bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-Swollen Cellulose by Two Endoglucanases (CelZ and CelY) from
Erwinia chrysanthemi
Authors: Zhou Shengde , Ingram Lonnie O. 
Published: July 2002

ABSTRACT

Erwinia chrysanthemi
produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of
E. chrysanthemi
. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.
Defects in
d
-Alanyl-Lipoteichoic Acid Synthesis in
Streptococcus mutans
Results in Acid Sensitivity
Authors: Boyd David A. , Cvitkovitch Dennis G. , Bleiweis Arnold S. , Kiriukhin Michael Y. , Debabov Dmitri V. , Neuhaus Francis C. , Hamilton Ian R. 
Published: July 2002

ABSTRACT

In the cariogenic organism,
Streptococcus mutans
, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:IS
S1
was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in
dltB
of the
dlt
operon encoding four proteins (DltA-DltD) involved in the synthesis of
d
-alanyl-lipoteichoic acid. Inactivation of the
dltC
gene, encoding the
d
-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate
d
-[
14
C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of
d
-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in
S. mutans.
Catalase-Peroxidases of
Legionella pneumophila
: Cloning of the
katA
Gene and Studies of KatA Function
Authors: Bandyopadhyay Purnima , Steinman Howard M. 
Published: July 2002

ABSTRACT

Legionella pneumophila
, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for
katA
was cloned and its function was studied in
L. pneumophila
and
Escherichia coli
and compared with prior studies of
katB
in this laboratory.
katA
is induced during exponential growth and is the predominant peroxidase in stationary phase. When
katA
is inactivated,
L. pneumophila
is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to
katB
. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when
katA
is inactivated, while no change from wild type is seen for the
katB
null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by
L. pneumophila
, further studies on the function and regulation of
katA
in stationary phase may give insights on the mechanisms of infectivity of this pathogen.
Secretion of an Acid Phosphatase (SapM) by
Mycobacterium tuberculosis
That Is Similar to Eukaryotic Acid Phosphatases
Authors: Saleh Mazen T. , Belisle John T. 
Published: July 2002

ABSTRACT

Mycobacterium tuberculosis
secretes a large number of polypeptides with broad biological and immunological functions. We describe here the characterization of a 28-kDa acid phosphatase of
M. tuberculosis
(SapM) localized to the culture filtrate. The mature protein demonstrated biochemical characteristics similar to those of the bacterial nonspecific acid phosphatases. However, SapM yielded significant sequence homology to fungal acid phosphatases and not those of bacteria. Thus, SapM may represent a new class of bacterial nonspecific acid phosphatases.
The 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in
Sphingomonas paucimobilis
SYK-6
Authors: Hara Hirofumi , Masai Eiji , Katayama Yoshihiro , Fukuda Masao 
Published: July 2002

ABSTRACT

Sphingomonas paucimobilis
SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the
O
-demethylase and converted into protocatechuate (PCA) and 3-
O
-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb
Eco
RI fragment carrying the genes for PCA 4,5-dioxygenase (
ligAB
), 2-pyrone-4,6-dicarboxylate hydrolase (
ligI
), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (
ligC
), we found the
ligJ
gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The
ligJ
gene is transcribed in the same direction as
ligABC
genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from
ligA
. The
ligJ
gene product (LigJ), expressed in
Escherichia coli
, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30°C. The

K
m

for OMA and the
V
max
were determined to be 138 μM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The
ligJ
gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the
ligJ
gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.
Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase ofEscherichia coliW: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily
Authors: Galán Beatriz , Díaz Eduardo , Prieto María A. , García José L. 
Published: July 2002

ABSTRACTEscherichia coliW uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by thehpaBandhpaCgenes, respectively, that form a single transcription unit. Overproduction of the small HpaC component inE. coliK-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochromecand ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin ofStreptococcus pneumoniaeallowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase ofE. coliW as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.
Native Plasmids of
Fusobacterium nucleatum
: Characterization and Use in Development of Genetic Systems
Authors: Haake Susan Kinder , Yoder Sean C. , Attarian Gwynne , Podkaminer Kara 
Published: July 2002

ABSTRACT

Three native plasmids of
Fusobacterium nucleatum
were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming
Escherichia coli
and
F. nucleatum
ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the
F. nucleatum
transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in
F. nucleatum
.
Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems
Authors: Grahn A. Marika , Haase Jana , Bamford Dennis H. , Lanka Erich 
Published: July 2002

ABSTRACT

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPα plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to
Escherichia coli
cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by
traF
of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two
E. coli
membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the
E. coli
cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.
Characterization and Evolution of Anthranilate 1,2-Dioxygenase from
Acinetobacter
sp. Strain ADP1
Authors: Eby D. Matthew , Beharry Zanna M. , Coulter Eric D. , Kurtz Donald M. , Neidle Ellen L. 
Published: July 2002

ABSTRACT

The two-component anthranilate 1,2-dioxygenase of the bacterium
Acinetobacter
sp. strain ADP1 was expressed in
Escherichia coli
and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O
2
and consumption of one NADH. The terminal oxygenase component formed an α
3
β
3
hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O
2
consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O
2
consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23°C) or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the α and β subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.
Characterization of Transmembrane Segments 3, 4, and 5 of MalF by Mutational Analysis
Authors: Steinke Angelika , Grau Sandra , Davidson Amy , Hofmann Eckhard , Ehrmann Michael 
Published: July 2002

ABSTRACT

MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in
Escherichia coli
. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L
323
Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.
Recombinant
Thermus aquaticus
RNA Polymerase, a New Tool for Structure-Based Analysis of Transcription
Authors: Minakhin Leonid , Nechaev Sergei , Campbell Elizabeth A. , Severinov Konstantin 
Published: July 2002

ABSTRACT

The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic
Thermus aquaticus
has recently been determined at 3.3 Å resolution. Currently, very little is known about
T. aquaticus
transcription and no genetic system to study
T. aquaticus
RNAP genes is available. To overcome these limitations, we cloned and overexpressed
T. aquaticus
RNAP genes in
Escherichia coli
. Overproduced
T. aquaticus
RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in
E. coli
. We used the recombinant
T. aquaticus
enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for
E. coli
RNAP can be adapted to study
T. aquaticus
transcription. However,
T. aquaticus
RNAP differs from the prototypical
E. coli
enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.
Genetic Characterization of theKlebsiella pneumoniae waaGene Cluster, Involved in Core Lipopolysaccharide Biosynthesis
Authors: Regué Miguel , Climent Núria , Abitiu Nihal , Coderch Núria , Merino Susana , Izquierdo Luis , Altarriba Maria , Tomás Juan M. 
Published: July 2002

ABSTRACTA recombinant cosmid containing genes involved inKlebsiella pneumoniaeC3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance toEscherichia coliK-12. The recombinant cosmid contains 12 genes, the wholewaagene cluster, flanked bykblandcoaDgenes, as was found inE. coliK-12. PCR amplification analysis showed that this cluster is conserved in representativeK. pneumoniaestrains. Partial nucleotide sequence determination showed that the same genes and gene order are found inK. pneumoniaesubsp.ozaenae, for which the core chemical structure is known. Complementation analysis of knownwaamutants fromE. coliK-12 and/orSalmonella entericaled to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of theEnterobacteriaceae. K. pneumoniae orf10mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructedK. pneumoniae waaEdeletion mutant suggests that the WaaE protein is involved in the transfer of the branch β-d-Glc to the O-4 position ofl-glycero-d-manno-heptose I, a feature shared byK. pneumoniae, Proteus mirabilis, andYersinia enterocolitica.
PhcS Represses Gratuitous Expression of Phenol-Metabolizing Enzymes in
Comamonas testosteroni
R5
Authors: Teramoto Maki , Harayama Shigeaki , Watanabe Kazuya 
Published: July 2002

ABSTRACT

We identified an open reading frame, designated
phcS
, downstream of the transcriptional activator gene (
phcR
) for the expression of multicomponent phenol hydroxylase (mPH) in
Comamonas testosteroni
R5. The deduced product of
phcS
was homologous to AphS of
C. testosteroni
TA441, which belongs to the GntR family of transcriptional regulators. The transformation of
Pseudomonas aeruginosa
PAO1c (phenol negative, catechol positive) with pROR502 containing
phcR
and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containing
phcS
,
phcR
, and mPH genes did not confer this ability. The disruption of
phcS
in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockout
phcS
gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the
phcS
gene. The disruption of
phcS
and/or
phcR
and the complementation in
trans
of these defects confirm that PhcS is a
trans
-acting repressor and that the unfavorable expression of mPH in the
phcS
knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.
Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon
Thermococcus
sp. Strain B1001
Authors: Hashimoto Yoshiteru , Yamamoto Tomoko , Fujiwara Shinsuke , Takagi Masahiro , Imanaka Tadayuki 
Published: July 2002

ABSTRACT

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon
Thermococcus
sp. strain B1001 produces predominantly (>85%) α-cyclomaltodextrin (α-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991–1997, 1999). Nucleotide sequencing of the CGTase gene (
cgtA
) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (
cgtB
), the gene encoding CGTase (
cgtA
), a gene homolog for a CD-binding protein (CBP) (
cgtC
), and a putative CBP-dependent ABC transporter involved in uptake of CDs (
cgtDE
). The CDase was expressed in
Escherichia coli
and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95°C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed β-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to α-CD. However, the CBP exhibited higher thermostability in the presence of α-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.