bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppression of an Hsp70 Mutant Phenotype in
Saccharomyces cerevisiae
through Loss of Function of the Chromatin Component Sin1p/Spt2p
Authors: Baxter Bonnie K. , Craig Elizabeth A. 
Published: December 2019

ABSTRACT

The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast
Saccharomyces cerevisiae
has four members, encoded by
SSA1
,
SSA2
,
SSA3
, and
SSA4
. Deletion of the two constitutively expressed genes,
SSA1
and
SSA2
, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of
ssa1 ssa2
strains,
EXA1-1
, is a loss-of-function mutation in
SIN1/SPT2
, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression of
SSA3
in the
ssa1 ssa2
mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for
SSA
genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of
SSA3
in the
ssa1 ssa2
mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of
SSA3
which is modulated by Sin1p. The
EXA1-1
suppressor mutation thus improves the growth of
ssa1 ssa2
strains by selectively increasing HSF-mediated expression of
SSA3
.
Analysis of Outgrowth of
Bacillus subtilis
Spores Lacking Penicillin-Binding Protein 2a
Authors: Murray Thomas , Popham David L. , Pearson Christine B. , Hand Arthur R. , Setlow Peter 
Published: December 2019

ABSTRACT

The loss of
Bacillus subtilis
penicillin-binding protein (PBP) 2a, encoded by
pbpA
, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in
pbpA
spore outgrowth have shown that (i) outgrowing
pbpA
spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of
pbpA
spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and
pbpA
spores once cell elongation had been initiated; (iii) outgrowth and elongation of
pbpA
spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.
Studies of Regulation of Expression of the Propionate (
prpBCDE
) Operon Provide Insights into How
Salmonella typhimurium
LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways
Authors: Tsang Allen W. , Horswill Alexander R. , Escalante-Semerena Jorge C. 
Published: December 2019

ABSTRACT

Expression of the
prpBCDE
operon of
Salmonella typhimurium
LT2 required (i) the synthesis of propionyl-coenzyme A (CoA) by the PrpE protein or the acetyl-CoA-synthesizing systems of the cell and (ii) the synthesis of 2-methylcitrate from propionyl-CoA and oxaloacetate by the PrpC protein. We propose that either 2-methylcitrate or a derivative of it signals the presence of propionate in the environment. This as yet unidentified signal is thought to serve as a coregulator of the activity of PrpR, the member of the sigma-54 family of transcriptional activators needed for activation of
prpBCDE
transcription. The CobB protein was also required for expression of the
prpBCDE
operon, but its role is less well understood. Expression of the
prpBCDE
operon in
cobB
mutants was restored to wild-type levels upon induction of the propanediol utilization (
pdu
) operon by 1,2-propanediol. This effect did not require catabolism of 1,2-propanediol, suggesting that a Pdu protein, not a catabolite of 1,2-propanediol, was responsible for the observed effect. We explain the existence of these redundant functions in terms of metabolic pathway integration. In an environment with 1,2-propanediol as the sole carbon and energy source, expression of the
prpBCDE
operon is ensured by the Pdu protein that has CobB-like activity. Since synthesis of this Pdu protein depends on the availability of 1,2-propanediol, the cell solves the problem faced in an environment devoid of 1,2-propanediol where propionate is the sole carbon and energy source by having
cobB
located outside of the
pdu
operon and its expression independent of 1,2-propanediol. At present, it is unclear how the CobB and Pdu proteins affect
prpBCDE
expression.
A Putative Multisubunit Na
+
/H
+
Antiporter from
Staphylococcus aureus
Authors: Hiramatsu Toshiaki , Kodama Kazuyo , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa 
Published: December 2019

ABSTRACT

We cloned several genes encoding an Na
+
/H
+
antiporter of
Staphylococcus aureus
from chromosomal DNA by using an
Escherichia coli
mutant, lacking all of the major Na
+
/H
+
antiporters, as the host.
E. coli
cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na
+
/H
+
antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na
+
/H
+
antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na
+
/H
+
antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na
+
/H
+
antiporter. We observed a large Na
+
extrusion activity, driven by respiration in
E. coli
cells harboring the plasmid carrying the genes. The Na
+
extrusion was sensitive to an H
+
conductor, supporting the idea that the system is not a respiratory Na
+
pump but an Na
+
/H
+
antiporter. Introduction of the plasmid into
E. coli
mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.
Expression of the
Bacillus subtilis acsA
Gene: Position and Sequence Context Affect
cre
-Mediated Carbon Catabolite Repression
Authors: Zalieckas Jill M. , Wray Lewis V. , Fisher Susan H. 
Published: December 2019

ABSTRACT

In
Bacillus subtilis
, carbon catabolite repression (CCR) of many genes is mediated at
cis
-acting carbon repression elements (
cre
) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor (
mfd
) partially relieve CCR at
cre
sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at
cre
sites which act as transcriptional roadblocks. Although the
acsA cre
is centered 44.5 bp downstream of the
acsA
transcriptional start site, CCR of
acsA
expression is not affected by an
mfd
mutation. When the
acsA cre
is centered 161.5 bp downstream of the transcriptional start site for the unregulated
tms
promoter, CCR is partially relieved by the
mfd
mutation. Since CCR mediated at an
acsA cre
centered 44.5 bp downstream of the
tms
start site is not affected by the
mfd
mutation, the inability of Mfd to modulate CCR of
acsA
expression most likely results from the location of the
acsA cre
. Higher levels of CCR were found to occur at
cre
sites flanked by A+T-rich sequences than at
cre
sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp
cre
consensus sequence participate in the formation of the CcpA catabolite repression complex at
cre
sites. Examination of CCR of
acsA
expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.
Functional Similarities between the
Listeria monocytogenes
Virulence Regulator PrfA and Cyclic AMP Receptor Protein: the PrfA* (Gly145Ser) Mutation Increases Binding Affinity for Target DNA
Authors: Vega Yolanda , Dickneite Carmen , Ripio María-Teresa , Böckmann Regine , González-Zorn Bruno , Novella Susana , Domínguez-Bernal Gustavo , Goebel Werner , Vázquez-Boland José A. 
Published: December 2019

ABSTRACT

Most
Listeria monocytogenes
virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene,
prfA
, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent
plcA
promoter. We have recently characterized
prfA
* mutants from
L. monocytogenes
which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress
prfA
and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a
prfA
* strain than in the wild type. Thus, the phenotype of
prfA
* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the
plcA
promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.
The Modified β-Ketoadipate Pathway in
Rhodococcus rhodochrous
N75: Enzymology of 3-Methylmuconolactone Metabolism
Authors: Cha Chang-Jun , Cain Ronald B. , Bruce Neil C. 
Published: December 2019

ABSTRACT

Rhodococcus rhodochrous
N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of
R. rhodochrous
N75 on
p
-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native
M
r
of 128,000, and a subunit
M
r
of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.
New Small, Acid-Soluble Proteins Unique to Spores of
Bacillus subtilis
: Identification of the Coding Genes and Regulation and Function of Two of These Genes
Authors: Bagyan Irina , Setlow Barbara , Setlow Peter 
Published: December 2019

ABSTRACT

Eleven small, acid-soluble proteins (SASP) which are present in spores but not in growing cells of
Bacillus subtilis
were identified by sequence analysis of proteins separated by acrylamide gel electrophoresis of acid extracts from spores which lack the three major SASP (α, β, and γ). Six of these proteins are encoded by open reading frames identified previously or by analysis of the complete sequence of the
B. subtilis
genome, including two minor α/β-type SASP (SspC and SspD) and a putative spore coat protein (CotK). Five proteins are encoded by short open reading frames that were not identified as coding regions in the analysis of the complete
B. subtilis
genomic sequence. Studies of the regulation of two of the latter genes, termed
sspG
and
sspJ
, showed that both are expressed only in sporulation. The
sspG
gene is transcribed in the mother cell compartment by RNA polymerase with the mother cell-specific sigma factor for RNA polymerase, ς
K
, and is cotranscribed with a downstream gene,
yurS
;
sspG
transcription also requires the DNA binding protein GerE. In contrast,
sspJ
is transcribed in the forespore compartment by RNA polymerase with the forespore-specific ς
G
and appears to give a monocistronic transcript. A mutation eliminating SspG had no effect on sporulation or spore properties, while loss of SspJ caused a slight decrease in the rate of spore outgrowth in an otherwise wild-type background.
Insertion Mutagenesis of the Ferric Pyoverdine Receptor FpvA of
Pseudomonas aeruginosa
: Identification of Permissive Sites and a Region Important for Ligand Binding
Authors: Kilburn Laurie , Poole Keith , Meyer Jean-Marie , Neshat Shadi 
Published: December 2019

ABSTRACT

Insertion of an 18-amino-acid-encoding sequence within the
fpvA
gene identified permissive sites at residues Y350, A402, R451, R521, and R558, consistent with these residues occurring in extramembranous loop regions of the protein. Insertions at R451, R521, and R558 did not adversely affect receptor function, although insertions at Y350 and A402 compromised ferric pyoverdine binding and uptake. The latter region likely contributes to or interacts with the ligand-binding site.
Nitrite-Specific Active Transport System of the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Maeda Shin-ichi , Okamura Masato , Kobayashi Masaki , Omata Tatsuo 
Published: December 2019

ABSTRACT

Studies on the nitrite uptake capability of a mutant of
Synechococcus
sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent

K
m

(NO
2
−
) of about 20 μM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium.
Intracellular Immunization of Prokaryotic Cells against a Bacteriotoxin
Authors: Chames Patrick , Fieschi Jacques , Baty Daniel , Duché Denis 
Published: December 2019

ABSTRACT

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of
Escherichia coli
. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of
E. coli
. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.
Transcriptional and Translational Regulation of Photosystem I and II Genes in Light-Dark- and Continuous-Light-Grown Cultures of the Unicellular Cyanobacterium
Cyanothece
sp. Strain ATCC 51142
Authors: Colón-López Milagros S. , Sherman Louis A. 
Published: December 2019

ABSTRACT

Cyanothece
sp. strain ATCC 51142, a unicellular, diazotrophic cyanobacterium, demonstrated extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N
2
-fixing conditions. This report describes the relationship of the biosynthesis of photosynthesis genes to changes in the oligomerization state of the photosystems. Transcripts of the
psbA
gene family, encoding the photosystem II (PSII) reaction center protein D1, accumulated primarily during the light period, and net transcription reached a peak between 2 to 6 h in the light in light-dark (LD) growth and between 4 to 10 h in the subjective light when grown under continuous light (LL). The relative amount of the D1 protein (form 1 versus form 2) appeared to change during this diurnal cycle, along with changes in the PSII monomer/dimer ratio. D1 form 1 accumulated at approximately equal levels throughout the 24-h cycle, whereas D1 form 2 accumulated at significantly higher levels at approximately 8 to 10 h in the light or subjective light. The
psbD
gene, encoding the reaction center protein D2, also demonstrated differences between the two copies of this gene, with one copy transcribed more heavily around 6 to 8 h in the light. Accumulation of the PSI reaction center proteins PsaA and PsaB was maximal in the dark or subjective-dark periods, a period during which PSI was primarily in the trimeric form. We conclude that photosystem organization changes during the diurnal cycle to favor either noncyclic electron flow, which leads to O
2
evolution and CO
2
fixation, or cyclic electron flow, which favors ATP synthesis.
Genetic Determinants of Immunity and Integration of Temperate
Myxococcus xanthus
Phage Mx8
Authors: Salmi Daniel , Magrini Vincent , Hartzell Patricia L. , Youderian Philip 
Published: December 2019

ABSTRACT

An 8.1-kb fragment of the temperate
Myxococcus xanthus
phage Mx8 genome, when cloned into a plasmid vector, permits site-specific integration of the plasmid and confers superinfection immunity. Sequence analysis of a 9.5-kb region of Mx8 DNA containing this fragment reveals 19 densely packed open reading frames, four of which have predicted products with known or suspected activities. The Mx8
imm
gene, required for superinfection immunity, has a sequence similar to that of
Arabidopsis thaliana
G-box-binding factor 1. Mx8 makes a DNA adenine methylase, Mox, and integrase, Int, related to other methylases and integrases. The
int
gene has two alternate translation initiation codons within the extensively overlapping
uoi
(upstream of int) gene. Comparison of the predicted product of the
uoi
gene with
Salmonella
phage P22 and
Streptomyces
plasmid Xis proteins shows that temperate phage excisionases may use variations of a helix-turn-helix motif to recognize specific DNA sequences.
Deletion of
algK
in Mucoid
Pseudomonas aeruginosa
Blocks Alginate Polymer Formation and Results in Uronic Acid Secretion
Authors: Jain Sumita , Ohman Dennis E. 
Published: December 2019

ABSTRACT

Chronic pulmonary infection with
Pseudomonas aeruginosa
is a common and serious problem in patients with cystic fibrosis (CF). The
P. aeruginosa
isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the
algD
operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the
algA
,
algC
, and
algD
genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called
algK
between
alg44
and
algE
. We sequenced
algK
from the mucoid CF isolate FRD1 and expressed it in
Escherichia coli
, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK
−
mutant, the
algK
coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of
algK
that are essential for polymer formation. The
algK
Δ mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK
−
mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing
algK
in
trans
. When the
algK
Δ mutation was placed in an
algJ
::Tn
501
background, where
algA
was not expressed due to polar transposon effects, the growth defect was not observed. AlgK
−
mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK
−
mutant. When AlgK
−
culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the
algKΔ algJ
::Tn
501
mutant produced no uronic acids. Thus, the alginate pathway in an AlgK
−
mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.