bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control by Nutrients of Growth and Cell Cycle Progression in Budding Yeast, Analyzed by Double-Tag Flow Cytometry
Authors: Alberghina Lilia , Smeraldi Carla , Ranzi Bianca Maria , Porro Danilo 
Published: December 2019

ABSTRACT

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual
Saccharomyces cerevisiae
cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing
S. cerevisiae
populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.
Isolation, Characterization, and Expression of the Gene Encoding the β Subunit of the Mitochondrial Processing Peptidase from
Blastocladiella emersonii
Authors: Rocha Cíntia Renata Costa , Gomes Suely Lopes 
Published: December 2019

ABSTRACT

A 2.3-kb
Bam
HI-
Kpn
I fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the β subunit of the
Blastocladiella
mitochondrial processing peptidase (β-MPP). The predicted β-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that β-MPP mRNA levels decrease significantly during
B. emersonii
sporulation, reaching basal levels in the zoospore stage. The amount of β-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.
The Succinyl and Acetyl Modifications of Succinoglycan Influence Susceptibility of Succinoglycan to Cleavage by the
Rhizobium meliloti
Glycanases ExoK and ExsH
Authors: York Gregory M. , Walker Graham C. 
Published: December 2019

ABSTRACT

In
Rhizobium meliloti
(
Sinorhizobium meliloti
) cultures, the endo-1,3-1,4-β-glycanases ExoK and ExsH depolymerize nascent high-molecular-weight (HMW) succinoglycan to yield low-molecular-weight (LMW) succinoglycan. We report here that the succinyl and acetyl modifications of succinoglycan influence the susceptibility of succinoglycan to cleavage by these glycanases. It was previously shown that
exoH
mutants, which are blocked in the succinylation of succinoglycan, exhibit a defect in the production of LMW succinoglycan. We have determined that
exoZ
mutants, which are blocked in the acetylation of succinoglycan, exhibit an increase in production of LMW succinoglycan. For both wild-type and
exoZ
mutant strains, production of LMW succinoglycan is dependent on the
exoK
+
and
exsH
+
genes, implying that the ExoK and ExsH glycanases cleave HMW succinoglycan to yield LMW succinoglycan. By supplementing cultures of glycanase-deficient strains with exogenously added ExoK or ExsH, we have demonstrated directly that the absence of the acetyl group increases the susceptibility of succinoglycan to cleavage by ExoK and ExsH, that the absence of the succinyl group decreases the susceptibility of succinoglycan to cleavage, and that the succinyl effect outweighs the acetyl effect for succinoglycan lacking both modifications. Strikingly, nonsuccinylated succinoglycan actually can be cleaved by ExoK and ExsH to yield LMW succinoglycan, but only when the glycanases are added to cultures at greater than physiologically relevant concentrations. Thus, we conclude that the molecular weight distribution of succinoglycan in
R. meliloti
cultures is determined by both the levels of ExoK and ExsH glycanase expression and the susceptibility of succinoglycan to cleavage.
Oligohistidine Tag Mutagenesis of the λ Holin Gene
Authors: Smith David L. , Young Ry 
Published: December 2019

ABSTRACT

Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. Recently, the holin
S
gene of phage λ was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D. L. Smith, D. K. Struck, J. M. Scholtz, and R. Young, J. Bacteriol. 180:2531–2540, 1998). Numerous locations within the
S
gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function. The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized. A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein. Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function. The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required.
One of Two OsmC Homologs in
Bacillus subtilis
Is Part of the ς
B
-Dependent General Stress Regulon
Authors: Völker Uwe , Andersen Kasper Krogh , Antelmann Haike , Devine Kevin M. , Hecker Michael 
Published: December 2019

ABSTRACT

In this report we present the identification and analysis of two
Bacillus subtilis
genes,
yklA
and
ykzA
, which are homologous to the partially RpoS-controlled
osmC
gene from
Escherichia coli
. The
yklA
gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter. Expression of
ykzA
is not medium dependent but increases dramatically when cells are exposed to stress and starvation. This stress-induced increase in
ykzA
expression is absolutely dependent on the alternative sigma factor ς
B
, which controls a large stationary-phase and stress regulon.
ykzA
is therefore another example of a gene common to the RpoS and ς
B
stress regulons of
E. coli
and
B. subtilis
, respectively. The composite complex expression pattern of the two
B. subtilis
genes is very similar to the expression profile of
osmC
in
E. coli.
Phosphate Assimilation in
Rhizobium
(
Sinorhizobium
)
meliloti
: Identification of a
pit
-Like Gene
Authors: Bardin Sylvie D. , Voegele Ralf T. , Finan Turlough M. 
Published: December 2019

ABSTRACT

Rhizobium meliloti
mutants defective in the
phoCDET
-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix
−
). We have previously reported that two classes of second-site mutations can suppress the Fix
−
phenotype of
phoCDET
mutants to Fix
+
. Here we show that one of these suppressor loci (
sfx1
) contains two genes,
orfA
and
pit
, which appear to form an operon transcribed in the order
orfA-pit
. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the
orfA
transcription start site. This mutation increased the level of
orfA-pit
transcription. These data, together with previous biochemical evidence, show that the
orfA-pit
genes encode a P
i
transport system that is expressed in wild-type cells grown with excess P
i
but repressed in cells under conditions of P
i
limitation. In
phoCDET
mutant cells,
orfA-pit
expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases
orfA-pit
expression compensating for the deficiencies in phosphate assimilation and symbiosis of the
phoCDET
mutants.
Regulation of
hepA
of
Anabaena
sp. Strain PCC 7120 by Elements 5′ from the Gene and by
hepK
Authors: Zhu Jinsong , Kong Renqiu , Wolk C. Peter 
Published: December 2019

ABSTRACT

In
Anabaena
spp., synthesis of the heterocyst envelope polysaccharide, required if the cell is to fix dinitrogen under aerobic conditions, is dependent on the gene
hepA
. A transcriptional start site of
hepA
was localized 104 bp 5′ from its translational initiation codon. A 765-bp open reading frame, denoted
hepC
, was found farther upstream. Inactivation of
hepC
led to constitutive expression of
hepA
and prevented the synthesis of heterocyst envelope polysaccharide. However, the glycolipid layer of the heterocyst envelope was synthesized. A
hepK
mutation blocked both the synthesis of the heterocyst envelope polysaccharide and induction of
hepA
. The predicted product of
hepK
resembles a sensory protein-histidine kinase of a two-component regulatory system. Analysis of the region between
hepC
and
hepA
indicated that DNA sequences required for the induction of
hepA
upon nitrogen deprivation are present between bp −574 and −440 and between bp −340 and −169 relative to the transcriptional start site of
hepA
. Gel mobility shift assays provided evidence that one or more proteins bind specifically to the latter sequence. The Fox box sequence downstream from
hepA
appeared inessential for the induction of
hepA
.
Analysis of the
puc
Operon Promoter from
Rhodobacter capsulatus
Authors: Nickens David G. , Bauer Carl E. 
Published: December 2019

ABSTRACT

Expression of the
Rhodobacter capsulatus puc
operon, which codes for structural polypeptides of the light-harvesting-II peripheral antenna complex, is highly regulated in response to alterations in oxygen tension and light intensity. To obtain an understanding of the
puc
promoter region we report the high-resolution 5′ mapping of the
puc
mRNA transcriptional start site and DNA sequence analysis of the
puc
upstream regulatory sequence (
puc
URS). A ς
70
-type promoter sequence was identified (
puc
P1) which has a high degree of sequence similarity with carotenoid and bacteriochlorophyll biosynthesis promoters. Inspection of the DNA sequence also indicated the presence of two CrtJ and four integration host factor (IHF) binding sites. Transcriptional fusions of the
puc
URS fused to
lacZ
also confirmed that
puc
promoter activity is regulated by the transcriptional regulators IHF, CrtJ, and RegA. Gel retardation analysis using cell extracts indicates that mutations in IHF and RegA disrupt protein binding to DNA fragments containing the
puc
URS.
Agrobacterium
VirE2 Proteins Can Form a Complex with T Strands in the Plant Cytoplasm
Authors: Gelvin Stanton B. 
Published: December 2019

ABSTRACT

Wild-type VirE2 and VirD2 proteins from
Agrobacterium tumefaciens
contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An
A. tumefaciens virE2 virD2
ΔNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.
Characterization of the Genes Encoding
d
-Amino Acid Transaminase and Glutamate Racemase, Two
d
-Glutamate Biosynthetic Enzymes of
Bacillus sphaericus
ATCC 10208
Authors: Fotheringham Ian G. , Bledig Stefan A. , Taylor Paul P. 
Published: December 2019

ABSTRACT

In
Bacillus sphaericus
and other
Bacillus
spp.,
d
-amino acid transaminase has been considered solely responsible for biosynthesis of
d
-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the
dat
gene encoding
d
-amino acid transaminase and the
glr
gene encoding a glutamate racemase from
B. sphaericus
ATCC 10208. The
glr
gene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of
Lactobacillus
,
Pediococcus
, and
Staphylococcus
species. The
dat
gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the
d
-amino acid transaminase of the thermophilic
Bacillus
sp. strain YM1.
Complete Nucleotide Sequence of pSK41: Evolution of Staphylococcal Conjugative Multiresistance Plasmids
Authors: Berg Tracey , Firth Neville , Apisiridej Sumalee , Hettiaratchi Anusha , Leelaporn Amornrut , Skurray Ronald A. 
Published: December 2019

ABSTRACT

The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from
Staphylococcus aureus
, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn
552
-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS
257
-mediated cointegrative capture of other plasmids.
A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in
Haemophilus influenzae
Rd
Authors: Macfadyen Leah P. , Ma Caixia , Redfield Rosemary J. 
Published: December 2019

ABSTRACT

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in
Haemophilus influenzae
. In
Escherichia coli
, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the
cpdA
gene. We have used several approaches to demonstrate that the homologous
icc
gene of
H. influenzae
encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In
E. coli
, expression of cloned
icc
reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for
cpdA
. In
H. influenzae
, an
icc
null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a
cya
mutation, an
icc
strain was 10,000-fold more sensitive to exogenous cAMP than an
icc
+
strain. The
icc
strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a
cya
strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the
H. influenzae
cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.
Contribution of the P

mra

Promoter to Expression of Genes in the
Escherichia coli mra
Cluster of Cell Envelope Biosynthesis and Cell Division Genes
Authors: Mengin-Lecreulx Dominique , Ayala Juan , Bouhss Ahmed , van Heijenoort Jean , Parquet Claudine , Hara Hiroshi 
Published: December 2019

ABSTRACT

Recently, a promoter for the essential gene
ftsI
, which encodes penicillin-binding protein 3 of
Escherichia coli
, was precisely localized 1.9 kb upstream from this gene, at the beginning of the
mra
cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (P

mra

) on the chromosome and its replacement by the
lac
promoter (P

mra

::P

lac

) led to isopropyl-β-
d
-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P

mra

to
ftsW
, the fifth gene downstream from
ftsI
, was provided in
trans
on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P

mra

::P

lac

promoter expression was repressed or fully induced. It was confirmed that the P

mra

promoter is required for expression of the first nine genes of the
mra
cluster:
mraZ
(
orfC
),
mraW
(
orfB
),
ftsL
(
mraR
),
ftsI
,
murE
,
murF
,
mraY
,
murD
, and
ftsW
. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P

mra

::P

lac

cells. This was correlated with an accumulation of the nucleotide precursors UDP–
N
-acetylglucosamine and UDP–
N
-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–
N
-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of
ftsZ
, the penultimate gene from this cluster, was significantly reduced when P

mra

expression was repressed. It was concluded that the transcription of the genes located downstream from
ftsW
in the
mra
cluster, from
murG
to
ftsZ
, is also mainly (but not exclusively) dependent on the P

mra

promoter.
Unique Regulation of Carbohydrate Chemotaxis in
Bacillus subtilis
by the Phosphoenolpyruvate-Dependent Phosphotransferase System and the Methyl-Accepting Chemotaxis Protein McpC
Authors: Garrity Liam F. , Schiel Stacey L. , Merrill Ronald , Reizer Jonathan , Saier Milton H. , Ordal George W. 
Published: December 2019

ABSTRACT

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of
Escherichia coli
to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in
Bacillus subtilis
is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for
E. coli
, a
B. subtilis ptsH
null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An
mcpC
null mutant showed no response to the stepwise addition or removal of
d
-glucose or
d
-mannitol, both of which are PTS substrates. Therefore, in contrast to
E. coli
PTS carbohydrate chemotaxis,
B. subtilis
PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate
d
-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that
B. subtilis
EnzymeI-P has the ability to inhibit
B. subtilis
CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of
d
-glucose, a decrease in
d
-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.
Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from
Streptomyces glaucescens
and Its Role in Initiation of Fatty Acid Biosynthesis
Authors: Han Lei , Lobo Sandra , Reynolds Kevin A. 
Published: December 2019

ABSTRACT

The
Streptomyces glaucescens fabH
gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in
Escherichia coli
, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an
M
r
of 37,000, while gel filtration analysis determined a native
M
r
of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a

K
m

value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with

K
m

values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the
S. glaucescens
FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in
Streptomyces
and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in
S. glaucescens
.