bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation of
CLN3
Expression by Glucose in
Saccharomyces cerevisiae
Authors: Parviz Fereshteh , Hall Duane D. , Markwardt David D. , Heideman Warren 
Published: December 2019

ABSTRACT

In
Saccharomyces cerevisiae
, the transition from the G
1
phase of the mitotic cycle into S phase is controlled by a set of G
1
cyclins that regulate the activity of the protein kinase encoded by
CDC28
. Yeast cells regulate progress through the G
1
/S boundary in response to nutrients, moving quickly through G
1
in glucose medium and more slowly in poorer medium. We have examined connections between glucose and the level of the message encoding Cln3, a G
1
cyclin. We found that glucose positively regulates
CLN3
mRNA levels through a set of repeated AAGAAAAA (A
2
GA
5
) elements within the
CLN3
promoter. Mutations in these sequences reduce both transcriptional activation and specific interaction between
CLN3
promoter elements and proteins in yeast extracts. Creation of five point mutations, replacing the G’s within these repeats with T’s, in the
CLN3
promoter substantially reduces
CLN3
expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose.
Peptidoglycan Structural Dynamics during Germination of
Bacillus subtilis
168 Endospores
Authors: Atrih Abdelmadjid , Zöllner Peter , Allmaier Günter , Williamson Michael P. , Foster Simon J. 
Published: December 2019

ABSTRACT

Peptidoglycan structural dynamics during endospore germination of
Bacillus subtilis
168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant
l
-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of
B. subtilis
168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic δ-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.
Cloning, Expression, and Catabolite Repression of a Gene Encoding β-Galactosidase of
Bacillus megaterium
ATCC 14581
Authors: Shaw Gwo-Chyuan , Kao Hsun-Sheng , Chiou Chih-Yung 
Published: December 2019

ABSTRACT

A gene encoding β-galactosidase, designated
mbgA
, was isolated from
Bacillus megaterium
ATCC 14581. Chromosomal β-galactosidase production could be dramatically induced by lactose but not by isopropyl-β-
d
-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of
mbgA
in the
B. megaterium
chromosome resulted in loss of lactose-inducible β-galactosidase production. A 27-bp inverted repeat was found to overlap the
mbgA
promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).
The
panE
Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to
apbA
in
Salmonella typhimurium
Authors: Frodyma Michael E. , Downs Diana 
Published: December 2019

ABSTRACT

In
Salmonella typhimurium
, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The
apbA
gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858–4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene
panE
. Although previous reports placed
panE
at 87 min on the
Escherichia coli
chromosome, we show herein that
apbA
and
panE
are allelic and map to 10 min on both the
S. typhimurium
and
E. coli
chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.
Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ghosh Mousumi , Grunden Amy M. , Dunn Dianne M. , Weiss Robert , Adams Michael W. W. 
Published: December 2019

ABSTRACT

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon
Pyrococcus furiosus
by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co
2+
ions (

K
d

, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co
2+
could be replaced by Mn
2+
(resulting in a 25% decrease in activity) but not by Mg
2+
, Ca
2+
, Fe
2+
, Zn
2+
, Cu
2+
, or Ni
2+
. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the
P. furiosus
genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in
Escherichia coli
and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from
P. furiosus
. Furthermore, the

K
m

values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater
V
max
value than the native protein. The amino acid sequence of
P. furiosus
prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The
P. furiosus
enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.
Escherichia coli tol-pal
Mutants Form Outer Membrane Vesicles
Authors: Bernadac Alain , Gavioli Marthe , Lazzaroni Jean-Claude , Raina Satish , Lloubès Roland 
Published: December 2019

ABSTRACT

Mutations in the
tol-pal
genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as
tolC
,
lpp
, and
rfa
mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the
tol-pal
genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in
tolC
and
rfaD
cells in the same conditions. A
tolA
deletion in three different
Escherichia coli
strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of
tol
strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.
Molecular Analysis of the Capsule Gene Region of Group A
Streptococcus
: the
hasAB
Genes Are Sufficient for Capsule Expression
Authors: Ashbaugh Cameron D. , Albertí Sebastián , Wessels Michael R. 
Published: December 2019

ABSTRACT

Enzymes directing the biosynthesis of the group A streptococcal hyaluronic acid capsule are encoded in the
hasABC
gene cluster. Inactivation of
hasC
, encoding UDP-glucose pyrophosphorylase in the heavily encapsulated group A streptococcal strain 87-282, had no effect on capsule production, indicating that
hasC
is not required for hyaluronic acid synthesis and that an alternative source of UDP-glucose is available for capsule production. Nucleotide sequence and deletion mutation analysis of the 5.5 kb of DNA upstream of
hasA
revealed that this region is not required for capsule expression. Many (10 of 23) group A streptococcal strains were found to contain insertion element IS
1239
′ approximately 50 nucleotides upstream of the −35 site of the
hasA
promoter. The presence of IS
1239
′ upstream of
hasA
did not prevent capsule expression. These results elucidate the molecular architecture of the group A streptococcal chromosomal region upstream of the
has
operon, indicate that
hasABC
are the sole components of the capsule gene cluster, and demonstrate that
hasAB
are sufficient to direct capsule synthesis in group A streptococci.
Isolation of
Candida glabrata
Homologs of the
Saccharomyces cerevisiae KRE9
and
KNH1
Genes and Their Involvement in Cell Wall β-1,6-Glucan Synthesis
Authors: Nagahashi Shigehisa , Lussier Marc , Bussey Howard 
Published: December 2019

ABSTRACT

The
Candida glabrata KRE9
(
CgKRE9
) and
KNH1
(
CgKNH1
) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a
Saccharomyces cerevisiae
mutant with the disrupted
KNH1
locus and the
KRE9
gene placed under the control of a tetracycline-responsive promoter. Although a
cgknh1Δ
mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of
CgKRE9
resulted in several phenotypes similar to those of the
S. cerevisiae kre9Δ
null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of β-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in
C. glabrata
of the cognate
CgKRE9
promoter with the tetracycline-responsive promoter in a
cgknh1Δ
background rendered cell growth tetracycline sensitive on media containing glucose or galactose.
cgkre9Δ
cells were shown to be sensitive to calcofluor white specifically on glucose medium. In
cgkre9
mutants grown on glucose medium, cellular chitin levels were massively increased.
Sucrose Is a Nonaccumulated Osmoprotectant in
Sinorhizobium meliloti
Authors: Gouffi Kamila , Pichereau Vianney , Rolland Jean-Paul , Thomas Daniel , Bernard Théophile , Blanco Carlos 
Published: December 2019

ABSTRACT

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of
Sinorhizobium meliloti
cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and
N
-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for
S. meliloti
. Overall, osmoregulation in
S. meliloti
appears to be strongly divergent from that in most bacteria.
Effect of Slow Growth on Metabolism of
Escherichia coli
, as Revealed by Global Metabolite Pool (“Metabolome”) Analysis
Authors: Tweeddale Helen , Notley-McRobb Lucinda , Ferenci Thomas 
Published: December 2019

ABSTRACT

Escherichia coli
growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions. The extent of pool changes was followed through two-dimensional thin-layer chromatography of all
14
C-glucose labelled compounds extracted from bacteria. The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions. Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates. The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly. The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis. Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate. As RpoS controls a number of metabolic genes in response to nutrient limitation, an
rpoS
mutant was also analyzed for metabolite pools. The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS. These results indicate that total metabolite pool (“metabolome”) analysis offers a means of revealing novel aspects of cellular metabolism and global regulation.
CheZ Has No Effect on Flagellar Motors Activated by CheY
13DK106YW
Authors: Scharf Birgit E. , Fahrner Karen A. , Berg Howard C. 
Published: December 2019

ABSTRACT

The behaviors of both
cheZ
-deleted and wild-type cells of
Escherichia coli
were found to be very sensitive to the level of expression of CheZ, a protein known to accelerate the dephosphorylation of the response regulator CheY-phosphate (CheY-P). However, cells induced to run and tumble by the unphosphorylated mutant protein CheY
13DK106YW
(CheY**) failed to respond to CheZ, even when CheZ was expressed at high levels. Therefore, CheZ neither affects the flagellar motors directly nor sequesters CheY**. In in vitro cross-linking studies, CheY** promoted trimerization of CheZ to the same extent as wild-type CheY but failed to induce the formation of complexes of higher molecular weight observed with CheY-P. Also, CheY** could be cross-linked to FliM, the motor receptor protein, nearly as well as CheY-P. Thus, to CheZ, CheY** looks like CheY, but to FliM, it looks like CheY-P.
hrcA
, Encoding the Repressor of the
groEL
Genes in
Streptomyces albus G
, Is Associated with a Second
dnaJ
Gene
Authors: Grandvalet Cosette , Rapoport Georges , Mazodier Philippe 
Published: December 2019

ABSTRACT

Expression of the principal chaperones of the heat shock stimulon of
Streptomyces albus
G are under the negative control of different repressors. The
dnaK
operon is regulated by
hspR
, the last gene of the operon (
dnaK-grpE-dnaJ-hspR
).
hsp18
, encoding a member of the small heat shock protein family, is regulated by
orfY
, which is in the opposite orientation upstream of
hsp18
. The
groES-groEL1
operon and the
groEL2
gene are regulated differently. They present tandem copies of the CIRCE element found in the 5′ region of many heat shock genes and shown to act in
Bacillus subtilis
as an operator for a repressor encoded by
hrcA
(
hrc
stands for heat regulation at CIRCE). We report the identification in
S. albus
of a new heat shock operon containing
hrcA
and
dnaJ
homologs. Disruption of
hrcA
increased the transcription of the
groES-groEL1
operon and of the
groEL2
gene. These features were lost when the mutant was complemented in
trans
by an intact copy of
hrcA
. Despite considerable accumulation of the GroE chaperones in the
hrcA
mutant, there was no effect on formation of the aerial mycelium and sporulation, indicating that neither
hrcA
nor the level of
groE
gene expression is directly involved in the regulation of
Streptomyces
morphological differentiation.
The Gene for 16S rRNA Methyltransferase (
ksgA
) Functions as a Multicopy Suppressor for a Cold-Sensitive Mutant of Era, an Essential RAS-Like GTP-Binding Protein in
Escherichia coli
Authors: Lu Qing , Inouye Masayori 
Published: December 2019

ABSTRACT

Era, a Ras-like GTP-binding protein in
Escherichia coli
, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an
E. coli
genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the
E. coli
genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the
ksgA
gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.
Two Amino Acid Residues of Transposase Contributing to Differential Transposability of IS
1
Elements in
Escherichia coli
Authors: Chen Jiann-Hwa , Hsu Wen-Ben , Hwang Jiing-Luen 
Published: December 2019

ABSTRACT

Escherichia coli
W3110 contains four types of IS
1
elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS
1
plasmid insertion mutants, which resulted from a minimum of 26 independent IS
1
insertion events. All of them had insertions of IS
1
of the IS
1
A (IS
1
E and IS
1
G) type. Inspection of the transposase sequences of the four IS
1
types and the IS
1
of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS
1
elements in W3110. The two amino acid residues of the transposase in IS
1
A (IS
1
E and IS
1
G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS
1
A (IS
1
E and IS
1
G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS
1
elements in W3110 was confirmed.
Catabolite Gene Activator Protein Mutations Affecting Activity of the
araBAD
Promoter
Authors: Zhang Xin , Schleif Robert 
Published: December 2019

ABSTRACT

We have studied catabolite gene activator protein (CAP) activation at the
araBAD
promoter,

p
BAD

, in the absence of DNA looping. We ruled out the two most plausible indirect activation mechanisms: CAP-induced folding of upstream DNA back upon RNA polymerase, and CAP-induced stabilization of AraC binding to DNA. Therefore, a direct CAP-RNA polymerase interaction seemed likely. We sought and found CAP mutants defective in transcription activation at

p
BAD

that retained normal DNA binding affinity. Some mutations altered residues in the interval from positions 150 to 164 that includes CAP activating region 1 (AR1), which has been shown to contact RNA polymerase at a number of promoters. Unexpectedly, additional mutations were found that altered residues in the region between positions 46 and 68 and at position 133. This includes the region known as activating region 3 (AR3). Mutations from both groups also affect the
araFGH
and
rhaBAD
promoters.
A DnaK Homolog in
Myxococcus xanthus
Is Involved in Social Motility and Fruiting Body Formation
Authors: Yang Zhaomin , Geng Yongzhi , Shi Wenyuan 
Published: December 2019

ABSTRACT

Myxococcus xanthus
is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of
M. xanthus
were isolated through Tn
5
transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the
dnaK
genes in other bacteria, this
M. xanthus
homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of
M. xanthus.
Growth-Independent Regulation of
CLN3
mRNA Levels by Nutrients in
Saccharomyces cerevisiae
Authors: Parviz Fereshteh , Heideman Warren 
Published: December 2019

ABSTRACT

Saccharomyces cerevisiae
cells regulate progress through the G
1
phase of the cell cycle in response to nutrients, moving quickly through G
1
in rich medium and slowly in poor medium. Recent work has shown that the levels of Cln3 protein, a G
1
cyclin, are low in cells growing in poor medium and high in cells growing rapidly in rich medium, consistent with the previously recognized role of Cln3 in promoting passage through Start. Cln3 protein levels appear to be regulated both transcriptionally and posttranscriptionally. We have worked to define the nutrient signals that regulate
CLN3
mRNA levels. We find that
CLN3
mRNA levels are high during log-phase growth in glucose medium, low in postdiauxic cells growing on ethanol, and slightly lower still in cells in stationary phase.
CLN3
mRNA levels are induced by glucose in a process that involves transcriptional control, requires metabolism of the glucose, and is independent of the Ras-cyclic AMP pathway.
CLN3
mRNA levels are also positively regulated by nitrogen sources, but phosphorus and sulfur limitation do not affect
CLN3
message levels.