bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Different-Size Transcripts from the
clpP-clpX
Operon of
Escherichia coli
during Carbon Deprivation
Authors: Li Chin , Tao Yi Ping , Simon Lee D. 
Published: July 2002

ABSTRACT

Transcription of the
clpP-clpX
operon of
Escherichia coli
leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entire
clpP-clpX
operon, whereas the shorter transcript contains the entire
clpP
gene but none of the
clpX
coding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς
32
-dependent promoter of the
clpP-clpX
operon within 15 min after the start of carbon starvation. Expression of the
clpP-clpX
operon from the promoters upstream of the
clpP
gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation.
E. coli rpoB
mutations that affect transcription termination efficiency alter the ratio of the shorter
clpP-clpX
transcript to the longer transcript. The
E. coli rpoB3595
mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the
rpoB8
mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter
clpP-clpX
transcript when
E. coli
is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.
Enzymatic Removal of Nitric Oxide Catalyzed by Cytochrome
c
′ in
Rhodobacter capsulatus
Authors: Cross Richard , Lloyd David , Poole Robert K. , Moir James W. B. 
Published: July 2002

ABSTRACT

Cytochrome
c
′ from
Rhodobacter capsulatus
has been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome
c
′ synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome
c
′ is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N
2
O), thus showing that cytochrome
c
′ is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome
c
′ is distinct from the NO reductase pathway that is involved in denitrification. Cytochrome
c
′ is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome
c
′ synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of
R. capsulatus
by NO suggests that one role for cytochrome
c
′ is to maintain oxidase activity when both NO and O
2
are present.
SdeK, a Histidine Kinase Required for
Myxococcus xanthus
Development
Authors: Pollack Jeffrey S. , Singer Mitchell 
Published: July 2002

ABSTRACT

The
sdeK
gene is essential to the
Myxococcus xanthus
developmental process. We reported previously, based on sequence analysis (A. G. Garza, J. S. Pollack, B. Z. Harris, A. Lee, I. M. Keseler, E. F. Licking, and M. Singer, J. Bacteriol. 180:4628–4637, 1998), that SdeK appears to be a histidine kinase. In the present study, we have conducted both biochemical and genetic analyses to test the hypothesis that SdeK is a histidine kinase. An SdeK fusion protein containing an N-terminal polyhistidine tag (His-SdeK) displays the biochemical characteristics of a histidine kinase. Furthermore, histidine 286 of SdeK, the putative site of phosphorylation, is required for both in vitro and in vivo protein activity. The results of these assays have led us to conclude that SdeK is indeed a histidine kinase. The developmental phenotype of a Δ
sdeK1
strain could not be rescued by codevelopment with wild-type cells, indicating that the defect is not due to the mutant's inability to produce an extracellular signal. Furthermore, the Δ
sdeK1
mutant was found to produce both A- and C-signal, based on A-factor and codevelopment assays with a
csgA
mutant, respectively. The expression patterns of several Tn
5lacZ
transcriptional fusions were examined in the Δ
sdeK1
-null background, and we found that all C-signal-dependent fusions assayed also required SdeK for full expression. Our results indicate that SdeK is a histidine kinase that is part of a signal transduction pathway which, in concert with the C-signal transduction pathway, controls the activation of developmental-gene expression required to progress past the aggregation stage.
Involvement of the TonB System in Tolerance to Solvents and Drugs inPseudomonas putidaDOT-T1E
Authors: Godoy Patricia , Ramos-González Marı́a Isabel , Ramos Juan L. 
Published: July 2002

ABSTRACTPseudomonas putidaDOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM)p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5′phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with14C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted theexbDgene, which forms part of theexbBD tonBoperon. Complementation by theexbBDandtonBgenes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.
Negative Control of
rpoS
Expression by Phosphoenolpyruvate:Carbohydrate Phosphotransferase System in
Escherichia coli
Authors: Ueguchi Chiharu , Misonou Naoko , Mizuno Takeshi 
Published: July 2002

ABSTRACT

The ς
S
(or ς
38
) subunit of RNA polymerase, encoded by the
rpoS
gene, is a crucial regulator in the transcriptional control of a set of genes under stressful conditions, such as nutrient starvation. The expression of
rpoS
is regulated in a complex manner at the levels of transcription, translation, and stability of the product. Although a number of factors involved in the regulation of
rpoS
expression have been identified, the underlying molecular mechanisms are not fully understood. In this study, we identified the Crr (or EIIA
Glc
) protein as a novel factor that plays an important role not only in the transcriptional control but also in the translational control of
rpoS
expression. Crr is an important component in glucose uptake through the well-characterized phosphoenolpyruvate:carbohydrate phosphotransferase system. The results of a series of genetic analyses revealed that Crr negatively controls
rpoS
translation and transcription. The observed transcriptional control by Crr appears to be mediated by cyclic AMP. However, it was found that Crr negatively controls
rpoS
translation rather directly. These results suggest a possible linkage between the control of
rpoS
expression and carbon metabolism.
Benzylsuccinate Synthase of
Azoarcus
sp. Strain T: Cloning, Sequencing, Transcriptional Organization, and Its Role in Anaerobic Toluene and
m
-Xylene Mineralization
Authors: Achong Gypsy R. , Rodriguez Ana M. , Spormann Alfred M. 
Published: July 2002

ABSTRACT

Biochemical studies in
Azoarcus
sp. strain T have demonstrated that anaerobic oxidation of both toluene and
m
-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from
Azoarcus
sp. strain T and examined the role of this enzyme in both anaerobic toluene and
m
-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase,
bssCAB
, together with two additional genes,
bssD
and
bssE
, were organized in an operon in the order
bssDCABE. bssD
is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase.
bssE
shows homology to
tutH
from
Thauera aromatica
strain T1, whose function is currently unknown. A second operon that is upstream of
bssDCABE
and divergently transcribed contains two genes,
tdiS
and
tdiR
. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null
bssA
mutant was constructed (the
bssA
gene encodes the α-subunit of benzylsuccinate synthase). This
bssA
null mutant strain was unable to grow under denitrifying conditions on either toluene or
m
-xylene, while growth on benzoate was unaffected. The growth phenotype of the Δ
bssA
mutant could be rescued by reintroducing
bssA
in
trans
. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and
m
-xylene.
Oversynthesis of a New
Escherichia coli
Small RNA Suppresses Export Toxicity of DsbA′-PhoA Unfoldable Periplasmic Proteins
Authors: Guigueno Agnès , Dassa Janie , Belin Pascal , Boquet Paul Louis 
Published: July 2002

ABSTRACT

In
Escherichia coli
, the DsbA′-PhoA hybrid proteins carrying an unfoldable DsbA′ fragment can be targeted to the envelope, where they exert their toxicity. Hybrid proteins stick to the periplasmic face of the inner membrane and paralyze the export mechanism, becoming lethal if sufficiently overproduced and if not degraded by the DegP protease (A. Guigueno, P. Belin, and P. L. Boquet, J. Bacteriol. 179:3260–3269, 1997). We isolated a multicopy suppressor that restores viability to a
degP
strain without modifying the expression level of the toxic fusion. Suppression does not involve activation of the known envelope stress-combative pathways, the Cpx pathway and the ς
E
regulon. Subclone analysis of the suppressor revealed a 195-bp DNA fragment that is responsible for toxicity suppression. The cloned gene, called
uptR
, is ≈130 bp long (including the promoter and a transcription termination signal) and is transcribed into a small RNA (92 nucleotides). Using site-directed mutagenesis, we found that UptR RNA does not require translation for toxicity suppression. UptR-mediated action reduces the amount of membrane-bound toxic hybrid protein. UptR RNA is the first example of a small RNA implicated in extracytoplasmic toxicity suppression. It appears to offer a new way of suppressing toxicity, and its possible modes of action are discussed.
Altered Pathway Routing in a Class of
Salmonella enterica
Serovar Typhimurium Mutants Defective in Aminoimidazole Ribonucleotide Synthetase
Authors: Zilles Julie L. , Kappock T. Joseph , Stubbe JoAnne , Downs Diana M. 
Published: July 2002

ABSTRACT

In
Salmonella enterica
serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the
purI
gene in
S. enterica
. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways,
purI
mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37–44, 1996). Unexpectedly, some mutant
purI
alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The

K
m

of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the
V
max
was decreased 2.5-fold. The

K
m

for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and
purR
null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.
Growth Inhibition Caused by Overexpression of the Structural Gene for Glutamate Dehydrogenase (
gdhA
) from
Klebsiella aerogenes
Authors: Janes Brian K. , Pomposiello Pablo J. , Perez-Matos Ana , Najarian David J. , Goss Thomas J. , Bender Robert A. 
Published: July 2002

ABSTRACT

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (
gdh-1
and
rev-2
) had been isolated at a locus near the
trp
cluster in
Klebsiella aerogenes
. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The
gdhA
gene from
K. aerogenes
was cloned and sequenced, and an insertion mutation was generated and shown to be linked to
trp
. A region of
gdhA
from a strain bearing
gdh-1
was sequenced and shown to have a single-base-pair change, confirming that the locus defined by
gdh-1
is the structural gene for GDH. Mutants with the same phenotype as
rev-2
were isolated, and their sequences showed that the mutations were located in the promoter region of the
gdhA
gene. The linkage of
gdhA
to
trp
in
K. aerogenes
was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of
gdhA
displayed an auxotrophy that was interpreted as a limitation for α-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive
gdhA1
allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the α-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.
Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from
Pyrococcus furiosus
Authors: Verhees Corné H. , Akerboom Jasper , Schiltz Emile , de Vos Willem M. , van der Oost John 
Published: July 2002

ABSTRACT

The
Pyrococcus furiosus fbpA
gene was cloned and expressed in
Escherichia coli
, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a

K
m

of 0.32 mM and a
V
max
of 12.2 U/mg. The
P. furiosus
fructose-1,6-bisphosphatase was strongly inhibited by Li
+
(50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the
P. furiosus
fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.
Identification and Characterization of Assembly Proteins of CS5 Pili from Enterotoxigenic
Escherichia coli
Authors: Duthy Thomas G. , Manning Paul A. , Heuzenroeder Michael W. 
Published: July 2002

ABSTRACT

This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic
Escherichia coli
. In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in
csfB
abolished CsfA major subunit accumulation in the periplasm, which could be restored by
trans
-complementation with a complete copy of the
csfB
gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the
csfE
gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of
csfE
expression in
trans
did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the
csfF
gene also resulted in an elongated pilus morphology identical to that of the
csfE
deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a
csfF
deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the
csfB
and
csfF
genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was
trans
-complemented with a wild-type copy of
csfB
or
csfF
, respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations.
A Turquoise Mutant Genetically Separates Expression of Genes Encoding Phycoerythrin and Its Associated Linker Peptides
Authors: Seib Laura Ort , Kehoe David M. 
Published: July 2002

ABSTRACT

During complementary chromatic adaptation (CCA), cyanobacterial light harvesting structures called phycobilisomes are restructured in response to ambient light quality shifts. Transcription of genes encoding components of the phycobilisome is differentially regulated during this process: red light activates
cpcB2A2
, whereas green light coordinately activates the
cpeCDE
and
cpeBA
operons. Three signal transduction components that regulate CCA have been isolated to date: a sensor-photoreceptor (RcaE) and two response regulators (RcaF and RcaC). Mutations in the genes encoding these components affect the accumulation of both
cpcB2A2
and
cpeBA
gene products. We have isolated and characterized a new pigmentation mutant called Turquoise 1. We demonstrate that this mutant phenotype is due to a dramatic decrease in
cpeBA
transcript abundance and results from a lesion in the
cpeR
gene. However, in this mutant
cpeCDE
RNA levels remain near those found in wild-type cells. Our results show that the coordinate regulation of
cpeBA
and
cpeCDE
by green light can be uncoupled by the loss of CpeR, and we furnish the first genetic evidence that different regulatory mechanisms control these two operons. Sequence analysis of CpeR reveals that it shares limited sequence similarity to members of the PP2C class of protein serine/threonine phosphatases. We also demonstrate that
cpeBA
and
cpeCDE
retain light quality responsiveness in a mutant lacking the RcaE photoreceptor. This provides compelling evidence for the partial control of CCA through an as-yet-uncharacterized second light quality sensing system.
Effect of
aniA
(Carbon Flux Regulator) and
phaC
(Poly-β-Hydroxybutyrate Synthase) Mutations on Pyruvate Metabolism in
Rhizobium etli
Authors: Dunn Michael F. , Araíza Gisela , Encarnación Sergio , del Carmen Vargas María , Mora Jaime 
Published: July 2002

ABSTRACT

The
Rhizobium etli
poly-β-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of
aniA
, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity,
pyc
gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA,
pyc
transcription, and reduced-nucleotide levels is discussed.
Isolation of an Intron-Containing Partial Sequence of the Gene Encoding Dermatophyte Actin (
ACT
) and Detection of a Fragment of the Transcript by Reverse Transcription-Nested PCR as a Means of Assessing the Viability of Dermatophytes in Skin Scales
Authors: Okeke Charles N. , Tsuboi Ryoji , Kawai Masaaki , Hiruma Masataro , Ogawa Hideoki 
Published: July 2002

ABSTRACT

An internal partial sequence of the gene encoding actin (
ACT
), 725 to 762 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced. An intron that is 56 to 93 bp in length was located along the
ACT
fragment of all of the dermatophytes at codon position 301 (−3) (a codon number followed by “−3” indicates that the intron directly follows the codon) with reference to the amino acid sequence of human α-smooth muscle actin. A primer pair that annealed to exon sequences flanking the
ACT
-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-PCR) of dermatophyte
ACT
mRNA. PCR primer pairs with antisense sequence based on the
ACT
intron sequence were species specific for dermatophytes, suggesting a potential for use in the identification of dermatophytes. The viability of dermatophytes in skin scales was subsequently assessed by the presence of
ACT
mRNA in total RNA extracted from a 48-h culture of scale samples in 250 μl of yeast carbon base broth. RT-nested PCR of dermatophyte-infected samples amplified an
ACT
fragment of the predicted size of 171 bp. The results of viability testing based on
ACT
mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics.