bluuebunny/crossref_metadata_2025_split huggingface dataset:
Leaf Vitamin C Contents Modulate Plant Defense Transcripts and Regulate Genes That Control Development through Hormone Signaling[W]
Authors: Pastori Gabriela M. , Kiddle Guy , Antoniw John , Bernard Stephanie , Veljovic-Jovanovic Sonja , Verrier Paul J. , Noctor Graham , Foyer Christine H. 
Published: April 2003

AbstractVitamin C deficiency in the Arabidopsis mutant vtc1 causes slow growth and late flowering. This is not attributable to changes in photosynthesis or increased oxidative stress. We have used the vtc1 mutant to provide a molecular signature for vitamin C deficiency in plants. Using statistical analysis, we show that 171 genes are expressed differentially in vtc1 compared with the wild type. Many defense genes are activated, particularly those that encode pathogenesis-related proteins. Furthermore, transcript changes indicate that growth and development are constrained in vtc1 by the modulation of abscisic acid signaling. Abscisic acid contents are significantly higher in vtc1 than in the wild type. Key features of the molecular signature of ascorbate deficiency can be reversed by incubating vtc1 leaf discs in ascorbate. This finding provides evidence that many of the observed effects on transcript abundance in vtc1 result from ascorbate deficiency. Hence, through modifying gene expression, vitamin C contents not only act to regulate defense and survival but also act via phytohormones to modulate plant growth under optimal conditions.
Subunit Oligomerization and Substrate Recognition of the
Escherichia coli
ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System
Authors: Lee Yi-Ying , Chang Chiung-Fang , Kuo Chueh-Ling , Chen Meng-Ching , Yu Chien Hung , Lin Pei-I , Wu Whi Fin 
Published: April 2003

ABSTRACT

The
Escherichia coli
ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpY
ΔI
) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.
Control of
rep
Gene Expression in Plasmid pGA1 from
Corynebacterium
glutamicum
Authors: Venkova-Canova Tatiana , Pátek Miroslav , Nešvera Jan 
Published: April 2003

ABSTRACT

The cryptic multicopy plasmid pGA1 (4,826 bp) from
Corynebacterium glutamicum
LP-6 belongs to the fifth group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was localized in the leader region of the
rep
gene coding for the initiator of plasmid replication. This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48. A promoter and a single transcriptional start site were found in the
rep
leader region in orientation opposite to the
rep
gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca. 89 nucleotides in length), which might inhibit translation of pGA1
rep
gene, is formed. Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria. Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters P
rep
and P-ctRNA with a potential to form secondary structures on both ctRNA and
rep
mRNA was found to cause low activity of the
rep
promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the
rep
leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1
rep
gene.
Genus-Specific Protein Binding to the Large Clusters of DNA Repeats (Short Regularly Spaced Repeats) Present in
Sulfolobus
Genomes
Authors: Peng Xu , Brügger Kim , Shen Biao , Chen Lanming , She Qunxin , Garrett Roger A. 
Published: April 2003

ABSTRACT

Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters. For the crenarchaeon
Sulfolobus solfataricus
P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The
Sulfolobus
conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the
S. solfataricus
chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from
S. solfataricus
. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also recognizes both main families of repeat sequences in
S. solfataricus
. The recombinant protein, expressed in
Escherichia coli
, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the
Sulfolobus
genus and in the closely related
Acidianus
genus. We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters.
Methionine Regeneration and Aminotransferases in
Bacillus subtilis
,
Bacillus cereus
, and
Bacillus anthracis
Authors: Berger Bradley J. , English Shane , Chan Gene , Knodel Marvin H. 
Published: April 2003

ABSTRACT

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of
Bacillus subtilis
has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in
B. subtilis
were found to be members of the If subfamily. These putative aspartate aminotransferases, the
yugH
,
ywfG
,
ykrV
,
aspB
, and
patA
gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of
B. subtilis
and
Bacillus cereus
utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in
B. subtilis
,
ybgE
and
ywaA
, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The
B. subtilis ybgE
gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of
B. cereus
and
Bacillus anthracis
genome data found that there were no subfamily IIIa homologues in these organisms. In both
B. cereus
and
B. anthracis
, two putative branched-chain aminotransferases and two putative
d
-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from
B. cereus
, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The
B. anthracis
homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of
B. subtilis
YbgE and also inhibited growth of
B. subtilis
and
B. cereus
in culture.
Signal Detection and Target Gene Induction by the CpxRA Two-Component System
Authors: DiGiuseppe Patricia A. , Silhavy Thomas J. 
Published: April 2003

ABSTRACT

The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the
cpxP
promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.
Transcription-Defective
soxR
Mutants of
Escherichia coli
: Isolation and In Vivo Characterization
Authors: Chander Monica , Raducha-Grace Laura , Demple Bruce 
Published: April 2003

ABSTRACT

The
soxRS
regulon protects
Escherichia coli
from superoxide and nitric oxide stress. SoxR protein, a transcription factor that senses oxidative stress via its [2Fe-2S] centers, transduces the signal to the
soxS
promoter to stimulate RNA polymerase. Here we describe 29 mutant alleles of
soxR
that cause defects in the activation of
soxS
transcription in response to paraquat, a superoxide stress agent. Owing to the selection and screen used in their isolation, most of these mutant alleles encode proteins that retained specific binding activity for the
soxS
promoter in vivo. The mutations were found throughout the SoxR polypeptide, although those closer to the N terminus typically exhibited greater defects in DNA binding. The degree of the defect in the transcriptional response to superoxide caused by each mutation was closely paralleled by its impaired response to nitric oxide. This work begins the general identification of the residues in the SoxR polypeptide that are critical for transducing oxidative stress signals into gene activation.
Identification and Characterization of a Mandelamide Hydrolase and an NAD(P)+-Dependent Benzaldehyde Dehydrogenase fromPseudomonas putidaATCC 12633
Authors: McLeish Michael J. , Kneen Malea M. , Gopalakrishna Kota N. , Koo Carolyn W. , Babbitt Patricia C. , Gerlt John A. , Kenyon George L. 
Published: April 2003

ABSTRACTThe enzymes of the mandelate metabolic pathway permitPseudomonas putidaATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denotedmdlX,mdlY, andmdlD. All were transcribed in the opposite direction from the genes of themdlABCoperon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)+-dependent dehydrogenase (mdlD). ThemdlYandmdlDgenes were isolated and expressed inEscherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase, respectively.
Effects of Overexpression of Nutrient Receptors on Germination of Spores of
Bacillus subtilis
Authors: Cabrera-Martinez Rosa-Martha , Tovar-Rojo Federico , Vepachedu Venkata Ramana , Setlow Peter 
Published: April 2003

ABSTRACT

The rates of germination of
Bacillus subtilis
spores with
l
-alanine were increased markedly, in particular at low
l
-alanine concentrations, by overexpression of the tricistronic
gerA
operon that encodes the spore's germinant receptor for
l
-alanine but not by overexpression of
gerA
operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K
+
(AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic
gerB
and
gerK
operons. Overexpression of the
gerB
or
gerK
operon or both the
gerB
and
gerK
operons also did not stimulate spore germination in AGFK. Overexpression of a mutant
gerB
operon, termed
gerB
*, that encodes a receptor allowing spore germination in response to either
d
-alanine or
l
-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with
d
-alanine or
l
-asparagine in spores overexpressing
gerB
* were well below the increases in the spore's levels of the GerBA protein. Germination of
gerB
* spores with
d
-alanine or
l
-asparagine did not require participation of the products of the
gerK
operon, but germination with these agents was decreased markedly in spores also overexpressing
gerA
. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with
d
-alanine or
l
-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the
gerA
or
gerK
operon (but not the
gerB
or
gerB*
operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of
gerA
or
gerK
overexpression that were used to generate spores for analysis of rates of germination.
Bacteriophage SPP1 Chu Is an Alkaline Exonuclease in the SynExo Family of Viral Two-Component Recombinases
Authors: Vellani Trina S. , Myers Richard S. 
Published: April 2003

ABSTRACTMany DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage λ and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized34.1gene fromBacillus subtilisphage SPP1, expressed it inEscherichia coli, purified the protein, and determined its enzymatic properties. Like λ exonuclease, Chu (the product of34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg2+-dependent reaction, and shows a preference for 5′-phosphorylated DNA ends. A model for viral recombination, based on the phage λ Red recombination system, is proposed.
Molecular Characterization of the Acid-InducibleasrGene ofEscherichia coliand Its Role in Acid Stress Response
Authors: Šeputienė Vaida , Motiejūnas Domantas , Sužiedėlis Kęstutis , Tomenius Henrik , Normark Staffan , Melefors Öjar , Sužiedėlienė Edita 
Published: April 2003

ABSTRACTEnterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified anEscherichia coligene,asr(acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shockedE. colicells harboring a plasmid-borneasrgene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of theasrgene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of theasrgene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for theasrnull mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of theasrgene from otherEnterobacteriaceaewere cloned and shown to be induced inE. coliunder acid shock conditions.
Substrate Specificity Classes and the Recognition Signal for
Salmonella
Type III Flagellar Export
Authors: Hirano Takanori , Minamino Tohru , Namba Keiichi , Macnab Robert M. 
Published: April 2003

ABSTRACT

Most flagellar proteins of
Salmonella
are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a
flgE
mutant (which is still at the hook-type specificity stage) were much higher than those from a
flgK
mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.