bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role and Mechanism of Action of C ·
Pvu
II, a Regulatory Protein Conserved among Restriction-Modification Systems
Authors: Vijesurier Roy M. , Carlock Leon , Blumenthal Robert M. , Dunbar Joan C. 
Published: July 2002

ABSTRACT

The
Pvu
II restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The
Pvu
II system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (
pvuIIC
) between the divergently oriented genes for the restriction endonuclease (
pvuIIR
) and modification methyltransferase (
pvuIIM
), with
pvuIIC
in the same orientation as and partially overlapping
pvuIIR
. The product of
pvuIIC
, C ·
Pvu
II, was found to act in
trans
and to be required for expression of
pvuIIR
. In this study we demonstrate that premature expression of
pvuIIC
prevents establishment of the
Pvu
II genes, consistent with the model that requiring C ·
Pvu
II for
pvuIIR
expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing
pvuIIC
and
pvuIIM
transcripts overlap by over 60 nucleotides at their 5′ ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C ·
Pvu
II, demonstrating that it is a sequence-specific DNA-binding protein that binds to the
pvuIIC
promoter and stimulates transcription of both
pvuIIC
and
pvuIIR
into a polycistronic mRNA. The apparent location of C ·
Pvu
II binding, overlapping the −10 promoter hexamer and the
pvuIICR
transcriptional starting points, is highly unusual for transcriptional activators.
Cloning and Characterization of the
str
Operon and Elongation Factor Tu Expression in
Bacillus stearothermophilus
Authors: Krásný Libor , Vacík Tomáš , Fučík Vladimír , Jonák Jiří 
Published: July 2002

ABSTRACT

The complete primary structure of the
str
operon of
Bacillus stearothermophilus
was determined. It was established that the operon is a five-gene transcriptional unit: 5′-
ybxF
(unknown function; homology to eukaryotic ribosomal protein L30)-
rpsL
(S12)-
rpsG
(S7)-
fus
(elongation factor G [EF-G])-
tuf
(elongation factor Tu [EF-Tu])-3′. The main operon promoter (
str
p) was mapped upstream of
ybxF
, and its strength was compared with the strength of the
tuf
-specific promoter (
tuf
p) located in the
fus-tuf
intergenic region. The strength of the
tuf
p region to initiate transcription is about 20-fold higher than that of the
str
p region, as determined in chloramphenicol acetyltransferase assays. Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting
cis
elements, identified upstream of
str
p (an inhibitory region) and
tuf
p (a stimulatory A/T-rich block). Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the
str
operon genes, as monitored by the expression of EF-Tu and EF-G.
Cooperative Action of the Catabolite Activator Protein and AraC In Vitro at the
araFGH
Promoter
Authors: Johnson Casonya M. , Schleif Robert F. 
Published: July 2002

ABSTRACT

Full activation of transcription of the
araFGH
promoter,

p
FGH

, requires both the catabolite activator protein (CAP) and AraC protein. At

p
FGH

, the binding site for CAP is centered at position −41.5, an essential binding site for AraC is centered at position −79.5, and a second, nonessential binding site is centered at position −154.5. In this work, we used the minimal promoter region required for in vivo activation of

p
FGH

to examine the roles of CAP and AraC in stimulating formation of open complexes at

p
FGH

. Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-

p
FGH

complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true

p
FGH

promoter.
Genetic Characterization and Evolutionary Implications of a
car
Gene Cluster in the Carbazole Degrader
Pseudomonas
sp. Strain CA10
Authors: Nojiri Hideaki , Sekiguchi Hiroyo , Maeda Kana , Urata Masaaki , Nakai Sei-Ichiro , Yoshida Takako , Habe Hiroshi , Omori Toshio 
Published: July 2002

ABSTRACT

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the
carAaAaBaBbCAc
(ORF7)
Ad
genes of carbazole-degrading
Pseudomonas
sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the
car
gene cluster was consequently revealed to consist of 10 genes (
carAaAaBaBbCAcAdDFE
) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between
carD
and
carFE
, makes it quite likely that the
carFE
genes were recruited from another locus. In the 21-kb region upstream from
carAa
, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated
antABC
. Four homologous insertion sequences, IS
5car1
to IS
5car4
, were identified in the neighboring regions of
car
and
ant
genes. IS
5car2
and IS
5car3
constituted the putative composite transposon containing
antABC
. One-ended transposition of IS
5car2
together with the 5′ portion of
antA
into the region immediately upstream of
carAa
had resulted in the formation of IS
5car1
and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the
car
gene cluster has been constructed. In addition, it was also revealed that the
car
and
ant
gene clusters are located on the megaplasmid pCAR1.
Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi
Authors: Navas Jesús , González-Zorn Bruno , Ladrón Néstor , Garrido Patricia , Vázquez-Boland José A. 
Published: July 2002

ABSTRACTThe virulence mechanisms of the facultative intracellular parasiteRhodococcus equiremain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, thechoEmonocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified inBrevibacterium sterolicumandStreptomycesspp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded byMycobacterium tuberculosisandMycobacterium leprae. Genetic tools for use withR. equiare poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (theaacC4apramycin resistance gene fromSalmonella), and homologous recombination. ThechoEallele was disrupted by insertion of theaacC4gene, cloned in pUC19 and introduced by electroporation inR. equi. choErecombinants were isolated at frequencies between 10−2and 10−3. Twelve percent of the recombinants were double-crossoverchoEmutants. ThechoEmutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression ofchoEfrom pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor forR. equi. The highly efficient targeted mutagenesis procedure that we used to generatechoEisogenic mutants will be a valuable tool for the molecular analysis ofR. equivirulence.
Methanococcus jannaschiiGeneratesl-Proline by Cyclization ofl-Ornithine
Authors: Graupner Marion , White Robert H. 
Published: July 2002

ABSTRACTCell extracts ofMethanococcus jannaschiihave been shown to readily convertl-ornithine tol-proline. This cyclization reaction proceeds with the loss of only the C-2 nitrogen, as has been documented for ornithine cyclodeaminase (EC4.3.1.12). Since no gene homologous to that coding for ornithine cyclodeaminase is present in the genome ofM. jannaschii, these results indicate that proline biosynthesis inM. jannaschiiis accomplished by a previously unrecognized enzyme.
Role of NtcB in Activation of Nitrate Assimilation Genes in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Aichi Makiko , Takatani Nobuyuki , Omata Tatsuo 
Published: July 2002

ABSTRACT

In
Synechocystis
sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units,
nrtABCD
-
narB
and
nirA
, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in
Synechococcus
sp. strain PCC 7942. The
slr0395
gene, which encodes a protein 47% identical to
Synechococcus
NtcB, was identified as the
Synechocystis ntcB
gene, on the basis of the inability of an
slr0395
mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While
Synechococcus
NtcB strictly requires nitrite for its action,
Synechocystis
NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the
Synechococcus ntcB
mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the
Synechocystis ntcB
mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in
Synechocystis
. Activities of the nitrate assimilation enzymes in the
Synechocystis ntcB
mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.
The Global Posttranscriptional Regulator RsmA Modulates Production of Virulence Determinants and
N-
Acylhomoserine Lactones in
Pseudomonas aeruginosa
Authors: Pessi Gabriella , Williams Faye , Hindle Zoë , Heurlier Karin , Holden Matthew T. G. , Cámara Miguel , Haas Dieter , Williams Paul 
Published: July 2002

ABSTRACT

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a
Pseudomonas aeruginosa
gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of
rsmA
resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of
rsmA
in
P. aeruginosa
had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on
N
-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of
N
-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and
N
-butanoylhomoserine lactone (C4-HSL) produced by the
rsmA
mutant and the
rsmA
-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using
lasI
and
rhlI
translational fusions. These data also highlighted the temporal expression control of the
lasI
gene, which was induced much earlier and to a higher level during the exponential growth phase in an
rsmA
mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control,
hcn
translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (
hcnABC
). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the
hcnA
ribosome-binding site. This suggests that, in
P. aeruginosa,
RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.
Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria
Authors: Leveau Johan H. J. , Lindow Steven E. 
Published: July 2002

ABSTRACT

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-
gfp
fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P

A1/O4/O3

in response to increasing concentrations of the inducer IPTG (isopropyl-
β
-
d
-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P

A1/O4/O3

. Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in
Erwinia herbicola
than in
Escherichia coli
. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-
gfp
fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.
Noncoupled NADH:Ubiquinone Oxidoreductase of
Azotobacter vinelandii
Is Required for Diazotrophic Growth at High Oxygen Concentrations
Authors: Bertsova Yulia V. , Bogachev Alexander V. , Skulachev Vladimir P. 
Published: July 2002

ABSTRACT

The gene encoding the noncoupled NADH:ubiquinone oxidoreductase (NDH II) from
Azotobacter vinelandii
was cloned, sequenced, and used to construct an NDH II-deficient mutant strain. Compared to the wild type, this strain showed a marked decrease in respiratory activity. It was unable to grow diazotrophically at high aeration, while it was fully capable of growth at low aeration or in the presence of NH
4
+
. This result suggests that the role of NDH II is as a vital component of the respiratory protection mechanism of the nitrogenase complex in
A. vinelandii
. It was also found that the oxidation of NADPH in the
A. vinelandii
respiratory chain is catalyzed solely by NDH II.
The LexA Protein from
Deinococcus radiodurans
Is Not Involved in RecA Induction following γ Irradiation
Authors: Narumi Issay , Satoh Katsuya , Kikuchi Masahiro , Funayama Tomoo , Yanagisawa Tadashi , Kobayashi Yasuhiko , Watanabe Hiroshi , Yamamoto Kazuo 
Published: July 2002

ABSTRACT

The involvement of LexA in induction of RecA was investigated in
Deinococcus radiodurans
. As in the wild-type strain, an increase in RecA protein synthesis following γ irradiation was detected in a
lexA
disruptant, indicating that LexA is not involved in the induction of RecA in
D. radiodurans
.
Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from
Ralstonia eutropha
Strain Bo
Authors: Zarnt Grit , Schräder Thomas , Andreesen Jan R. 
Published: July 2002

ABSTRACT

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from
Ralstonia eutropha
strain Bo was investigated for its catalytic properties. The apparent
k
cat
/

K
m

and
K
i
values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with
n
-pentanol exhibiting a
k
cat
/

K
m

value of 788 × 10
4
M
−1
s
−1
. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from
R. eutropha
strain Bo (
tfaA
) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.
The
ner
Gene of
Photorhabdus
: Effects on Primary-Form-Specific Phenotypes and Outer Membrane Protein Composition
Authors: O'Neill Keith H. , Roche Declan M. , Clarke David J. , Dowds Barbara C. A. 
Published: July 2002

ABSTRACT

The nematode-bacterium complex of
Heterorhabditis-Photorhabdus
is pathogenic to insect larvae. The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes. The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries. Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as
ner
, a regulatory gene in gram-negative bacteria and their phages. Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form. These and other pieces of evidence indicate that the endogenous
ner
gene is not responsible for the secondary phenotype, but that
ner
, when overexpressed, can repress expression of primary phenotypes at stationary phase. Inactivation of the endogenous
ner
gene in the primary form affected the outer membrane protein profile. A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the
ner
primary strain.
Atypical Processing in Domain III of 23S rRNA of
Rhizobium leguminosarum
ATCC 10004
T
at a Position Homologous to an rRNA Fragmentation Site in Protozoa
Authors: Klein Franziska , Samorski Regina , Klug Gabriele , Evguenieva-Hackenberg Elena 
Published: July 2002

ABSTRACT

For still unknown reasons, the 23S rRNA of many α-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The α-proteobacterium
Rhizobium leguminosarum
ATCC 10004
T
possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5′ end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.
The Products of the
spoVA
Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of
Bacillus subtilis
Authors: Tovar-Rojo Federico , Chander Monica , Setlow Barbara , Setlow Peter 
Published: July 2002

ABSTRACT

Bacillus subtilis
cells with mutations in the
spoVA
operon do not complete sporulation. However, a
spoVA
strain with mutations that remove all three of the spore’s functional nutrient germinant receptors (termed the
ger3
mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation.
ger3 spoVA
and
sleB spoVA
spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or
ger3
spores. These properties of
ger3 spoVA
and
sleB spoVA
spores are identical to those of
ger3 spoVF
and
sleB spoVF
spores that lack DPA due to deletion of the
spoVF
operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in
ger3 spoVF
spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into
ger3 spoVA
spores. These data indicate that one or more products of the
spoVA
operon are involved in DPA transport into the developing forespore during sporulation.