bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
nif
Gene Operon of the Methanogenic Archaeon
Methanococcus maripaludis
Authors: Kessler Peter S. , Blank Carrine , Leigh John A. 
Published: December 2019

ABSTRACT

Nitrogen fixation occurs in two domains,
Archaea
and
Bacteria
. We have characterized a
nif
(nitrogen fixation) gene cluster in the methanogenic archaeon
Methanococcus maripaludis
. Sequence analysis revealed eight genes, six with sequence similarity to known
nif
genes and two with sequence similarity to
glnB
. The gene order,
nifH
,
ORF105
(similar to
glnB
),
ORF121
(similar to
glnB
),
nifD
,
nifK
,
nifE
,
nifN
, and
nifX
, was the same as that found in part in other diazotrophic methanogens and except for the presence of the
glnB
-like genes, also resembled the order found in many members of the
Bacteria
. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the
nif
gene cluster. Northern analysis revealed the presence of either a single 7.6-kb
nif
mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ to
nifH
. Distinctive features of the
nif
gene cluster include the presence of the six primary
nif
genes in a single operon, the placement of the two
glnB
-like genes within the cluster, the apparent physical separation of the cluster from any other
nif
genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens.
Sensory Rhodopsin II Transducer HtrII Is Also Responsible for Serine Chemotaxis in the Archaeon
Halobacterium salinarum
Authors: Hou Shaobin , Brooun Alexei , Suk Yu Hyung , Freitas Tracey , Alam Maqsudul 
Published: December 2019

ABSTRACT
Previously, we demonstrated that the methyl-accepting protein HtrII is the transducer for photoreceptor sensory rhodopsin II. Here, we provide experimental evidence that HtrII is also a chemotransducer. Using an agarose-in-plug bridge method, we show that an HtrII overexpression strain has a quicker response to serine than does an HtrII deletion strain. Furthermore, an in vivo flow assay demonstrates that the deletion strain is unable to modulate methylesterase activity after serine addition or photostimulation, while the overexpression strain shows distinct methanol peaks following both types of stimuli.
The
Saccharomyces cerevisiae SCS2
Gene Product, a Homolog of a Synaptobrevin-Associated Protein, Is an Integral Membrane Protein of the Endoplasmic Reticulum and Is Required for Inositol Metabolism
Authors: Kagiwada Satoshi , Hosaka Kohei , Murata Masayuki , Nikawa Jun-ichi , Takatsuki Akira 
Published: December 2019

ABSTRACT

The
Saccharomyces cerevisiae SCS2
gene has been cloned as a suppressor of inositol auxotrophy of
CSE1
and
hac1/ire15
mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39–45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from
Aplysia californica
(P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580–1583, 1995). In this study we have characterized an
SCS2
gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the
SCS2
gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34°C. Genetic studies reveal that the overexpression of the
INO1
gene rescues the inositol auxotrophy of the
SCS2
disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the
SCS2
disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.
Identification of a Novel
Salmonella
Invasion Locus Homologous to
Shigella ipgDE
Authors: Hong K. Heran , Miller Virginia L. 
Published: December 2019

ABSTRACT

Genes essential for
Salmonella typhimurium
invasion have been localized to
Salmonella
pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn
10
dTc insertion mutants of
S. typhimurium
in the tissue culture invasion assay. One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to the
ipgDEF
operon of the
Shigella flexneri
and
Shigella sonnei
virulence plasmid. A second mutant, SVM168, has an insertion in an IS
3
-type element with homology to the
Salmonella enteritidis
IS
1351
element and
Yersinia enterocolitica
IS
1400
element from a high-pathogenicity island. Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system. In addition, transcription of this operon,
sigDE
(
Salmonella
invasion gene), is dependent on the presence of
sirA
, an activator of
hilA
expression. HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the
sigDE
promoter.
Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in
Escherichia coli
Authors: Ault-Riché Dana , Fraley Cresson D. , Tzeng Chi-Meng , Kornberg Arthur 
Published: December 2019

ABSTRACT

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of
Escherichia coli
mutants revealed that polyP accumulation depends on several regulatory genes,
glnD
(NtrC),
rpoS
,
relA
, and
phoB.
An Exported Inducer Peptide Regulates Bacteriocin Production in
Enterococcus faecium
CTC492
Authors: Nilsen Trine , Nes Ingolf F. , Holo Helge 
Published: December 2019

ABSTRACT

Production of the bacteriocins enterocin A and enterocin B in
Enterococcus faecium
CTC492 was dependent on the presence of an extracellular peptide produced by the strain itself. This induction factor (EntF) was purified, and amino acid sequencing combined with DNA sequencing of the corresponding gene identified it as a peptide of 25 amino acids. The gene encodes a prepeptide of 41 amino acids, including a 16-amino-acid leader peptide of the double-glycine type. Environmental factors influenced the level of bacteriocin production in
E. faecium
CTC492. The optimal pH for bacteriocin production was 6.2. At pH 5.5, growth was slow, and very little bacteriocin was formed. The presence of NaCl or ethanol (EtOH) was also inhibitory to bacteriocin production, and at high concentrations of these solutes, no bacteriocin production was observed. The induction factor induced its own synthesis, and by dilution of the culture 10
6
times or more, nonproducing cultures were obtained. Bacteriocin production was induced in these cultures by addition of EntF. The response was linear, and low bacteriocin production could be induced by about 10
−17
M EntF. This response was attenuated by low pH or the presence of high concentrations of NaCl or EtOH, and 300 times more EntF was needed to induce detectable bacteriocin production in the presence of 6.5% NaCl. High levels of bacteriocin production in cultures grown at low pH or in the presence of high concentrations of NaCl or EtOH were obtained by addition of sufficient amounts of EntF.
A Small Protein (Ags1p) and the Pho80p-Pho85p Kinase Complex Contribute to Aminoglycoside Antibiotic Resistance of the Yeast
Saccharomyces cerevisiae
Authors: Wickert Stephan , Finck Markus , Herz Britta , Ernst Joachim F. 
Published: December 2019

ABSTRACT

We identified the
AGS1
and
AGS3
genes by their ability to partially complement an
ags
mutant (RC1707) which is supersensitive to various aminoglycoside antibiotics (J. F. Ernst and R. K. Chan, J. Bacteriol. 163:8–14, 1985).
AGS1
is located in proximity to the centromere of chromosome III and encodes a small protein of 88 amino acids. The size of the
AGS1
transcript, which in wild-type cells is 1 kb, is reduced to 0.75 kb in mutant RC1707. Disruption of
AGS1
rendered strains supersensitive to hygromycin B and increased their resistance to vanadate. In addition,
ags1
Δ strains underglycosylated invertase but had normal carboxypeptidase Y glycosylation, suggesting that Ags1p is required for the elaboration of outer N-glycosyl chains.
AGS3
was found to be identical to
PHO80
(
TUP7
), which encodes a cyclin activating the Pho85p protein kinase. Deletion of either
PHO80
or
PHO85
led to aminoglycoside supersensitivity;
pho80Δ ags1
Δ strains showed an enhanced-sensitivity phenotype compared to single mutants.
pho80
and
pho85
mutants were rendered resistant by deletion of
PHO4
, indicating that activation of the Pho4p transcription factor is required for increased aminoglycoside sensitivity. Thus, both the Pho80p-Pho85p kinase complex (by Pho4p phosphorylation) and a novel component of the N glycosylation pathway contribute to basal levels of aminoglycoside resistance in
Saccharomyces cerevisiae.
An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to
Lactococcus lactis
Authors: Poquet Isabelle , Ehrlich S. Dusko , Gruss Alexandra 
Published: December 2019

ABSTRACT

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the
Staphylococcus aureus
secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Δ
SP
Nuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in
Lactococcus lactis
, thus demonstrating the suitability of Δ
SP
Nuc to report protein export. The shuttle vector pFUN was designed to construct Δ
SP
Nuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Δ
SP
Nuc to reveal and identify exported proteins was tested by generating an
L. lactis
genomic library in pFUN and by screening for Nuc activity directly in
L. lactis
. All Δ
SP
Nuc fusions displaying a strong Nuc
+
phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in
L. lactis
. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with
L. lactis
show that Δ
SP
Nuc is well suited to report both protein export and membrane protein topology.
The Atrazine Catabolism GenesatzABCAre Widespread and Highly Conserved
Authors: de Souza Mervyn L. , Seffernick Jennifer , Martinez Betsy , Sadowsky Michael J. , Wackett Lawrence P. 
Published: December 2019

ABSTRACTPseudomonasstrain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genesatzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous toatzA, -B, and -C. The sequence identities of theatzgenes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.
Evidence for a Regulatory Link of Nitrogen Fixation and Photosynthesis in
Rhodobacter capsulatus
via HvrA
Authors: Kern Monika , Kamp Paul-Bertram , Paschen Annette , Masepohl Bernd , Klipp Werner 
Published: December 2019

ABSTRACT

A
Rhodobacter capsulatus
reporter strain, carrying a constitutively expressed
nifA
gene and a
nifH-lacZ
gene fusion, was used for random transposon Tn
5
mutagenesis to search for genes required for the NtrC-independent ammonium repression of NifA activity. A mutation in
hvrA
, which is known to be involved in low-light activation of the photosynthetic apparatus, released both ammonium and oxygen control of
nifH
expression in this reporter strain, demonstrating a regulatory link of nitrogen fixation and photosynthesis via HvrA. In addition, a significant increase in bacteriochlorophyll
a
(BChl
a
) content was found in cells under nitrogen-fixing conditions. HvrA was not involved in this up-regulation of BChl
a
. Instead, the presence of active nitrogenase seemed to be sufficient for this process, since no increase in BChl
a
content was observed in different
nif
mutants.
Characterization of the
Aspergillus nidulans nmrA
Gene Involved in Nitrogen Metabolite Repression
Authors: Andrianopoulos Alex , Kourambas Sophie , Sharp Julie A. , Davis Meryl A. , Hynes Michael J. 
Published: December 2019

ABSTRACT

The gene
nmrA
of
Aspergillus nidulans
has been isolated and found to be a homolog of the
Neurospora crassa
gene
nmr-1
, involved in nitrogen metabolite repression. Deletion of
nmrA
results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting
areA
gene.
Expression and Regulation of the
sodF
Gene Encoding Iron- and Zinc-Containing Superoxide Dismutase in
Streptomyces coelicolor
Müller
Authors: Kim Eun-Ja , Chung Hye-Jung , Suh Bumsu , Hah Yung Chil , Roe Jung-Hye 
Published: December 2019

ABSTRACT

Streptomyces coelicolor
Müller contains two superoxide dismutases (SODs), nickel-containing (NiSOD) and iron- and zinc-containing SOD (FeZnSOD). The
sodF
gene encoding FeZnSOD was isolated by using PCR primers corresponding to the N-terminal peptide sequence of the purified FeZnSOD and a C-terminal region conserved among known FeSODs and MnSODs. The deduced amino acid sequence exhibited highest similarity to Mn- and FeSODs from
Propionibacterium shermanii
and
Mycobacterium
spp. The transcription start site of the
sodF
gene was determined by primer extension. When the
sodF
gene was cloned in pIJ702 and introduced into
Streptomyces lividans
TK24, it produced at least 30 times more FeZnSOD than the control cells. We disrupted the
sodF
gene in
S. lividans
TK24 and found that the disruptant did not produce any FeZnSOD enzyme activity but produced more NiSOD. The expression of the cloned
sodF
gene in TK24 cells was repressed significantly by Ni, consistent with the regulation pattern in nonoverproducing cells. This finding suggests that the cloned
sodF
gene contains the
cis
-acting elements necessary for Ni regulation. When the
sodF
mRNA in
S. coelicolor
Müller cells was analyzed by S1 mapping of both 5′ and 3′ ends, we found that Ni caused a reduction in the level of monocistronic
sodF
transcripts. Ni did not affect the stability of
sodF
mRNA, indicating that it regulates transcription.
S. lividans
TK24 cells overproducing FeZnSOD became more resistant to oxidants such as menadione and lawsone than the control cells, suggesting the protective role of FeZnSOD. However, the
sodF
disruptant survived as well as the wild-type strain in the presence of these oxidants, suggesting the complementing role of NiSOD increased in the disruptant.
The Periplasmic, Group III Catalase ofVibrio fischeriIs Required for Normal Symbiotic Competence and Is Induced Both by Oxidative Stress and by Approach to Stationary Phase
Authors: Visick Karen L. , Ruby Edward G. 
Published: December 2019

ABSTRACTThe catalase gene,katA, of the sepiolid squid symbiontVibrio fischerihas been cloned and sequenced. The predicted amino acid sequence of KatA has a high degree of similarity to the recently defined group III catalases, including those found inHaemophilus influenzae,Bacteroides fragilis, andProteus mirabilis. Upstream of the predicted start codon ofkatAis a sequence that closely matches the consensus sequence for promoters regulated inEscherichia coliby the alternative sigma factor encoded byrpoS. Further, the level of expression of the clonedkatAgene in anE. coli rpoSmutant is much lower than in wild-typeE. coli. Catalase activity is induced three- to fourfold both as growingV. fischericells approach stationary phase and upon the addition of a small amount of hydrogen peroxide during logarithmic growth. The catalase activity was localized in the periplasm of wild-typeV. fischericells, where its role could be to detoxify hydrogen peroxide coming from the external environment. No significant catalase activity could be detected in akatAnull mutant strain, demonstrating that KatA is the predominately expressed catalase inV. fischeriand indicating thatV. fischericarries only a single catalase gene. The catalase mutant was defective in its ability to competitively colonize the light organs of juvenile squids in coinoculation experiments with the parent strain, suggesting that the catalase enzyme plays an important role in the symbiosis betweenV. fischeriand its squid host.
Generation and Properties of a
Streptococcus pneumoniae
Mutant Which Does Not Require Choline or Analogs for Growth
Authors: Yother Janet , Leopold Klaus , White Johanna , Fischer Werner 
Published: December 2019

ABSTRACT

A mutant (JY2190) of
Streptococcus pneumoniae
Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (
N
-acetylmuramyl-
l
-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86–93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.
PP
i
-Dependent Phosphofructokinase from
Thermoproteus tenax
, an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution
Authors: Siebers Bettina , Klenk Hans-Peter , Hensel Reinhard 
Published: December 2019

ABSTRACT

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC
2.7.1.11
), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP
i
-dependent PFK (PP
i
-PFK; EC
2.7.1.90
), which uses PP
i
instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP
i
-PFK from the anaerobic, hyperthermophilic archaeon
Thermoproteus tenax
, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP
i
-PFK from
T. tenax
consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from
Bacteria
and
Eucarya
; PFK group II contains only PP
i
-PFKs from the genus
Propionibacterium
, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP
i
-dependent enzymes from
T. tenax
and
Amycolatopsis methanolica
and the ATP-PFK from
Streptomyces coelicolor
. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP
i
as a cosubstrate.
Molecular Analysis of the Gene Encoding F
420
-Dependent Glucose-6-Phosphate Dehydrogenase from
Mycobacterium smegmatis
Authors: Purwantini Endang , Daniels Lacy 
Published: December 2019

ABSTRACT

The gene
fgd
, which codes for F
420
-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from
Mycobacterium smegmatis
, and its sequence was determined and analyzed. A homolog of FGD which has a very high similarity to the
M. smegmatis
FGD-derived amino acid sequence was identified in
Mycobacterium tuberculosis
. FGD showed significant homology with F
420
-dependent
N
5
,
N
10
-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from
M. tuberculosis
and
Archaeoglobus fulgidus
, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases. Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences. Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences. Moderately high levels of FGD were expressed in
Escherichia coli
BL21(DE3) carrying
fgd
in pBluescript.