bluuebunny/crossref_metadata_2025_split huggingface dataset:
ilvIH
Operon Expression in
Escherichia coli
Requires Lrp Binding to Two Distinct Regions of DNA
Authors: Jafri Samina , Chen Shaolin , Calvo Joseph M. 
Published: February 2003

ABSTRACT

The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in
Escherichia coli
, including the
ilvIH
operon. Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the
ilvIH
promoter, an upstream region (−260 to −190) and a downstream region (−150 to −40). The effect of mutations in these regions on
ilvIH
promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6. Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q. Wang and J. M. Calvo, J. Mol. Biol. 229:306-318, 1993). Here we present data that require a reevaluation of the above conclusion. Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position −160 showed no
ilvIH
promoter activity in vivo. These results unambiguously establish that DNA at or upstream of position −160 is required for
ilvIH
promoter expression. Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation. In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations. Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others. These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp. Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter.
Regulatory Response of
Methanococcus maripaludis
to Alanine, an Intermediate Nitrogen Source
Authors: Lie Thomas J. , Leigh John A. 
Published: February 2003

ABSTRACT

In the methanogenic archaeon
Methanococcus maripaludis
, growth with ammonia results in conditions of nitrogen excess. Complete repression of nitrogen fixation (
nif
) gene transcription occurs, and glutamine synthetase (
glnA
) gene transcription falls to a basal constitutive level. In addition, ammonia completely switches off nitrogenase enzyme activity. In contrast, growth with dinitrogen as the sole nitrogen source results in nitrogen starvation, full expression of
nif
and
glnA
, and high activity of nitrogenase. Here we report that a third nitrogen source, alanine, results in an intermediate regulatory response. Growth with alanine resulted in intermediate transcription of
nif
and
glnA
, and addition of alanine to a nitrogen-fixing (diazotrophic) culture caused partial switch-off of nitrogenase. This uniformity of response occurred despite differences in regulatory mechanisms. Nitrogenase switch-off requires the nitrogen sensor homologs NifI
1
and NifI
2
, while transcriptional regulation of
nif
and
glnA
relies on a different, unknown sensor mechanism. In addition, although
nif
and
glnA
transcription are governed by a common repressor, the numbers and arrangements of repressor binding sites differ. Thus, the
nif
promoter region contains two operators situated downstream of the transcription start site, while the
glnA
promoter region contains only one operator just upstream of two closely spaced transcription start sites. In a previous study of
nif
expression using ammonia, we were able to detect a role only for the first
nif
operator in repression. Here we show that
nif
repression by alanine requires the second operator as well. In contrast, in the case of
glnA
the single operator was sufficient for repression by ammonia or alanine. These results suggest a uniform cellular response to nitrogen that is mediated by a different mechanism in each case.
Adaptation of Sucrose Metabolism in the
Escherichia coli
Wild-Type Strain EC3132†
Authors: Jahreis Knut , Bentler Lars , Bockmann Jürgen , Hans Stephan , Meyer Astrid , Siepelmeyer Jörg , Lengeler Joseph W. 
Published: February 2003

ABSTRACT

Although
Escherichia coli
strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding
csc
genes and adjacent genes. The
csc
regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes
cscB
,
cscK
, and
cscA
, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore,
csc
gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the
E. coli
strains EC3132, K-12, and O157:H7 in addition to
Salmonella enterica
serovar Typhimurium LT2 revealed that the
csc
genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the
csc
genes might have been transferred relatively recently to the
E. coli
wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene
argW
for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the
csc
genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised
cscR
(Con) mutations that expressed all
csc
genes constitutively. The second class constituted a
cscKo
operator mutation, which became inducible for
csc
gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.
Structural Maintenance of Chromosomes Protein of
Bacillus
subtilis
Affects Supercoiling In Vivo
Authors: Lindow Janet C. , Britton Robert A. , Grossman Alan D. 
Published: February 2003

ABSTRACT

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion.
Bacillus
subtilis
SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an
smc
null mutation on DNA supercoiling in vivo. We found that an
smc
null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an
smc
null mutant. Plasmid DNA isolated from an
smc
null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.
MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase inEscherichia coli
Authors: Zhang Junjie , Inouye Masayori 
Published: February 2003

ABSTRACTEra is an essential GTPase inEscherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using theeragene as bait in the yeast two-hybrid system to screenE. coligenomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPγS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi, with a preference for deoxynucleotides. AmazGdeletion strain ofE. coliwas constructed by replacing themazGgene with a kanamycin resistance gene. UnlikemutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, themazGdeletion did not result in a mutator phenotype inE. coli.
Interdependent Expression of the
ccoNOQP
-
rdxBHIS
Loci in
Rhodobacter sphaeroides
2.4.1
Authors: Roh Jung Hyeob , Kaplan Samuel 
Published: February 2003

ABSTRACT

The
rdxBHIS
gene cluster of
Rhodobacter sphaeroides
2.4.1, located downstream of the
ccoNOQP
operon encoding the
cbb
3
cytochrome
c
oxidase, is required for the posttranscriptional modification of the
cbb
3
cytochrome
c
oxidase. The
cbb
3
cytochrome
c
oxidase is the main terminal oxidase under microaerobic conditions, as well as a component of the signal transduction pathway controlling photosynthesis gene expression. Because of the intimate functional and positional relationships of the
ccoNOQP
operon and the
rdxBHIS
gene cluster, we have examined the transcriptional activities of this DNA region in order to understand their expression and regulation. Northern blot analysis and reverse transcription-PCR, together with earlier complementation analysis, suggested that the
ccoNOQP
-
rdxBHIS
cluster is transcribed as
ccoNOQP
-,
ccoNOQP
-
rdxBH
-,
rdxBH
-, and
rdxIS
-specific transcripts. Multiple transcriptional start sites have been identified by primer extension analyses: five for
ccoN
, four for
rdxB
, and one for
rdxI
. Transcription from P1
N
of
ccoN
and P1
B
of
rdxB
is dependent on the presence of FnrL. LacZ fusion analysis support the above-described studies, especially the importance of FnrL. Expression of the
cco
-
rdx
cluster is closely related to photosynthesis gene expression, suggesting that transcript stoichiometry and presumably the stoichiometry of the gene products are critical factors in controlling photosynthesis gene expression.
Stimulation of Menaquinone-Dependent Electron Transfer in the Respiratory Chain ofBacillus subtilisby Membrane Energization
Authors: Azarkina N. , Konstantinov A. A. 
Published: February 2003

ABSTRACTAt a pH of ≤7, respiration ofBacillus subtiliscells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanidem-chlorophenylhydrazone [CCCP]) and a K+-ionophore (valinomycin). The same effect was observed with cell spheroplasts lacking the cell wall. The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+-valinomycin was below 100 nM. Either CCCP or valinomycin alone was much less efficient than the combination of the two. The inhibitory effect was easily reversible and depended specifically on the H+and K+concentrations in the medium. Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) andN,N,N′,N′-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol. Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling. The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores. We conclude that the menaquinone-dependent electron transfer in theB. subtilisrespiratory chain is facilitated, thermodynamically or kinetically, by membrane energization. A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant ofB. subtilislacking succinate:quinone reductase as well as for substrates other than succinate. Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration inB. subtilisare discussed.
Three Modifications in the D and T Arms of tRNA Influence Translation in
Escherichia coli
and Expression of Virulence Genes in
Shigella flexneri
Authors: Urbonavičius Jaunius , Durand Jérôme M. B. , Björk Glenn R. 
Published: February 2003

ABSTRACT

The modified nucleosides 2′-
O
-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m
5
U54), and pseudouridine, present at position 55 (Ψ55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium
Escherichia coli
. The formation of these modified nucleosides is catalyzed by the products of genes
trmH
(Gm18),
trmA
(m
5
U54), and
truB
(Ψ55). The combination of
trmH
,
trmA
, and
truB
mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-
dl
-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Ψ55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Ψ55 in tRNA of human pathogen
Shigella flexneri
leads to a reduced expression of several virulence-associated genes.
Activation of the
glnA
,
glnK
, and
nac
Promoters as
Escherichia coli
Undergoes the Transition from Nitrogen Excess Growth to Nitrogen Starvation
Authors: Atkinson Mariette R. , Blauwkamp Timothy A. , Bondarenko Vladamir , Studitsky Vasily , Ninfa Alexander J. 
Published: February 2003

ABSTRACT

The nitrogen-regulated genes and operons of the Ntr regulon of
Escherichia coli
are activated by the enhancer-binding transcriptional activator NRI∼P (NtrC∼P). Here, we examined the activation of the
glnA
,
glnK
, and
nac
promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does
glnA
. A diauxic growth pattern was obtained when
E. coli
was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than
glnA
become activated only upon amplification of the NRI concentration.
Context-Dependent Functions of the PII and GlnK Signal Transduction Proteins in
Escherichia coli
Authors: Atkinson Mariette R. , Blauwkamp Timothy A. , Ninfa Alexander J. 
Published: February 2003

ABSTRACT

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated
glnK
promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated
glnA
promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.
Purification, Substrate Range, and Metal Center of AtzC: the
N
-Isopropylammelide Aminohydrolase Involved in Bacterial Atrazine Metabolism
Authors: Shapir Nir , Osborne Jeffrey P. , Johnson Gilbert , Sadowsky Michael J. , Wackett Lawrence P. 
Published: February 2003

ABSTRACT

N
-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in
Pseudomonas
sp. strain ADP, catalyzes the stoichiometric hydrolysis of
N
-isopropylammelide to cyanuric acid and isopropylamine. The
atzC
gene was cloned downstream of the
tac
promoter and expressed in
Escherichia coli
, where the expressed enzyme comprised 36% of the soluble protein. AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography. It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000. The

K
m

and
k
cat
values for AtzC with
N
-isopropylammelide were 406 μM and 13.3 s
−1
, respectively. AtzC hydrolyzed other
N
-substituted amino dihydroxy-
s
-triazines, and those with linear
N
-alkyl groups had higher
k
cat
values than those with branched alkyl groups. Native AtzC contained 0.50 eq of Zn per subunit. The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts. Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Δε = 84 M
−1
cm
−1
) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values:
g

(
x
)

= 5.18,
g

(
y
)

= 3.93, and
g

(
z
)

= 2.24. Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to
g

(
x
)

= 5.11,
g

(
y
)

= 4.02, and
g

(
z
)

= 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW. Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of
s
-triazine herbicides.
Control of Inducer Accumulation Plays a Key Role in Succinate-Mediated Catabolite Repression in
Sinorhizobium
meliloti
Authors: Bringhurst Ryan M. , Gage Daniel J. 
Published: February 2003

ABSTRACT

The symbiotic, nitrogen-fixing bacterium
Sinorhizobium
meliloti
favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the α-galactosides raffinose and stachyose. We isolated
lacR
mutants in a genetic screen designed to find
S
.
meliloti
mutants that had abnormal succinate-mediated catabolite repression of the
melA-agp
genes, which are required for the utilization of raffinose and other α-galactosides. The loss of catabolite repression in
lacR
mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of β-galactoside utilization genes in
lacR
mutants. However, the inactivation of
lacR
did not cause the constitutive expression of α-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which
lacR
mutants overproduce β-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced β-galactoside transporters and cause the induction of α-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a
lacF
lacR
double mutant (
lacF
encodes a β-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.
Forespore Signaling Is Necessary for Pro-σKProcessing duringBacillus subtilisSporulation Despite the Loss of SpoIVFA upon Translational Arrest
Authors: Kroos Lee , Yu Yuen-Tsu Nicco , Mills Denise , Ferguson-Miller Shelagh 
Published: February 2003

ABSTRACTThe σKcheckpoint coordinates gene expression in the mother cell with signaling from the forespore duringBacillus subtilissporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-σK, and the resulting σKRNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-σKprocessing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-σKprocessing began during sporulation. In extracts of aspoIVBmutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-σKwas observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent ofspoIVB. The inability to process pro-σKunder these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-σK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-σKprocessing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.