bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the
n
-Alkane-Assimilating Yeast
Yarrowia lipolytica
Authors: Wang Huijie J. , Le Dall Marie-Thérèse , Waché Yves , Laroche Céline , Belin Jean-Marc , Gaillardin Claude , Nicaud Jean-Marc 
Published: December 2019

ABSTRACT

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the
n
-alkane-assimilating yeast
Yarrowia lipolytica
, encoded by the
POX1
through
POX5
genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of
POX1
only, strains did not grow on fatty acids, whereas
POX4
alone elicited partial growth, and the growth of the double
POX2-POX3
-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the
POX
genotype.
Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation
Authors: Anthony Karen G. , Klimke William A. , Manchak Jan , Frost Laura S. 
Published: December 2019

ABSTRACT

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (
trbA
to
traS
) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F
+
cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.
The Dual-Specificity Protein Phosphatase Yvh1p Acts Upstream of the Protein Kinase Mck1p in Promoting Spore Development in
Saccharomyces cerevisiae
Authors: Beeser Alexander E. , Cooper Terrance G. 
Published: December 2019

ABSTRACT

Diploid
Saccharomyces cerevisiae
cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen. We show that
yvh1
disruption causes a defect in spore maturation; overexpression of
MCK1
or
IME1
suppresses this
yvh1
phenotype. While
mck1
mutations are epistatic to those in
yvh1
relative to spore maturation, overexpression of
MCK1
does not suppress the
yvh1
slow-vegetative-growth phenotype. We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth.
Translation of Two Nested Genes in Bacteriophage P4 Controls Immunity-Specific Transcription Termination
Authors: Forti Francesca , Polo Simona , Lane Kirk B. , Six Erich W. , Sironi Gianpiero , Dehò Gianni , Ghisotti Daniela 
Published: December 2019

ABSTRACT

In phage P4, transcription of the left operon may occur from both the constitutive P
LE
promoter and the regulated P
LL
promoter, about 400 nucleotides upstream of P
LE
. A strong Rho-dependent termination site,

t
imm

, is located downstream of both promoters. When P4 immunity is expressed, transcription starting at P
LE
is efficiently terminated at

t
imm

, whereas transcription from P
LL
is immunity insensitive and reads through

t
imm

. We report the identification of two nested genes,
kil
and
eta
, located in the P4 left operon. The P4
kil
gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the P
LE
promoter, and its expression is controlled by P4 immunity. Overexpression of
kil
causes cell killing. This gene is the terminal part of a longer open reading frame,
eta
, which begins upstream of P
LE
. The
eta
gene is expressed when transcription starts from the P
LL
promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both
kil
and
eta
overlap the

t
imm

site. By cloning
kil
upstream of a tRNA reporter gene, we demonstrated that translation of the
kil
region prevents premature transcription termination at

t
imm

. This suggests that P4 immunity might negatively control
kil
translation, thus enabling transcription termination at

t
imm

. Transcription starting from P
LL
proceeds through

t
imm

. Mutations that create nonsense codons in
eta
caused premature termination of transcription starting from P
LL
. Suppression of the nonsense mutation restored transcription readthrough at

t
imm

. Thus, termination of transcription from P
LL
is prevented by translation of
eta
.
Analysis of BvgA Activation of the Pertactin Gene Promoter in
Bordetella pertussis
Authors: Kinnear Susan M. , Boucher Philip E. , Stibitz Scott , Carbonetti Nicholas H. 
Published: December 2019

ABSTRACT

Bordetella pertussis
, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the
bvg
regulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated.
fha
is transcribed at 10 min following an inducing signal, while
ptx
is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that
prn
is transcribed at 1 h, an intermediate time compared to those of
fha
and
ptx
. We have identified
cis
-acting sequences necessary for expression of
prn
in
B. pertussis
by using
prn-lac
fusions containing alterations in the sequence upstream of the
prn
open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of
prn
to activate transcription from the promoter. Our genetic data indicate that the region critical for
prn
activation extends upstream to position −84. However, these data do not support the location of the
prn
transcription start site as previously published. We used a number of methods, including
prn-lac
fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify the
bvg
-dependent 5′ end of the
prn
transcript to the cytosine at −125 with respect to the published start site.
HWP1
Functions in the Morphological Development of
Candida albicans
Downstream of
EFG1
,
TUP1
, and
RBF1
Authors: Sharkey Laura L. , McNemar Mark D. , Saporito-Irwin Susan M. , Sypherd Paul S. , Fonzi William A. 
Published: December 2019

ABSTRACT

The morphological plasticity of
Candida albicans
is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent.
HWP1
, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of
HWP1
resulted in a medium-conditional defect in hyphal development.
HWP1
expression was blocked in a Δ
efg1
mutant, reduced in an Δ
rbf1
mutant, and derepressed in a Δ
tup1
mutant. Therefore,
HWP1
functions downstream of the developmental regulators
EFG1
,
TUP1
, and
RBF1
. Mutation of
CPH1
had no effect on
HWP1
expression, suggesting that the positive regulators of hyphal development,
CPH1
and
EFG1
, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene,
ECE1
, was similarly regulated by
EFG1
. Since
ECE1
is not required for hyphal development, the regulatory role of
EFG1
apparently extends beyond the control of cell shape determinants. However, expression of
ECE1
was not influenced by
TUP1
, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.
Identification of a Two-Component Signal Transduction System from
Corynebacterium diphtheriae
That Activates Gene Expression in Response to the Presence of Heme and Hemoglobin
Authors: Schmitt Michael P. 
Published: December 2019

ABSTRACT

Corynebacterium diphtheriae
, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The
C. diphtheriae hmuO
gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the
hmuO
gene in
C. diphtheriae
is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a
C. diphtheriae
chromosomal library were shown to activate transcription from the
hmuO
promoter in
Escherichia coli
. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the
C. diphtheriae
chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (
chrA
) and its cognate sensor kinase (
chrS
), was shown to strongly activate transcription from the
hmuO
promoter in a heme-dependent manner in
E. coli
. A mutation in
chrA
(
chrAD50N
), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the
hmuO
promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.
Cold Shock Response of
Bacillus subtilis
: Isoleucine-Dependent Switch in the Fatty Acid Branching Pattern for Membrane Adaptation to Low Temperatures
Authors: Klein Wolfgang , Weber Michael H. W. , Marahiel Mohamed A. 
Published: December 2019

ABSTRACT

Bacillus subtilis
has developed sophisticated mechanisms to withstand fluctuations in temperature. Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane’s function at all temperatures. The fatty acid profile of
B. subtilis
is characterized by a high content of branched fatty acids irrespective of the growth medium. Here, we report on the importance of isoleucine for
B. subtilis
to survive cold shock from 37 to 15°C. Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis. Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective. Fatty acid profiles of different
B. subtilis
wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock. There were no significant changes in the fatty acid saturation or acyl chain length. The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile. The switch to a fatty acid profile dominated by anteiso-C
15:0
and C
17:0
at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of
B. subtilis
in the cold.
Regulation of Cell Component Production by Growth Rate in the Group BStreptococcus
Authors: Ross Robin A. , Madoff Lawrence C. , Paoletti Lawrence C. 
Published: December 2019

ABSTRACTGroup BStreptococcus(GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-htd) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at atdof 1.4 h than at atdof 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at atdof 1.4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.
Two Different Types of Dehalogenases, LinA and LinB, Involved in γ-Hexachlorocyclohexane Degradation in
Sphingomonas paucimobilis
UT26 Are Localized in the Periplasmic Space without Molecular Processing
Authors: Nagata Yuji , Futamura Akiko , Miyauchi Keisuke , Takagi Masamichi 
Published: December 2019

ABSTRACT

γ-Hexachlorocyclohexane (γ-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a γ-HCH-degrading bacterium,
Sphingomonas paucimobilis
UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (γ-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of γ-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.
Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from
Pseudomonas aeruginosa
Authors: Ochs Martina M. , Lu Chung-Dar , Hancock Robert E. W. , Abdelal Ahmed T. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of
oprD
, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the
oprD
gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of
oprD
is linked to both carbon and nitrogen metabolism of
Pseudomonas aeruginosa
.
Regulation of Synthesis of the
Bacillus subtilis
Transition-Phase, Spore-Associated Antibacterial Protein TasA
Authors: Stöver Axel G. , Driks Adam 
Published: December 2019

ABSTRACT

Previously, we identified a novel component of
Bacillus subtilis
spores, called TasA, which possesses antibacterial activity. TasA is made early in spore formation, as cells enter stationary phase, and is secreted into the medium as well as deposited into the spore. Here, we show that
tasA
expression can occur as cells enter stationary phase even under sporulation-repressing conditions, indicating that TasA is a transition-phase protein.
tasA
and two upstream genes,
yqxM
and
sipW
, likely form an operon, transcription of which is under positive control by the transition-phase regulatory genes
spo0A
and
spo0H
and negative control by the transition phase regulatory gene
abrB
. These results are consistent with the suggestion that
yqxM
,
sipW
, and
tasA
constitute a transition phase operon that could play a protective role in a variety of cellular responses to stress during late-exponential-phase and early-stationary-phase growth in
B. subtilis
.
A Unique α-1,3 Mannosyltransferase of the Pathogenic Fungus
Cryptococcus neoformans
Authors: Doering Tamara L. 
Published: December 2019

ABSTRACT

The major virulence factor of the pathogenic fungus
Cryptococcus neoformans
is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear α-1,3-linked mannan substituted with
d
-xylose and
d
-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an α-1,3-dimannoside acceptor, forming a second α-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast
Saccharomyces cerevisiae
, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from
C. neoformans
membranes adds mannose in α-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the α-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.
Characterization of
Pseudomonas aeruginosa
Enoyl-Acyl Carrier Protein Reductase (FabI): a Target for the Antimicrobial Triclosan and Its Role in Acylated Homoserine Lactone Synthesis
Authors: Hoang Tung T. , Schweizer Herbert P. 
Published: December 2019

ABSTRACT

The
Pseudomonas aeruginosa fabI
structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that
fabI
is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal
fabI
gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The
fabI
-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the
fabI
mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly
95
-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified
P. aeruginosa N
-butyryl-
l
-homoserine lactone (C
4
-HSL) synthase, RhlI, could synthesize C
4
-HSL from crotonyl-ACP and
S
-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C
4
-HSL and
N
-(3-oxo)-dodecanoyl-
l
-homoserine lactones were reduced 50% in a
fabI
mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.