bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium
Agrobacterium radiobacter
Strain S2
Authors: Contzen Matthias , Stolz Andreas 
Published: July 2002

ABSTRACT

The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacterium
Agrobacterium radiobacter
strain S2 (DSMZ 5681). The
pcaH1G1
genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strain
Agrobacterium tumefaciens
A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the
pcaH2G2
genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.
Fate of the SpoIIAB*-ADP Liberated after SpoIIAB Phosphorylates SpoIIAA of
Bacillus subtilis
Authors: Lee Chung-Sheng , Lucet Isabelle , Yudkin Michael D. 
Published: July 2002

ABSTRACT

Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific ς factor, ς
F
, of
Bacillus subtilis
. The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state. Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP. We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized. In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.
A Gene of
Synechocystis
sp. Strain PCC 6803 Encoding a Novel Iron Transporter
Authors: Katoh Hirokazu , Grossman Arthur R. , Hagino Natsu , Ogawa Teruo 
Published: July 2002

ABSTRACT

A mutant of
Synechocystis
sp. strain PCC 6803 disrupted for
sll1878
exhibited greatly reduced Fe
3+
transport activity. The

K
m

value of
sll1878
-dependent Fe
3+
transport in cells grown in iron-replete medium was 0.5 μM. Both the maximal rate and

K
m

value were increased in iron-starved cells.
Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens
Authors: Correia Jason D. , Jarrell Ken F. 
Published: July 2002

ABSTRACTMethanococcus voltaeis a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits.Escherichia colimembranes containing the expressedM. voltaepreflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species.M. voltaepreflagellin peptidase activity was optimal at 37°C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.
Crc Is Involved in Catabolite Repression Control of the
bkd
Operons of
Pseudomonas putida
and
Pseudomonas aeruginosa
Authors: Hester Kathryn L. , Lehman Jodi , Najar Fares , Song Lin , Roe Bruce A. , MacGregor Carolyn H. , Hager Paul W. , Phibbs Paul V. , Sokatch John R. 
Published: July 2002

ABSTRACT

Crc (catabolite repression control) protein of
Pseudomonas aeruginosa
has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in
Pseudomonas putida
. The
bkd
operons of
P. putida
and
P. aeruginosa
encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the
P. putida crc
gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the
bkd
operon, were isolated and identified as
crc
and
vacB
(
rnr
) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.
Feruloyl Esterase Activity of the
Clostridium thermocellum
Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ
Authors: Blum David L. , Kataeva Irina A. , Li Xin-Liang , Ljungdahl Lars G. 
Published: July 2002

ABSTRACT

The cellulosome of
Clostridium thermocellum
is a multiprotein complex with endo- and exocellulase, xylanase, β-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungus
Orpinomyces
. Purified cellulosomes from
C. thermocellum
were found to hydrolyze FAXX (
O
-{5-
O
-[(
E
)-feruloyl]-α-
l
-arabinofuranosyl}-(1→3)-
O
-β-
d
-xylopyranosyl-(1→4)-
d
-xylopyranose) and FAX
3
(5-
O
-[(
E
)-feruloyl]-[
O
-β-
d
-xylopyranosyl-(1→2)]-
O
-α-
l
-arabinofuranosyl-[1→3]}-
O
-β-
d
-xylopyranosyl-(1→4)-
d
-xylopyranose), yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into
Escherichia coli
, and the expressed proteins hydrolyzed FAXX and FAX
3
. The recombinant feruloyl esterase domain of XynZ alone (FAE
XynZ
) and with the adjacent cellulose binding domain (FAE-CBD
XynZ
) were characterized. FAE-CBD
XynZ
had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene.

K
m

and
V
max
values for FAX
3
were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX
3
, a substrate similar to FAX
3
but with a
p
-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70°C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount of
p
-coumaric acid. FAE
XynZ
had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE
XynZ
resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the
C. thermocellum
cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.
Expression of
ykdA
, Encoding a
Bacillus subtilis
Homologue of HtrA, Is Heat Shock Inducible and Negatively Autoregulated
Authors: Noone David , Howell Alistair , Devine Kevin M. 
Published: July 2002

ABSTRACT

There are three members of the HtrA family of serine proteases, YkdA, YvtA, and YyxA, encoded in the chromosome of
Bacillus subtilis
. In this study, we report on the promoter structure and regulation of
ykdA
expression. The
ykdA
gene is heat inducible, exhibiting a biphasic pattern of expression during a 60-min interval after heat shock. Increased expression after heat shock occurs at the transcriptional level. The heat-shock-inducible promoter has a single mismatch with a SigA-type −10 motif, but does not exhibit similarity to a SigA −35 region. There are six octamer repeats with a consensus TTTTCACA positioned at, and upstream of, the normal position of a −35 region. While repeats V and VI appear dispensable, repeat IV is essential for normal thermoinducible expression. This promoter structure is also found in the control region of
yvtA
, encoding a second member of this family of proteases. Expression of
ykdA
is negatively autoregulated both during the growth cycle and during heat shock. Our evidence suggests that YkdA protease activity is not required for this form of regulation. Null mutants of
ykdA
display increased tolerance to heat and are 80-fold more resistant to 10 mM hydrogen peroxide than wild-type cells. However,
ykdA
expression is not induced by hydrogen peroxide. These results indicate that the regulon to which YkdA belongs is linked to the oxidative stress response in
B. subtilis
.
A Two-Component Multidrug Efflux Pump, EbrAB, in
Bacillus subtilis
Authors: Masaoka Yoko , Ueno Yasuhiro , Morita Yuji , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa 
Published: July 2002

ABSTRACT

Genes (
ebrAB
) responsible for ethidium resistance were cloned from chromosomal DNA of
Bacillus subtilis
ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in
Escherichia coli
but also in
B. subtilis
. It also caused an elevated energy-dependent efflux of ethidium in
E. coli
. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither
ebrA
nor
ebrB
was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.
High Spontaneous Mutation Rate in the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Is Mediated by Transposable Elements
Authors: Martusewitsch Erika , Sensen Christoph W. , Schleper Christa 
Published: July 2002

ABSTRACT

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon
Sulfolobus solfataricus
in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10
−4
and 10
−5
per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal
pyrEF
locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS
4
-IS
5
IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different
S. solfataricus
strains, no homologous sequences were identified in DNA of other well-characterized strains of the order
Sulfolobales
.
Escherichia coli
Strains Blocked in Tat-Dependent Protein Export Exhibit Pleiotropic Defects in the Cell Envelope
Authors: Stanley Nicola R. , Findlay Kim , Berks Ben C. , Palmer Tracy 
Published: July 2002

ABSTRACT

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that
tat
strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the
lpxC
(
envA
) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.
Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4-Type Bacteriophages
Authors: Tétart Françoise , Desplats Carine , Kutateladze Mzia , Monod Caroline , Ackermann Hans-Wolfgang , Krisch H. M. 
Published: July 2002

ABSTRACT

We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria,
Aeromonas
,
Burkholderia,
and
Vibrio
. Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4. A few of them had more elongated heads and larger genomes. All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene
23
), tail sheath gene (gene
18
), and tail tube gene (gene
19
) of T4. On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens. In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts. However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups. The compilation of a number of gene
23
sequences reveals a pattern of conserved motifs separated by sequences that differ in the T4-type subgroups. Such variable patches in the gene
23
sequences may determine the size of the virion head and consequently the viral genome length. This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches.
Some
Lactobacillus
l
-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate
Authors: Arai Kazuhito , Kamata Takeo , Uchikoba Hiroyuki , Fushinobu Shinya , Matsuzawa Hiroshi , Taguchi Hayao 
Published: July 2002

ABSTRACT

The nonallosteric and allosteric
l
-lactate dehydrogenases of
Lactobacillus pentosus
and
L. casei
, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate

K
m

values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the
L. pentosus
enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.
Regulation of Expression of the
vanD
Glycopeptide Resistance Gene Cluster from
Enterococcus faecium
BM4339
Authors: Casadewall Barbara , Reynolds Peter E. , Courvalin Patrice 
Published: July 2002

ABSTRACT

A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the
vanD
glycopeptide resistance cluster in
Enterococcus faecium
BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644–3648, 1999). In this cluster, genes coding for the VanR
D
-VanS
D
two-component regulatory system were cotranscribed from the
P

R
D

promoter, whereas transcription of the

vanY
D
, vanH
D
, vanD, vanX
D

, and
intD
genes was initiated from the
P

Y
D

promoter located between

vanS
D

and

vanY
D

(the D subscript indicates that the gene is part of the
vanD
operon). The VanR
D
-VanS
D
regulatory system is likely to activate transcription of the resistance genes from the promoter
P

Y
D

. Glycopeptide-susceptible derivatives of BM4339 were obtained by
trans
complementation of the frameshift mutation in the
ddl
gene, restoring functional
d
-alanine:
d
-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only
d
-alanyl-
d
-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY
D
d
,
d
-carboxypeptidase, the VanH
D
dehydrogenase, the VanD ligase, the VanX
D
d
,
d
-dipeptidase, and also the IntD integrase, although the regulatory region of the
vanD
cluster was still transcribed. In BM4409, the absence of VanR
D
-VanS
D
, apparently dependent, transcription from promoter
P

Y
D

correlated with the lack of
d
-alanyl-
d
-lactate-terminating precursors. The

vanX
D

gene was transcribed in BM4339, but detectable amounts of VanX
D
d
,
d
-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in
Escherichia coli
, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize
d
-alanyl-
d
-alanine.
Requirement of Novel Competence Genes
pilT
and
pilU
of
Pseudomonas stutzeri
for Natural Transformation and Suppression of
pilT
Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI
Authors: Graupner Stefan , Weger Nicole , Sohni Monika , Wackernagel Wilfried 
Published: July 2002

ABSTRACT

The ubiquitous species
Pseudomonas stutzeri
has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed
pilT
. The deduced amino acid sequence has identity with PilT of
Pseudomonas aeruginosa
(94%),
Neisseria gonorrhoeae
(67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [
3
H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of
pilT
a gene, termed
pilU
, coding for a putative protein with 88% amino acid identity with PilU of
P. aeruginosa
was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable
P. aeruginosa
. When the
pilAI
gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in
pilAI
::Gm
r
cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the
pilT
mutation. Considering the hypothesized role of
pilT
+
in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the
pilT
-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.
Monitoring Intracellular Levels of XylR inPseudomonas putidawith a Single-Chain Antibody Specific for Aromatic-Responsive Enhancer-Binding Proteins
Authors: Fraile Sofı́a , Roncal Fernando , Fernández Luis A. , de Lorenzo Vı́ctor 
Published: July 2002

ABSTRACTWe have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the ς54-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons ofPseudomonasspp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRΔA) ofPseudomonas stutzeriOX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage inPseudomonas putidaunder various conditions. Growth stage, the physical form of the protein produced (XylR or XylRΔA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of ∼30 molecules (monomers) per cell during exponential phase to ∼140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRΔA were higher, fluctuating between ∼90 and ∼570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.