bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyanobacterial Sulfide-Quinone Reductase: Cloning and Heterologous Expression
Authors: Bronstein Michal , Schütz Michael , Hauska Günter , Padan Etana , Shahak Yosepha 
Published: July 2002

ABSTRACT

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.′), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium
Oscillatoria limnetica
, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the
Rhodobacter capsulatus
SQR and in an open reading frame found in the genome of
Aquifex aeolicus
. SQR activity was also detected in the unicellular cyanobacterium
Aphanothece halophytica
following sulfide induction, with a
V
max
of 180 μmol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent

K
m

values of 20 and 40 μM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding
A. halophytica
SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for
O. limnetica
SQR and 437 amino acids for
A. halophytica
SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and −14 for
O. limnetica
SQR and
A. halophytica
SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium
Anabaena
PCC7120 as well as the chemolithotrophic bacteria
Shewanella putrefaciens
and
Thiobacillus ferrooxidans
. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in
Escherichia coli
under the control of the T7 promoter. Membranes isolated from
E. coli
cells expressing
A. halophytica
SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2
n
-nonyl-4-hydroxyquinoline-
N
-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.
Virulence of the Phytopathogen
Pseudomonas syringae
pv. Maculicola Is
rpoN
Dependent
Authors: Hendrickson Erik L. , Guevera Pablo , Peñaloza-Vàzquez Alejandro , Shao Jing , Bender Carol , Ausubel Frederick M. 
Published: July 2002

ABSTRACT

We cloned the
rpoN
(
ntrA
and
glnF
) gene encoding ς
54
from the phytopathogen
Pseudomonas syringae
pv. maculicola strain ES4326. The
P. syringae
ES4326
rpoN
gene complemented
Pseudomonas aeruginosa
,
Escherichia coli
, and
Klebsiella aerogenes rpoN
mutants for a variety of
rpoN
mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the
P. syringae
ES4326
rpoN
gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the ς
54
protein encoded by the
Pseudomonas putida rpoN
gene. A marker exchange protocol was used to construct an ES4326
rpoN
insertional mutation,
rpoN
::Km
r
. In contrast to wild-type ES4326, ES4326
rpoN
::Km
r
was nonmotile and could not utilize nitrate, urea, C
4
-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources.
rpoN
was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326
rpoN
::Km
r
did not multiply or elicit disease symptoms when infiltrated into
Arabidopsis thaliana
leaves, did not elicit the accumulation of several
Arabidopsis
defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (
Nicotiana tabacum
) leaves. Furthermore, whereas
P. syringae
ES4326 carrying the avirulence gene
avrRpt2
elicited an HR when infiltrated into
Arabidopsis
ecotype Columbia leaves, ES4326
rpoN
::Km
r
carrying
avrRpt2
elicited no response. Constitutive expression of ES4326
hrpL
in ES4326
rpoN
::Km
r
partially restored defense-related mRNA accumulation, showing a direct role for the
hrp
cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of
hrpL
in ES4326
rpoN
::Km
r
did not restore coronatine production, showing that coronatine biosynthesis requires factors other than
hrpL
.
Fur Positive Regulation of Iron Superoxide Dismutase in
Escherichia coli
: Functional Analysis of the
sodB
Promoter
Authors: Dubrac Sarah , Touati Danièle 
Published: July 2002

ABSTRACT

In
Escherichia coli
, the expression of
sodB
, which encodes iron superoxide dismutase, has been suggested to be activated by Fur, the iron-responsive global regulator initially characterized as a transcriptional repressor. We investigated
sodB
regulation by functional analysis of the
sodB
promoter using
sodB-lac
fusions with various truncated and mutated promoters. Several
cis
- and
trans
-acting elements involved in
sodB
regulation have been identified. The β-galactosidase activity of
sodB-lacZ
reporter fusions and RNA analysis showed sevenfold iron-dependent, Fur-mediated activation of expression. A region just downstream from −10, including a large palindromic sequence encompassing the +1 position followed by a 14-bp AT-rich motif, is the site of Fur positive regulation, and the integrity of both sequences was required for full Fur-mediated activation. The life span of
sodB
mRNA was three times longer in a
fur
+
strain, indicating that Fur-mediated activation proceeds, at least in part, at the posttranscriptional level. The H-NS and IHF histone-like factors also affected
sodB
expression. IHF slightly repressed
sodB
expression independently of Fur regulation. In contrast, H-NS negative regulation operated only in the absence of Fur. Remarkably, p
sodB
behaved like a “pure extended -10” promoter. Deletion of the −35 region did not affect expression, whereas expression was totally abolished by a TG-to-CC mutation in the extended −10 sequence TGcTACCCT.
Identification, Expression, and Characterization of
Escherichia coli
Guanine Deaminase
Authors: Maynes Jason T. , Yuan Richard G. , Snyder Floyd F. 
Published: July 2002

ABSTRACT

Using the human cDNA sequence corresponding to guanine deaminase, the
Escherichia coli
genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a

K
m

of 15 μM with guanine and a
k
cat
of 3.2 s
−1
. The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The
E. coli
guanine deaminase locus is 3′ from an open reading frame which shows homology to a bacterial purine base permease.
Plasmid pGS5 from the Hyperthermophilic Archaeon
Archaeoglobus profundus
Is Negatively Supercoiled
Authors: López-García Purificación , Forterre Patrick , van der Oost John , Erauso Gaël 
Published: July 2002

ABSTRACT

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from
Archaeoglobus profundus
is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative
A. fulgidus
, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells.
Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene
cis
-Dihydroxylation: Control by Phenylalanine 352 in the α Subunit
Authors: Parales Rebecca E. , Resnick Sol M. , Yu Chi-Li , Boyd Derek R. , Sharma Narain D. , Gibson David T. 
Published: July 2002

ABSTRACT

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by
Pseudomonas
sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including
cis
-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the α subunit of NDO altered the stereochemistry of naphthalene
cis
-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl
cis
-3,4-dihydrodiol [77 and 60% (−)-(3
S
,4
R
), respectively] to that formed by wild-type NDO [>98% (+)-(3
R
,4
S
)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene
cis
-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from
Sphingomonas yanoikuyae
B8/36. A recombinant
Escherichia coli
strain expressing the F352V variant of NDO and the enantioselective toluene
cis
-dihydrodiol dehydrogenase from
Pseudomonas putida
F1 was used to produce enantiomerically pure (−)-biphenyl
cis
-(3
S
,4
R
)-dihydrodiol and (−)-phenanthrene
cis
-(1
S
,2
R
)-dihydrodiol from biphenyl and phenanthrene, respectively.
The
Synechococcus
Strain PCC 7942
glnN
Product (Glutamine Synthetase III) Helps Recovery from Prolonged Nitrogen Chlorosis
Authors: Sauer Jörg , Dirmeier Ulrike , Forchhammer Karl 
Published: July 2002

ABSTRACT

We report the cloning and sequencing of the
glnN
gene encoding a class III glutamine synthetase from the cyanobacterium
Synechococcus
strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of
glnN
is impaired in NtcA- and P
II
-deficient mutants. The only parameter which was negatively affected in the
glnN
mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.
Polyprenyl Phosphate Biosynthesis in
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
Authors: Crick Dean C. , Schulbach Mark C. , Zink Erin E. , Macchia Marco , Barontini Silvia , Besra Gurdyal S. , Brennan Patrick J. 
Published: July 2002

ABSTRACT

Mycobacterium smegmatis
has been shown to contain two forms of polyprenyl phosphate (Pol-P), while
Mycobacterium tuberculosis
contains only one. Utilizing subcellular fractions from
M. smegmatis
and
M. tuberculosis
, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in
M. tuberculosis
are 10- to 100-fold lower than those in
M. smegmatis
. In
M. smegmatis
decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in
M. tuberculosis
only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes ω,
E
,
Z
-farnesyl diphosphate and the other present in the cytosol that synthesizes ω,
E
,
E
,
E
-geranylgeranyl diphosphate. In
M. smegmatis
, the ω,
E
,
Z
-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the ω,
E
,
E
,
E
-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In
M. tuberculosis
, however, ω,
E
,
E
,
E
-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of
M. smegmatis
and
M. tuberculosis
can be attributed to distinct enzymatic differences between these two organisms.
Evidence of a Role for LytB in the Nonmevalonate Pathway of Isoprenoid Biosynthesis
Authors: Cunningham Francis X. , Lafond Toulouse P. , Gantt Elisabeth 
Published: July 2002

ABSTRACT

It is proposed that the
lytB
gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium
Synechocystis
strain PCC 6803 with an insertion in the promoter region of
lytB
grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of
lytB
were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The
Synechocystis lytB
gene and a
lytB
cDNA from the flowering plant
Adonis aestivalis
were each found to significantly enhance accumulation of carotenoids in
Escherichia coli
engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (
dxs
), the initial enzyme of the DOXP pathway, the individual salutary effects of
lytB
and
dxs
were multiplied. In contrast, the combination of
lytB
and a cDNA encoding IPP isomerase (
ipi
) was no more effective in enhancing carotenoid accumulation than
ipi
alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.
Inhibitory Effect of Heterologous Ribosome Recycling Factor on Growth of
Escherichia coli
Authors: Atarashi Kenji , Kaji Akira 
Published: July 2002

ABSTRACT

Ribosome recycling factor (RRF) of
Thermotoga maritima
was expressed in
Escherichia coli
from the cloned
T. maritima
RRF gene and purified. Expression of
T. maritima
RRF inhibited growth of the
E. coli
host in a dose-dependent manner, an effect counteracted by the overexpression of
E. coli
RRF.
T. maritima
RRF also inhibited the
E. coli
RRF reaction in vitro. Genes encoding RRFs from
Streptococcus pneumoniae
and
Helicobacter pylori
have been cloned, and they also impair growth of
E. coli
, although the inhibitory effect of these RRFs was less pronounced than that of
T. maritima
RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in
T. maritima
RRF differed totally from that of
E. coli
RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible for
T. maritima
RRF inhibition of the
E. coli
RRF reaction.
Evidence for a Role of
rpoE
in Stressed and Unstressed Cells of Marine
Vibrio angustum
Strain S14
Authors: Hild Erika , Takayama Kathy , Olsson Rose-Marie , Kjelleberg Staffan 
Published: July 2002

ABSTRACT

We report the cloning, sequencing, and characterization of the
rpoE
homolog in
Vibrio angustum
S14. The
rpoE
gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis. The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of
Sphingomonas aromaticivorans
, ς
24
, displaying sequence similarity and identity of 63 and 43%, respectively. Northern blot analysis demonstrated the induction of
rpoE
6, 12, and 40 min after a temperature shift to 40°C. An
rpoE
mutant was constructed by gene disruption. There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the
rpoE
mutant strain. In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation. The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30°C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30°C, compared to the wild-type strain. Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain. It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of
V. angustum
S14.
Function of a Principal Na
+
/H
+
Antiporter, ShaA, Is Required for Initiation of Sporulation in
Bacillus subtilis
Authors: Kosono Saori , Ohashi Yoshiaki , Kawamura Fujio , Kitada Makio , Kudo Toshiaki 
Published: July 2002

ABSTRACT

ShaA (sodium/hydrogen antiporter, previously termed YufT [or NtrA]), which is responsible for Na
+
/H
+
antiporter activity, is considered to be the major Na
+
excretion system in
Bacillus subtilis
. We found that a
shaA
-disrupted mutant of
B. subtilis
shows impaired sporulation but normal vegetative growth when the external Na
+
concentration was increased in a low range. In the
shaA
mutant, ς
H
-dependent expression of
spo0A
(P
S
) and
spoVG
at an early stage of sporulation was sensitive to external NaCl. The level of ς
H
protein was reduced by the addition of NaCl, while the expression of
spo0H
, which encodes ς
H
, was little affected, indicating that posttranscriptional control of ς
H
rather than
spo0H
transcription is affected by the addition of NaCl in the
shaA
mutant. Since this mutant is considered to have a diminished ability to maintain a low internal Na
+
concentration, an increased level of internal Na
+
may affect posttranscriptional control of ς
H
. Bypassing the phosphorelay by introducing the
sof-1
mutation into this mutant did not restore
spo0A
(P
S
) expression, suggesting that disruption of
shaA
affects ς
H
accumulation, but does not interfere with the phosphorylation and phosphotransfer reactions of the phosphorelay. These results suggest that ShaA plays a significant role at an early stage of sporulation and not only during vegetative growth. Our findings raise the possibility that fine control of cytoplasmic ion levels, including control of the internal Na
+
concentration, may be important for the progression of the sporulation process.
The TorR High-Affinity Binding Site Plays a Key Role in Both
torR
Autoregulation and
torCAD
Operon Expression in
Escherichia coli
Authors: Ansaldi Mireille , Simon Gwénola , Lepelletier Michèle , Méjean Vincent 
Published: July 2002

ABSTRACT

In the presence of trimethylamine
N
-oxide (TMAO), the TorS-TorR two-component regulatory system induces the
torCAD
operon, which encodes the TMAO respiratory system of
Escherichia coli
. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of
torCAD
. The
torR
gene and the
torCAD
operon are divergently transcribed, and the short
torR-torC
intergenic region contains four direct repeats (the
tor
boxes) which proved to be TorR binding sites. The
tor
box 1-box 2 region covers the
torR
transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using
torR-lacZ
operon fusions in different genetic backgrounds, we showed that the
torR
gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a
torS
mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the
torR
promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the
torCAD
operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.
Gene Families Encoding Phase- and Size-Variable Surface Lipoproteins of
Mycoplasma hyorhinis
Authors: Citti Christine , Watson-McKown Robyn , Droesse Martina , Wise Kim S. 
Published: July 2002

ABSTRACT

A prototype family of seven genes encoding the variable surface lipoproteins (Vlps) of
Mycoplasma hyorhinis
is characterized in the pathogenic SK76 strain, using long-range PCR to amplify and analyze the single chromosomal region containing expressed genes
vlpA
to -
G
, each of which is subject to phase and size variation. Smaller families of
vlp
genes in subclones of SK76 or in another strain of
M. hyorhinis
, GDL, can be attributed to deletions of specific
vlp
genes from the prototype array described here. Two genes,
vlpA
and the newly revealed
vlpG
, contain repeat motifs in their 3′ coding regions that differ from the short tandem repeats in other
vlp
genes yet retain structural features common to all
vlp
gene products. SK76 and GDL
vlp
gene families are similarly organized and show sequence similarity between corresponding individual
vlp
genes. In light of the extensive potential for diversity within the
vlp
gene system, such conservation provides a provisional basis to hypothesize that
vlp
genes may exist in specific arrays that endow selected functions while retaining common structural features required during phase-variable expression of this set of gene products.
Reinvestigation of the Proteolytic Activity of Neocarzinostatin
Authors: Heyd Bernadette , Lerat Guilhem , Adjadj Elisabeth , Minard Philippe , Desmadril Michel 
Published: July 2002

ABSTRACT

Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species. It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily. With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in
Escherichia coli
was constructed. The recombinant protein was properly folded and functional. Its histone-specific proteolytic activity was similar to the activity described for the natural protein. Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations. The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results. These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.