal structure is the leading end in gliding motility, and its duplication is thought to precede cell division, raising the possibility that mutations affecting cytadherence also confer a defect in motility or cell development. Mycoplasma surface protein P30 is associated with the attachment organelle, and P30 mutants II-3 and II-7 do not cytadhere. In this study, the recombinant wild-type but not the mutant II-3
p30
allele restored cytadherence when transformed into P30 mutants by recombinant transposon delivery. The mutations associated with loss of P30 in mutant II-3 and reacquisition of P30 in cytadhering revertants thereof were identified by nucleotide sequencing of the
p30
gene. Morphological abnormalities that included ovoid or multilobed cells having a poorly defined tip structure were associated with loss of P30. Digital image analysis confirmed quantitatively the morphological differences noted visually. Transformation of the P30 mutants with the wild-type
p30
allele restored a normal morphology, as determined both visually and by digital image analysis, suggesting that P30 plays a role in mycoplasma cell development. Finally, the P30 mutants localized the adhesin protein P1 to the terminal organelle, indicating that P30 is not involved in P1 trafficking but may be required for its receptor-binding function.
Analysis of Phosphorylated Sphingolipid Long-Chain Bases Reveals Potential Roles in Heat Stress and Growth Control in
Saccharomyces
Authors: Skrzypek Marek S. , Nagiec M. Marek , Lester Robert L. , Dickson Robert C. 
Published: December 2019

ABSTRACT

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that
Saccharomyces cerevisiae
cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in
S. cerevisiae
cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25°C, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37°C causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of
DPL1
and
LCB3
, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44°C heat stress 10-fold better than parental cells. These and other data for
dpl1
or
lcb3
single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.
Conditional Stability of the HemA Protein (Glutamyl-tRNA Reductase) Regulates Heme Biosynthesis in
Salmonella typhimurium
Authors: Wang Liying , Elliott Meenal , Elliott Thomas 
Published: December 2019

ABSTRACT

In many bacteria, including the enteric species
Salmonella typhimurium
and
Escherichia coli
, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the
hemA
gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of
S. typhimurium
, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for
E. coli
. The near absence of regulation of
hemA-lac
operon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is ≃20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a
lon clpP
double mutant of
E. coli
. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the
lon clpP
double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.
Impact of either Elevated or Decreased Levels of Cytochrome
bd
Expression on
Shigella flexneri
Virulence
Authors: Way Sing Sing , Sallustio Sandra , Magliozzo Richard S. , Goldberg Marcia B. 
Published: December 2019

ABSTRACT

Shigella
spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that
S. flexneri
cytochrome
bd
expression is necessary for normal intracellular survival and virulence. Cytochrome
bd
is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain.
S. flexneri
mutants that contain a disruption in the
cydC
locus, which leads to defective cytochrome
bd
expression, or in the riboflavin (
ribE
) or ubiquinol-8 (
ubiH
) biosynthetic pathway, which leads to elevated cytochrome
bd
expression, were evaluated in intracellular survival and virulence assays. The
cydC
mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The
ribE
and
ubiH
mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome
bd
is required for
S. flexneri
intracellular survival and virulence.