bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Novel Member of the DegS-DegU Regulon Affected by Salt Stress in
Bacillus subtilis
Authors: Dartois Véronique , Débarbouillé Michel , Kunst Frank , Rapoport Georges 
Published: December 2019

ABSTRACT

As a soil bacterium also found in estuarine and marine habitats,
Bacillus subtilis
has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of
wapA
, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of
wapA
transcription by salt stress was shown to require the phosphorylated form of DegU. Moreover, DegU-mediated repression of
wapA
occurred only in high-salt medium. Alignment between the control region of
wapA
and other DegU-regulated promoters allowed the identification of a putative DegU target sequence, AGAAN
11
TTCAG. Mutation/deletion analyses of the
wapA
promoter region confirmed the role of the putative DegU control site in repression of
wapA
transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of DegU, it seems likely that an additional repressor acts on the
wapA
control region to further downregulate
wapA
transcription under salt stress conditions.
Regulation of
ntcA
Expression and Nitrite Uptake in the Marine
Synechococcus
sp. Strain WH 7803
Authors: Lindell Debbie , Padan Etana , Post Anton F. 
Published: December 2019

ABSTRACT

NtcA is a transcriptional activator involved in global nitrogen control in cyanobacteria. In the absence of ammonium it regulates the transcription of a series of genes encoding proteins required for the uptake and assimilation of alternative nitrogen sources (I. Luque, E. Flores, and A. Herrero, EMBO J. 13:2862–2869, 1994).
ntcA
, present in a single copy in the marine
Synechococcus
sp. strain WH 7803, was cloned and sequenced. The putative amino acid sequence shows a high degree of identity to NtcA from freshwater cyanobacteria in two functional domains. The expression of
ntcA
was negatively regulated by ammonium from a putative transcription start point located downstream of an NtcA consensus recognition sequence. Addition of either rifampin or ammonium led to a rapid decline in
ntcA
transcript levels with half-lives of less than 2 min in both cases. Nitrate-grown cells showed high
ntcA
transcript levels, as well as the capacity for active nitrite uptake. However, ammonium-grown cells showed low levels of the
ntcA
transcript and did not utilize nitrite. The addition of ammonium to nitrite uptake-active cells resulted in a gradual decline in the rate of uptake over a 24-h period. Active nitrite uptake was not induced in cells transferred to medium lacking a nitrogen source despite evidence of elevated expression of
ntcA
, indicating that
ntcA
expression is not sufficient for uptake capacity to develop. Nitrate and nitrite addition led to the development of nitrite uptake, whereas the addition of leucine did not. Furthermore, nitrite addition triggered the de novo protein synthesis required for uptake capacity to develop. These data suggest that nitrite and nitrate act as specific inducers for the synthesis of proteins required for nitrite uptake.
A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in
Lactococcus lactis
Authors: Le Loir Y. , Gruss A. , Ehrlich S. D. , Langella P. 
Published: December 2019

ABSTRACT

Lactococcus lactis
, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of
L. lactis
by using the
Staphylococcus aureus
nuclease (Nuc) as a heterologous model protein. When expressed in
L. lactis
from an efficient lactococcal promoter and its native signal peptide, only ∼60% of total Nuc was present in a secreted form at ∼5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ∼30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ∼80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ∼20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase of
Bacillus stearothermophilus
, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in
L. lactis.
Sterol Uptake in
Saccharomyces cerevisiae
Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification
Authors: Ness Frédérique , Achstetter Tilman , Duport Catherine , Karst Francis , Spagnoli Roberto , Degryse Eric 
Published: December 2019

ABSTRACT

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in
erg
auxotrophs.
Escherichia coli mrsC
Is an Allele of
hflB
, Encoding a Membrane-Associated ATPase and Protease That Is Required for mRNA Decay
Authors: Wang Rong-fu , O’Hara Eileen B. , Aldea Marti , Bargmann Cornelia I. , Gromley Heather , Kushner Sidney R. 
Published: December 2019

ABSTRACT

The
mrsC
gene of
Escherichia coli
is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive
mrsC505
allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920–1928, 1998). The cloned
mrsC
gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain.
mrsC
is identical to the independently identified
ftsH
gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344–1351, 1993). The purified protein had a

K
m

of 28 μM for ATP and a
V
max
of 21.2 nmol/μg/min. An amino-terminal glutathione
S
-transferase–MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (
mrsC201
) abolished the complementation of the
mrsC505
mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, the
mrsC505
allele conferred a temperature-sensitive HflB phenotype, while the
hflB29
mutation promoted mRNA stability at both 30 and 44°C, suggesting that the inviability associated with the
mrsC505
allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in
E. coli.
The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of
Escherichia coli
Are Important but Not Essential for Cytochrome
c
Maturation In Vivo
Authors: Fabianek Renata A. , Hennecke Hauke , Thöny-Meyer Linda 
Published: December 2019

ABSTRACT

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome
c
maturation in
Escherichia coli
. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in
ccmG
resulted in the complete absence of all
c
-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of
Bradyrhizobium japonicum
holocytochrome
c
550
expressed in
E. coli
. This defect, but not that of the
ccmG
null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.
Frequency of Pilin Antigenic Variation in
Neisseria gonorrhoeae
Authors: Serkin Carla D. , Seifert H. Steven 
Published: December 2019

ABSTRACT

Variation of the pilus of
Neisseria gonorrhoeae
occurs by the recombination of silent pilin DNA sequences into the pilin expression locus. We have developed a quantitative, competitive reverse transcription-PCR assay which measures the frequency of pilin antigenic variation independently of changes in gonococcal colony morphology and have determined this frequency within a gonococcal population. We have also studied the frequency of antigenic variation during growth and have concluded that growth does not dramatically influence the frequency of pilin antigenic variation, although a reproducible, twofold increase is observed upon the transition into late log/stationary phase.
Occurrence of Homologs of the
Escherichia coli lytB
Gene in Gram-Negative Bacterial Species
Authors: Potter Sheila , Yang Xiaoming , Boulanger Martin J. , Ishiguro Edward E. 
Published: December 2019

ABSTRACT

The
Escherichia coli
LytB protein regulates the activity of guanosine 3′,5′-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the
E. coli lytB
gene as a probe revealed the presence of
lytB
homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The
lytB
homologs from
Enterobacter aerogenes
and
Pseudomonas fluorescens
complemented the
E. coli lytB44
mutant allele.
Growth Rate-Dependent Accumulation of RNA from Plasmid-Borne rRNA Operons in
Escherichia coli
Authors: Stevenson Bradley S. , Schmidt Thomas M. 
Published: December 2019

ABSTRACT

Inadequate regulation of the expression of additional plasmid-borne rRNA operons in
Escherichia coli
was exaggerated at slow growth rates, resulting in increases of approximately 100% for RNA concentration and 33% for doubling time. These observations are consistent with the hypothesis that multiple rRNA operons constitute a metabolic burden at slow growth rates.
Enterocins L50A and L50B, Two Novel Bacteriocins from
Enterococcus faecium
L50, Are Related to Staphylococcal Hemolysins
Authors: Cintas Luis M. , Casaus Pilar , Holo Helge , Hernandez Pablo E. , Nes Ingolf F. , Håvarstein Leiv Sigve 
Published: December 2019

ABSTRACT

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodrı́guez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643–2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by
Enterococcus faecium
L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that
entL50A
and
entL50B
are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.
Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69
Authors: Yeh Lu-Shu , Hsu Tien , Karam Jim D. 
Published: December 2019

ABSTRACT

The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene
46-45.2-
(
rpbA
)
-45-44-62
cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the
rpbA
region (which encodes a nonessential protein in T4) is highly diverged (∼49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.
Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism
Authors: Jung Kwang-Hwan , Spudich John L. 
Published: December 2019

ABSTRACT

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon
Halobacterium salinarum
. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (
K
eq
) for the conformational transition in different mutants and mutant-suppressor pairs.
The
katX
Gene, Which Codes for the Catalase in Spores of
Bacillus subtilis
, Is a Forespore-Specific Gene Controlled by ς
F
, and KatX Is Essential for Hydrogen Peroxide Resistance of the Germinating Spore
Authors: Bagyan Irina , Casillas-Martinez Lilliam , Setlow Peter 
Published: December 2019

ABSTRACT

Previous work has shown that the
katX
gene encodes the major catalase in dormant spores of
Bacillus subtilis
but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a
katX-lacZ
fusion began at approximately h 2 of sporulation, and >75% of the
katX
-driven β-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor ς
F
abolished
katX-lacZ
expression, while mutations in genes encoding ς
E
, ς
G
, and ς
K
did not. Induction of ς
F
synthesis in vegetative cells also resulted in
katX-lacZ
expression, while induction of ς
G
expression did not; the
katX-lacZ
fusion was also not induced by hydrogen peroxide. Upstream of the in vivo
katX
transcription start site there are sequences with good homology to those upstream of known ς
F
-dependent start sites. These data indicate that
katX
is an additional member of the forespore-specific ς
F
regulon. A mutant in the
katA
gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while a
katX
mutant was not. However, outgrowth of
katX
spores, but not
katA
spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.
Temperature Sensitivity of Bacteriolysis Induced by β-Lactam Antibiotics in Amino Acid-Deprived
Escherichia coli
Authors: Rodionov Dmitrii G. , Ishiguro Edward E. 
Published: December 2019

ABSTRACT

The temperature-sensitive penicillin tolerance response previously reported in amino acid-deprived
Escherichia coli
(W. Kusser and E. E. Ishiguro, J. Bacteriol. 169:2310–2312, 1987) was not due to the induction of the heat shock response resulting from a temperature upshift and was therefore unrelated to the findings of another report (J. K. Powell and K. D. Young, J. Bacteriol. 173:4021–4026, 1991) indicating a positive correlation between the expression of heat shock proteins and penicillin tolerance. The thermosensitive event occurred in the lysis induction stage.
Specific Binding of
Escherichia coli
Ribosomal Protein S1 to
boxA
Transcriptional Antiterminator RNA
Authors: Mogridge Jeremy , Greenblatt Jack 
Published: December 2019

ABSTRACT

We show that ribosomal protein S1 specifically binds the
boxA
transcriptional antiterminator RNAs of bacteriophage λ and the
Escherichia coli
ribosomal RNA operons. Although S1 competes with the NusB-S10 antitermination complex for binding to
boxA
, it does not affect antitermination by the λ N protein in vitro, and its role, if any, in rRNA synthesis is still unknown.
Physiological Regulation of the Derepressible Phosphate Transporter inSaccharomyces cerevisiae
Authors: Martinez Paula , Zvyagilskaya Renata , Allard Peter , Persson Bengt L. 
Published: December 2019

ABSTRACTThe extracellular phosphate concentration permissive for the expression of different amounts of the active high-affinity Pho84 phosphate transporter in the plasma membrane as well as thePHO84messenger RNA levels in low-phosphate-grownSaccharomyces cerevisiaecells is very narrow and essential for a tight regulation of the transporter. The Pho84 transporter undergoes a rapid degradation once the supply of phosphate and/or carbon source is exhausted.
The
yhhP
Gene Encoding a Small Ubiquitous Protein Is Fundamental for Normal Cell Growth of
Escherichia coli
Authors: Yamashino Takafumi , Isomura Mototoshi , Ueguchi Chiharu , Mizuno Takeshi 
Published: December 2019

ABSTRACT

H-NS is a major constituent of the
Escherichia coli
nucleoid, whereas ς
S
is a stress-induced sigma factor. An
hns
null mutation affects the cellular content of ς
S
in such a way that a remarkable accumulation of ς
S
is observed in the logarithmic growth phase, which results in enhanced expression of a number of ς
S
-dependent genes, including the
katE
gene. We isolated an extragenic mutation that affects the expression of the
katE-lacZ
fusion gene in the Δ
hns
background. The relevant gene was identified as
yhhP
, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of ς
S
. A deletion analysis of
yhhP
revealed that this small protein plays a fundamental role in the general physiology of
E. coli
. The
yhhP
-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.