bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcription of the
nfrA-ywcH
Operon from
Bacillus subtilis
Is Specifically Induced in Response to Heat
Authors: Moch Christine , Schrögel Oliver , Allmansberger Rudolf 
Published: July 2002

ABSTRACT

The NfrA protein, an oxidoreductase from the soil bacterium
Bacillus subtilis
, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the
nfrA
gene belongs to the class III heat shock genes in
B. subtilis
. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that
nfrA
and the gene downstream of
nfrA
are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding
nfrA
and
ywcH
. The
nfrA-ywcH
operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.
Metabolic Pathways for Cytotoxic End Product Formation from Glutamate- and Aspartate-Containing Peptides by
Porphyromonas gingivalis
Authors: Takahashi Nobuhiro , Sato Takuichi , Yamada Tadashi 
Published: July 2002

ABSTRACT

Metabolic pathways involved in the formation of cytotoxic end products by
Porphyromonas gingivalis
were studied. The washed cells of
P. gingivalis
ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of
P. gingivalis. P. gingivalis
cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.
Genetic and Biochemical Characterization of 4-Carboxy-2-Hydroxymuconate-6-Semialdehyde Dehydrogenase and Its Role in the Protocatechuate 4,5-Cleavage Pathway in
Sphingomonas paucimobilis
SYK-6
Authors: Masai Eiji , Momose Kiyotaka , Hara Hirofumi , Nishikawa Seiji , Katayama Yoshihiro , Fukuda Masao 
Published: July 2002

ABSTRACT

Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of
Sphingomonas paucimobilis
SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (
ligC
). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that
ligC
was located 295 bp downstream of
ligB
, which encodes the large subunit of the PCA 4,5-dioxygenase. The
ligC
gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of
ligC
showed 19 to 20% identity with 3-chlorobenzoate
cis
-dihydrodiol dehydrogenase of
Alcaligenes
sp. strain BR60 and phthalate
cis
-dihydrodiol dehydrogenases of
Pseudomonas putida
NMH102-2 and
Burkholderia cepacia
DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13–56, 1994). The
ligC
gene was expressed in
Escherichia coli
and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP
+
by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25°C, respectively. The

K
m

for NADP
+
was estimated to be 24.6 ± 1.5 μM, which was approximately 10 times lower than that for NAD
+
(252 ± 3.9 μM). The

K
m

s for CHMS in the presence of NADP
+
and NAD
+
are 26.0 ± 0.5 and 20.6 ± 1.0 μM, respectively. Disruption of
ligC
in
S. paucimobilis
SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the
ligC
insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of
ligC
into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for
ligAB
gene expression.
Interdependence of Activation at
rhaSR
by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR
Authors: Holcroft Carolyn C. , Egan Susan M. 
Published: July 2002

ABSTRACT

The
Escherichia coli rhaSR
operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of
l
-rhamnose. β-Galactosidase assays of various
rhaS-lacZ
promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of
rhaSR
expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at
rhaSR
, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of
rhaSR
. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.
CTX Prophages in Classical Biotype
Vibrio cholerae
: Functional Phage Genes but Dysfunctional Phage Genomes
Authors: Davis Brigid M. , Moyer Kathryn E. , Boyd E. Fidelma , Waldor Matthew K. 
Published: July 2002

ABSTRACT

CTXφ is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by
Vibrio cholerae
. CTX prophages in O1 El Tor and O139 strains of
V. cholerae
are found within arrays of genetically related elements integrated at a single locus within the
V. cholerae
large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXφ. In contrast, O1 classical strains of
V. cholerae
do not produce CTXφ, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXφ. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXφ and is often interspersed with CTX prophages in El Tor strains, was not detected in classical
V. cholerae
. Our model for CTXφ production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of
V. cholerae
to produce CTXφ is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.
Characterization of a Major Cluster ofnif,fix, and Associated Genes in a Sugarcane Endophyte,Acetobacter diazotrophicus
Authors: Lee Sunhee , Reth Alexander , Meletzus Dietmar , Sevilla Myrna , Kennedy Christina 
Published: July 2002

ABSTRACTA major 30.5-kb cluster ofnifand associated genes ofAcetobacter diazotrophicus(syn.Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguousnif-fixand associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of thenif-fixcluster inAzospirillum brasilense, while the individual gene products are more similar to those in species ofRhizobiaceaeor inRhodobacter capsulatus.
Cytochrome
c
′ from
Rhodobacter capsulatus
Confers Increased Resistance to Nitric Oxide
Authors: Cross Richard , Aish Joanne , Paston Samantha J. , Poole Robert K. , Moir James W. B. 
Published: July 2002

ABSTRACT

We report the cloning and sequencing of the gene containing cytochrome
c
′ (
cycP
) from the photosynthetic purple bacterium
Rhodobacter capsulatus
and the regions flanking that gene. Mutant strains unable to synthesize cytochrome
c
′ had increased sensitivity to nitrosothiols and to nitric oxide (which binds to the heme moiety of cytochrome
c
′).
Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of
Haemophilus ducreyi
Authors: Sun Shuhua , Schilling Birgit , Tarantino Laurie , Tullius Michael V. , Gibson Bradford W. , Munson Robert S. 
Published: July 2002

ABSTRACT

Haemophilus ducreyi
is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of
H. ducreyi
to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the
N
-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an
H. ducreyi
35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the
N
-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of
Haemophilus influenzae
,
Haemophilus somnus
,
Neisseria
species, and
Pasteurella haemolytica
. The putative
H. ducreyi
glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the
N
-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.
Identification of
ccdA
in
Paracoccus pantotrophus
GB17: Disruption of
ccdA
Causes Complete Deficiency in
c
-Type Cytochromes
Authors: Bardischewsky Frank , Friedrich Cornelius G. 
Published: July 2002

ABSTRACT

A transposon Tn
5-mob
insertional mutant of
Paracoccus pantotrophus
GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional
c-
type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a
c
-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn
5-mob
-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA,
ccdA
, and ORFB, with the latter being oriented divergently. ORFA and
ccdA
were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn
5-mob
was inserted into
ccdA
. The deduced ORFA product showed no similarity to any protein in databases. However, the
ccdA
gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome
c
biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of
P. pantotrophus
is 42% identical to a protein predicted by ORF2, and its location within the
sox
gene cluster coding for lithotrophic sulfur oxidation suggested a different function.
In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon
Sulfolobus solfataricus
Authors: Ciammaruconi Andrea , Londei Paola 
Published: July 2002

ABSTRACT

In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon
Sulfolobus solfataricus
, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5′ external transcribed spacer of the
S. solfataricus
pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of
Saccharomyces cerevisiae
and vertebrates. This site is present in other members of the kingdom
Crenarchaeota
but apparently not in the
Euryarchaeota
. Furthermore,
S. solfataricus
pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the
Crenarchaeota
and the
Euryarchaeota
. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5′ or the 3′ end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.
Transcription-Induced Cytosine-to-Thymine Mutations Are Not Dependent on Sequence Context of the Target Cytosine
Authors: Beletskii Anton , Bhagwat Ashok S. 
Published: July 2002

ABSTRACT

We showed previously that transcription of a plasmid-borne
kan
allele increases C-to-T mutations in the nontranscribed strand. Using two new plasmid-borne
kan
alleles, one
cmp
allele, and a chromosomal
kan
allele, we found in this study that transcription-induced mutations are not limited to specific genes, alleles, or locations and are likely to be a general property of transcript elongation in
Escherichia coli
.
Molecular Characterization and Regulation of the
aguBA
Operon, Responsible for Agmatine Utilization in
Pseudomonas aeruginosa
PAO1
Authors: Nakada Yuji , Jiang Ying , Nishijyo Takayuki , Itoh Yoshifumi , Lu Chung-Dar 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by
aguA
; also called agmatine iminohydrolase) and
N
-carbamoylputrescine amidohydrolase (encoded by
aguB
). The
aguBA
and adjacent
aguR
genes were cloned and characterized. The predicted AguB protein (
M
r
32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (
M
r
41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to
Arabidopsis
. The
aguR
gene encoded a putative regulatory protein (
M
r
24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and
N
-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and
N
-carbamoylputrescine on expression of the
aguBA
operon. The presence of an inducible promoter for the
aguBA
operon in the
aguR
-
aguB
intergenic region was demonstrated by
lacZ
fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of
aguB
by S1 nuclease mapping. Experiments with knockout mutants of
aguR
established that expression of the
aguBA
operon became constitutive in the
aguR
background. Interaction of AguR overproduced in
Escherichia coli
with the
aguBA
regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the
aguBA
operon, and binding of agmatine and
N
-carbamoylputrescine to AguR would antagonize its repressor function.
Stability and Subcellular Localization of Cytadherence-Associated Protein P65 in
Mycoplasma pneumoniae
Authors: Jordan Jarrat L. , Berry Karen M. , Balish Mitchell F. , Krause Duncan C. 
Published: July 2002

ABSTRACT

The surface protein P65 is a constituent of the
Mycoplasma pneumoniae
cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2. Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2. P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1. P65 exhibited a polar localization like that in wild-type
M. pneumoniae
in all mutants having normal levels of HMW1 and HMW2. Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.
A Putatively Phase Variable Gene (
dca
) Required for Natural Competence in
Neisseria gonorrhoeae
but Not
Neisseria meningitidis
Is Located within the Division Cell Wall (
dcw
) Gene Cluster
Authors: Snyder Lori A. S. , Saunders Nigel J. , Shafer William M. 
Published: July 2002

ABSTRACT

A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in
Neisseria gonorrhoeae
and
Neisseria meningitidis
. The three additional ORFs, internal to the
dcw
cluster, are not homologous to
dcw
-related genes present in other bacterial species. Analysis of the
N. meningitidis
strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial
dcw
gene cluster. Reverse transcription-PCR analysis of RNA extracted from
N. gonorrhoeae
strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (
dca
, for division cluster competence associated), located between
murE
and
murF
, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that
dca
encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains
dca
is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of
N. meningitidis
, suggesting that
dca
is phase variable in this species. A deletion and insertional mutation was made in the
dca
gene of
N. gonorrhoeae
strain FA19 and
N. meningitidis
strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the
dca
-encoded gene product is an essential competence factor for gonococci.
Interaction ofProteus mirabilisUrease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology
Authors: Heimer Susan R. , Mobley Harry L. T. 
Published: July 2002

ABSTRACTProteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions amongP. mirabilisaccessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo,P. mirabilisUreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.