bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered Na
+
and Li
+
Homeostasis in
Saccharomyces cerevisiae
Cells Expressing the Bacterial Cation Antiporter NhaA
Authors: Ros Roc , Montesinos Consuelo , Rimon Abraham , Padan Etana , Serrano Ramón 
Published: December 2019

ABSTRACT

The bacterial Na
+
(Li
+
)/H
+
antiporter NhaA has been expressed in the yeast
Saccharomyces cerevisiae
. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na
+
, Li
+
) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H
+
ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na
+
, Li
+
) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H
+
ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na
+
, Li
+
) ATPase and vacuolar H
+
ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na
+
, Li
+
) ATPase and vacuolar H
+
ATPase.
Transcriptional Regulation of
Alcaligenes eutrophus
Hydrogenase Genes
Authors: Schwartz Edward , Gerischer Ulrike , Friedrich Bärbel 
Published: December 2019

ABSTRACT

Alcaligenes eutrophus
H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H
2
, supplying the organism with energy for autotrophic growth. The promoters of the structural genes for the SH and the MBH, P
SH
and P
MBH
, respectively, were identified by means of the primer extension technique. Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under conditions which repressed hydrogenase synthesis. The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Both promoters were dependent on the minor sigma factor ς
54
and on the hydrogenase regulator HoxA in vivo. P
SH
was stronger than P
MBH
under both heterotrophic and autotrophic growth conditions. The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures. The response regulator HoxA mediated low-level activation of P
SH
and P
MBH
in a heterologous system.
TheArcanobacterium(Actinomyces)pyogenesPlasmid pAP1 Is a Member of the pIJ101/pJV1 Family of Rolling Circle Replication Plasmids
Authors: Billington Stephen J. , Jost B. Helen , Songer J. Glenn 
Published: December 2019

ABSTRACTThe 2.4-kb plasmid pAP1 fromArcanobacterium(Actinomyces)pyogeneshad sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate inEscherichia coliandCorynebacterium pseudotuberculosis, as well as inA. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.
The
Bacillus subtilis
AraE Protein Displays a Broad Substrate Specificity for Several Different Sugars
Authors: Krispin Oliver , Allmansberger Rudolf 
Published: December 2019

ABSTRACT

Bacillus subtilis
168 is unable to grow on xylose and galactose as sole carbon sources, owing to the lack of specific transporters. We show that they are imported into the cell by the activity of AraE, an arabinose transporter whose synthesis is induced by
l
-arabinose.
The FixK2Protein Is Involved in Regulation of Symbiotic Hydrogenase Expression inBradyrhizobium japonicum
Authors: Durmowicz Meredith C. , Maier Robert J. 
Published: December 2019

ABSTRACTThe roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2in the symbiotic regulation of hydrogenase structural gene expression inBradyrhizobium japonicumhave been investigated. Bacteroids from FixJ and FixK2mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1mutant exhibit wild-type levels of hydrogenase activity. In β-galactosidase transcriptional assays with NifA and FixK2expression plasmids, the FixK2protein induces transcription from thehuppromoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2is involved in the transcriptional activation of symbiotic hydrogenase expression. By using β-galactosidase transcriptional fusion constructs containing successive truncations of thehuppromoter, the region of thehuppromoter required for regulation by FixK2was determined to be between 29 and 44 bp upstream of the transcription start site.
Activation of Bacteriophage Mu
mom
Transcription by C Protein Does Not Require Specific Interaction with the Carboxyl-Terminal Region of the α or ς
70
Subunit of
Escherichia coli
RNA Polymerase
Authors: Sun Weiyong , Hattman Stanley , Fujita Noboyuki , Ishihama Akira 
Published: December 2019

ABSTRACT

Late in its growth cycle, transcription of the phage Mu
mom
promoter (P

mom

) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the α or ς
70
subunit of
Escherichia coli
RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both α and ς
70
.
Transcriptional Analysis of Essential Genes of the
Escherichia coli
Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous
Salmonella typhimurium
Gene Cluster
Authors: Zhang Yan , Cronan John E. 
Published: December 2019

ABSTRACT

The genes encoding several key fatty acid biosynthetic enzymes (called the
fab
cluster) are clustered in the order
plsX-fabH-fabD-fabG-acpP-fabF
at min 24 of the
Escherichia coli
chromosome. A difficulty in analysis of the
fab
cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614–3620, 1996) is that several of these genes are essential for the growth of
E. coli
. We overcame this complication by use of the
fab
gene cluster of
Salmonella typhimurium
, a close relative of
E. coli
, to provide functions necessary for growth. The
S. typhimurium fab
cluster was isolated by complementation of an
E. coli fabD
mutant and was found to encode proteins with >94% homology to those of
E. coli
. However, the
S. typhimurium
sequences cannot recombine with the
E. coli
sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the
plsX
transcripts initiate at promoters located far upstream and include the upstream
rpmF
ribosomal protein gene, a promoter located upstream of the
plsX
coding sequence (probably within the upstream gene,
rpmF
) is sufficient for normal growth. We have also found that the
fabG
gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (Ω-Cm cassette) between the
fabD
and
fabG
genes of the
E. coli
chromosome abolished
fabG
transcription and blocked cell growth, thus providing the first indication that
fabG
is an essential gene. Insertion of the Ω-Cm cassette between
fabH
and
fabD
caused greatly decreased transcription of the
fabD
and
fabG
genes and slower cellular growth, indicating that
fabD
has only a weak promoter(s).
Induction of the SOS Response Increases the Efficiency of Global Nucleotide Excision Repair of Cyclobutane Pyrimidine Dimers, but Not 6-4 Photoproducts, in UV-Irradiated
Escherichia coli
Authors: Crowley David J. , Hanawalt Philip C. 
Published: December 2019

ABSTRACT

Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair. In
Escherichia coli
, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response. We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin. Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively. These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage.
Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate
Authors: Montrone Marco , Eisenbach Michael , Oesterhelt Dieter , Marwan Wolfgang 
Published: December 2019

ABSTRACT

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered
Escherichia coli
. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with
cheA
to
cheZ
deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.
Sed1p Is a Major Cell Wall Protein of
Saccharomyces cerevisiae
in the Stationary Phase and Is Involved in Lytic Enzyme Resistance
Authors: Shimoi Hitoshi , Kitagaki Hiroshi , Ohmori Hisanobu , Iimura Yuzuru , Ito Kiyoshi 
Published: December 2019

ABSTRACT

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of
Saccharomyces cerevisiae
by incubation with
Rarobacter faecitabidus
protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the
SED1
gene.
SED1
was formerly identified as a multicopy suppressor of
erd2
, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of
SED1
had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.
The
fnr
Gene of
Bacillus licheniformis
and the Cysteine Ligands of the C-Terminal FeS Cluster
Authors: Klinger Anette , Schirawski Jan , Glaser Philippe , Unden Gottfried 
Published: December 2019

ABSTRACT

In the facultatively anaerobic bacterium
Bacillus licheniformis
a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from
Bacillus subtilis
, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from
B. subtilis
, this cluster (Cys226-X
2
-Cys229-X
4
-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the
B. licheniformis
gene to an
fnr
mutant of
B. subtilis
complemented the ability for synthesis of nitrate reductase during anaerobic growth.
Regulation of Biosynthesis of Individual Cellulases in
Thermomonospora fusca
Authors: Spiridonov Nikolay A. , Wilson David B. 
Published: December 2019

ABSTRACT

Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium
Thermomonospora fusca
ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.
Roles of the Carboxy-Terminal Half of
Pseudomonas aeruginosa
Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association
Authors: Rawling Eileen G. , Brinkman Fiona S. L. , Hancock Robert E. W. 
Published: December 2019

ABSTRACT

OprF, the major outer membrane protein of
Pseudomonas aeruginosa
, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on
P. aeruginosa
cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of
P. aeruginosa
in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.
Lack of
S
-Adenosylmethionine Results in a Cell Division Defect in
Escherichia coli
Authors: Newman E. B. , Budman L. I. , Chan E. C. , Greene R. C. , Lin R. T. , Woldringh C. L. , D’Ari R. 
Published: December 2019

ABSTRACT

The enzyme
S
-adenosylmethionine (SAM) synthetase, the
Escherichia coli metK
gene product, produces SAM, the cell’s major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried
metK
gene prevents filamentation and restores normal growth to the
metK
mutant. This indicates that lack of SAM results in a division defect.
Low Ubiquinone Content in
Escherichia coli
Causes Thiol Hypersensitivity
Authors: Zeng H. , Snavely I. , Zamorano P. , Javor G. T. 
Published: December 2019

ABSTRACT

Thiol hypersensitivity in a mutant of
Escherichia coli
(IS16) was reversed by complementation with a plasmid that carried the
ubiX
gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal
ubiX
genes indicated that both parent and mutant strains were
ubiX
mutants. The low ubiquinone content of IS16 was possibly caused by a
ubiD ubiX
genotype. A
ubiA
mutant also exhibited thiol hypersensitivity. Neither IS16 nor the
ubiA
mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb
−
phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery.
Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium
Pseudomonas putida
GM73
Authors: Kim Kwang , Lee Sungjin , Lee Kyunghee , Lim Dongbin 
Published: December 2019

ABSTRACT

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium,
Pseudomonas putida
GM73, we carried out Tn
5
mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10
−6
). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn
5
-inserted genes, and the DNA sequence flanking Tn
5
was determined. From comparison with a sequence database, putative protein products encoded by
ttg
genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from
ttg8
did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.
Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product from
Bacillus subtilis
Authors: Inaoka Takashi , Matsumura Yoshinobu , Tsuchido Tetsuaki 
Published: December 2019

ABSTRACT

Bacillus subtilis
was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated
sodA
, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of
sodA
. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant
Escherichia coli sodA sodB
, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of
B. subtilis sodA
.
Identification of a New Site for Ferrichrome Transport by Comparison of the FhuA Proteins of
Escherichia coli
,
Salmonella paratyphi
B,
Salmonella typhimurium
, and
Pantoea agglomerans
Authors: Killmann Helmut , Herrmann Christina , Wolff Helga , Braun Volkmar 
Published: December 2019

ABSTRACT

The
fhuA
genes of
Salmonella paratyphi
B,
Salmonella typhimurium
, and
Pantoea agglomerans
were sequenced and compared with the known
fhuA
sequence of
Escherichia coli
. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in
E. coli
controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and φ80. All the FhuA proteins contained the region in the gating loops required in
E. coli
for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of
S. paratyphi
B which is infected by the
E. coli
phages, whereas two of the subdomains were deleted in
S. typhimurium
and
P. agglomerans
which are resistant to the
E. coli
phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated
E. coli
FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to φ80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of
S. paratyphi
B and
S. typhimurium
displayed
S. paratyphi
B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of
S. paratyphi
B and displayed
S. typhimurium
FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the
S. typhimurium
FhuA. The central segment of the
S. paratyphi
B FhuA flanked on both sides by
S. typhimurium
FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.