bluuebunny/crossref_metadata_2025_split huggingface dataset:
General Nitrogen Regulation of Nitrate Assimilation Regulatory Gene
nasR
Expression in
Klebsiella oxytoca
M5al
Authors: Wu Stephen Qitu , Chai Weihang , Lin Janine T. , Stewart Valley 
Published: December 2019

ABSTRACT

Klebsiella oxytoca
can assimilate nitrate and nitrite by using enzymes encoded by the
nasFEDCBA
operon. Expression of the
nasF
operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of
nasR
gene expression. We constructed strains bearing single-copy Φ(
nasR-lacZ
) operon fusions within the chromosomal
rhaBAD-rhaSR
locus. The expression of Δ
rhaBS
::[Φ(
nasR-lacZ
)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both
ntrC
and
rpoN
null mutants, indicating that Ntr control of
nasR
gene expression requires the NtrC and ς
N
(ς
54
) proteins. Sequence inspection of the
nasR
control region reveals an apparent ς
N
-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the ς
N
-dependent
nasR
promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of β-galactosidase expression indistinguishable from Φ(
nasR-lacZ
) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of
nasR
transcription. Together, these results indicate that
nasR
gene expression does not require specific upstream NtrC-binding sequences, as previously noted for
argT
gene expression in
Salmonella typhimurium
(G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107–117, 1988).
Negative Control of Flagellum Synthesis in
Pseudomonas aeruginosa
Is Modulated by the Alternative Sigma Factor AlgT (AlgU)
Authors: Garrett Edward S. , Perlegas Demetra , Wozniak Daniel J. 
Published: December 2019

ABSTRACT

Many respiratory isolates of
Pseudomonas aeruginosa
from cystic fibrosis patients are mucoid (alginate producing) yet lack flagella. It was hypothesized that an alginate regulator inhibits flagellar gene expression. Mutations in
algB
,
algR
, and
algT
resulted in nonmucoid derivatives, yet
algT
mutants expressed flagella. AlgT-dependent control of flagellum synthesis occurred through inhibition of
fliC
but not
rpoN
transcription.
Characterization of a Novel, Antifungal, Chitin-Binding Protein from
Streptomyces tendae
Tü901 That Interferes with Growth Polarity
Authors: Bormann Christiane , Baier Daniel , Hörr Ingmar , Raps Claudia , Berger Jürgen , Jung Günther , Schwarz Heinz 
Published: December 2019

ABSTRACT

The
afp1
gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing
Streptomyces tendae
Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against
Paecilomyces variotii
in agar diffusion assays. The 384-bp
afp1
gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for
Streptomyces
genes. AFP1 was purified from culture filtrate of
S. tendae
carrying the
afp1
gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of
P. variotii
but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of
Aspergillus
species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.
Molecular Analysis of the
Candida albicans
Homolog of
Saccharomyces cerevisiae MNN9
, Required for Glycosylation of Cell Wall Mannoproteins
Authors: Southard Susan B. , Specht Charles A. , Mishra Chitra , Chen-Weiner Joan , Robbins Phillips W. 
Published: December 2019

ABSTRACT

The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the
Candida albicans
homolog of
Saccharomyces cerevisiae MNN9
, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption.
CaMNN9
has significant homology with
S. cerevisiae MNN9
, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in
Candida. CaMNN9
resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of
CaMNN9
leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with β-1,3-glucanase. Like all members of the
S. cerevisiae MNN9
gene family the
Camnn9
Δ strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the
Camnn9
Δ strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of
MNN9
gene family members
CaVAN1
and
CaANP1
in the
Camnn9
Δ strain showed that transcription of those genes is not affected in the absence of
CaMNN9
transcription. Our results suggest that, while the role
MNN9
plays in glycosylation in both
Candida
and
Saccharomyces
is conserved, loss of
MNN9
function in
C. albicans
leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target.
Citrate Synthase Mutants of
Sinorhizobium meliloti
Are Ineffective and Have Altered Cell Surface Polysaccharides
Authors: Mortimer Michael W. , McDermott Timothy R. , York Gregory M. , Walker Graham C. , Kahn Michael L. 
Published: December 2019

ABSTRACT

The
gltA
gene, encoding
Sinorhizobium meliloti
104A14 citrate synthase, was isolated by complementing an
Escherichia coli gltA
mutant. The
S. meliloti gltA
gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type
gltA
with the
gltA
::
kan
allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0.2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.
Mutations in Catabolite Control Protein CcpA Showing Glucose-Independent Regulation in
Bacillus megaterium
Authors: Küster-Schöck Elke , Wagner Andrea , Völker Uwe , Hillen Wolfgang 
Published: December 2019

ABSTRACT
We identified five single amino acid exchanges in CcpA that lead to permanent repression of the xylose utilization genes in the absence of glucose. Other proteins from the CcpA regulon also show glucose-independent regulation in the mutants. The mutant CcpA proteins bind to the DNA target catabolite responsive elements without the corepressor HPr-Ser-P.
The Periplasmic 9.6-Kilodaltonc-Type Cytochrome ofGeobacter sulfurreducensIs Not an Electron Shuttle to Fe(III)
Authors: Lloyd Jon R. , Blunt-Harris Elizabeth L. , Lovley Derek R. 
Published: December 2019

ABSTRACTGeobacter sulfurreducenscontains a 9.6-kDac-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions ofG. sulfurreducensit did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellularc-type cytochromes in cultures ofG. sulfurreducensdemonstrated that the dominantc-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.
Topology of RbsC, a Membrane Component of the Ribose Transporter, Belonging to the AraH Superfamily
Authors: Park Yongkyu , Park Chankyu 
Published: December 2019

ABSTRACT

RbsC of
Escherichia coli
is the hydrophobic membrane component of ribose uptake system classified as the ATP-binding cassette transporter. To understand the structure and function of RbsC, its transmembrane topology was investigated by using 64 RbsC-PhoA fusions isolated either specifically or randomly. In order to confirm the cytoplasmic location of the short C-terminal region (5 amino acids), inside-out or right-side-out membrane vesicles were generated, and the C-terminal region was found to be digested by carboxypeptidase A only in inside-out vesicles. This result is consistent with the model, based on the results of alkaline phosphatase fusions, in which the protein traverses the membrane six times and the N and C termini are exposed to the cytoplasm.
Escherichia coli
Cells Exposed to Streptomycin Display a Mutator Phenotype
Authors: Ren Li , Rahman M. Sayeedur , Humayun M. Zafri 
Published: December 2019

ABSTRACT

Mistranslation mediated by the
mutA
and
mutC
tRNA alleles elicits a strong mutator phenotype (H. S. Murphy and M. Z. Humayun, J. Bacteriol. 179:7507–7514, 1997; M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380–4385, 1996). Here, we show that exposure to streptomycin, an antibiotic known to promote mistranslation, induces a
recA
- and
umuDC
-independent mutator phenotype detected as enhanced mutagenesis at a 3,N
4
-ethenocytosine lesion borne on transfected M13 single-stranded DNA.
The Presence of Glutamate Dehydrogenase Is a Selective Advantage for the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 under Nonexponential Growth Conditions
Authors: Chávez S. , Lucena J. M. , Reyes J. C. , Florencio F. J. , Candau P. 
Published: December 2019

ABSTRACT

The unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to
Synechocystis
sp. strain PCC 6803 in late stages of growth.
The
mutK
Gene of
Vibrio cholerae
: a New Gene Involved in DNA Mismatch Repair
Authors: Bhakat Kishor K. , M. Sudarshana Sharma , Das Jyotirmoy 
Published: December 2019

ABSTRACT

A new gene,
mutK
, of
Vibrio cholerae
, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the
mutK
gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of
Escherichia coli mutS
,
mutL
,
mutU
, and
dam
mutants. The spontaneous mutation frequency of a chromosomal
mutK
knockout mutant was almost identical to that of wild-type
V. cholerae
cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the
mutK
gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the
mutK
gene product. The
mutK
gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of
mutK
, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the
E. coli vsr
gene. ORF1, located farther upstream of
mutK
, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of
mutK
, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The
mutK
gene has recently been positioned in the ordered cloned DNA map of the genome of the
V. cholerae
strain from which the gene was isolated (10).
Recovery of DNA Replication in UV-Irradiated
Escherichia coli
Requires both Excision Repair and RecF Protein Function
Authors: Courcelle Justin , Crowley David J. , Hanawalt Philip C. 
Published: December 2019

ABSTRACT

After UV doses that disrupt DNA replication, the recovery of replication at replication forks in
Escherichia coli
requires a functional copy of the
recF
gene. In
recF
mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that
recF
function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of
recF
to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair,
recF
+
cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either
uvr
or
recF
result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that
recF
and excision repair contribute to a common pathway in the recovery of replication.
Phenotypic Analysis of Random
hns
Mutations Differentiate DNA-Binding Activity from Properties of
fimA
Promoter Inversion Modulation and Bacterial Motility
Authors: Donato Gina M. , Kawula Thomas H. 
Published: December 2019

ABSTRACT

H-NS is a major
Escherichia coli
nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in
hns
result in constitutive expression of the
proU
and
fimB
genes, increased
fimA
promoter inversion rates, and repression of the
flhCD
master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned
hns
gene into a mutator strain and collected a series of mutant alleles that failed to repress
proU
expression. Each of these isolated
hns
mutant alleles also failed to repress
fimB
expression, suggesting that H-NS-specific repression of
proU
and
fimB
occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type
fimA
promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various
hns
mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.
High-Molecular-Mass Multi-
c
-Heme Cytochromes from
Methylococcus capsulatus
Bath
Authors: Bergmann David J. , Zahn James A. , DiSpirito Alan A. 
Published: December 2019

ABSTRACT

The polypeptide and structural gene for a high-molecular-mass
c
-type cytochrome, cytochrome
c
553O
, was isolated from the methanotroph
Methylococcus capsulatus
Bath. Cytochrome
c
553O
is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme
c
concentration was estimated to be 8.2 ± 0.4 mol of heme
c
per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome
c
553O
was used to identify a DNA fragment from
M. capsulatus
Bath that contains
occ
, the gene encoding cytochrome
c
553O
.
occ
is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic
c
-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with
occ
and contains nine
c
-heme-binding motifs. ORF3 encodes a putative periplasmic
c
-type cytochrome with a molecular mass of 94,000 Da and contains seven
c
-heme-binding motifs but shows no sequence homology to
occ
or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome
c
553O
and the gene products of ORF1 and ORF3 constitute a new class of
c
-type cytochrome.
Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic
Crenarchaeota
and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation
Authors: Menendez Castor , Bauer Zsuzsa , Huber Harald , Gad’on Nasser , Stetter Karl-Otto , Fuchs Georg 
Published: December 2019

ABSTRACT

The pathway of autotrophic CO
2
fixation was studied in the phototrophic bacterium
Chloroflexus aurantiacus
and in the aerobic thermoacidophilic archaeon
Metallosphaera sedula
. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO
2
-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either
14
CO
2
or [
14
C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for
C. aurantiacus
. The investigation was extended to the autotrophic archaea
Sulfolobus metallicus
and
Acidianus infernus
, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as
C. aurantiacus
, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea
M. sedula
,
S. metallicus
, and
A. infernus
use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO
2
fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in
C. aurantiacus
.