bluuebunny/crossref_metadata_2025_split huggingface dataset:
A New Thermoactive Pullulanase from
Desulfurococcus mucosus
: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in
Bacillus subtilis
Authors: Duffner Fiona , Bertoldo Costanzo , Andersen Jens T. , Wagner Karen , Antranikian Garabed 
Published: July 2002

ABSTRACT

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon
Desulfurococcus mucosus
(
apuA
) was cloned in
Escherichia coli
and sequenced.
apuA
from
D. mucosus
showed 45.4% pairwise amino acid identity with the pullulanase from
Thermococcus aggregans
and contained the four regions conserved among all amylolytic enzymes.
apuA
encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The
apuA
gene was then expressed in
Bacillus subtilis
and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a
Bacillus
strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from
Archaea
are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from
D. mucosus
should be considered an archaeal pullulanase type II with a wider substrate specificity.
ADP-Ribosylation of Variants of
Azotobacter vinelandii
Dinitrogenase Reductase by
Rhodospirillum rubrum
Dinitrogenase Reductase ADP-Ribosyltransferase
Authors: Grunwald Sandra K. , Ryle Matthew J. , Lanzilotta William N. , Ludden Paul W. 
Published: July 2002

ABSTRACT

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from
Azotobacter vinelandii
is known. In this study, mutant forms of dinitrogenase reductase from
A. vinelandii
that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from
Rhodospirillum rubrum
. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe
4
S
4
cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073–2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.
Characterization of a Putative Pathogenicity Island from Bovine
Staphylococcus aureus
Encoding Multiple Superantigens
Authors: Fitzgerald J. Ross , Monday Steven R. , Foster Timothy J. , Bohach Gregory A. , Hartigan Patrick J. , Meaney William J. , Smyth Cyril J. 
Published: July 2002

ABSTRACT

Previous studies have demonstrated that a proportion of
Staphylococcus aureus
isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (
tst
), the SEC bovine variant (
sec
-bovine), and a gene (
sel
) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3′ end of the GMP synthase gene (
gmps
) in the
S. aureus
chromosome. SaPIbov contains a central region of sequence identity with the previously characterized
tst
pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527–543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of
S. aureus
strain RF122 were constructed by allele replacement. In vitro bovine Vβ-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of
tst
and
sec
-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.
Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product
Authors: Saegusa Haruhisa , Shiraki Mari , Kanai Chie , Saito Terumi 
Published: July 2002

ABSTRACTAn intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutrophaH16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract ofEscherichia colicontaining the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomericd(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed inR. eutrophacells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant ofR. eutrophawhosephaZgene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the clonedphaZgene encodes an intracellular PHB depolymerase inR. eutropha.
Novel Type of Glucose-6-Phosphate Isomerase in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Hansen Thomas , Oehlmann Margitta , Schönheit Peter 
Published: July 2002

ABSTRACT

Glucose-6-phosphate isomerase (phosphoglucose isomerase [PGI]) (EC
5.3.1.9
) from the hyperthermophilic archaeon
Pyrococcus furiosus
was purified 500-fold to homogeneity. The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (α
2
) structure. Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80°C. Rate dependence on both substrates followed Michaelis-Menten kinetics. The apparent

K
m

values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparent
V
max
values were 800 and 130 U/mg. The enzyme had a temperature optimum of 96°C and showed a significant thermostability up to 100°C, which is in accordance with its physiological function under hyperthermophilic conditions. Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of
P. furiosus
. The ORF was characterized by functional overexpression in
Escherichia coli
as a gene,
pgi
, encoding glucose-6-phosphate isomerase. The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from
P. furiosus
. The deduced amino acid sequence of
P. furiosus
PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya. This is the first description of an archaeal PGI, which represents a novel type of PGI.
Functional Analysis ofrelAandrshA, TworelA/spoTHomologues ofStreptomyces coelicolorA3(2)
Authors: Sun Jongho , Hesketh Andrew , Bibb Mervyn 
Published: July 2002

ABSTRACTDeletion of the (p)ppGpp synthetase gene,relA, ofStreptomyces coelicolorA3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives ofS. coelicolor relAthat could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducibletipApromoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in arelAnull mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion ofrelAhad little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A secondS. coelicolorRelA homologue (RshA, with 42% identity toS. coelicolorRelA) was identified in the genome sequence. However, deletion ofrshAhad no effect on the ability of therelAmutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction oftipAp::rshAin therelAmutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in therelAmutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression ofrshA.
Activity of the
Kluyveromyces lactis
Pdr5 Multidrug Transporter Is Modulated by the Sit4 Protein Phosphatase
Authors: Chen Xin Jie 
Published: July 2002

ABSTRACT

A possible role for posttranslational modifications in regulating the activity of ATP-binding cassette (ABC) transporters has not been well established. In this study, the drug efflux ABC transporter gene
KlPDR5
was isolated from the budding yeast
Kluyveromyces lactis
, and it was found that the encoded KlPdr5 drug pump is posttranslationally regulated by the type 2A-related Ser/Thr protein phosphatase, Sit4p. The KlPdr5 transporter is a protein of 1,525 amino acids sharing 63.8% sequence identity with its
Saccharomyces cerevisiae
counterpart, ScPdr5p. Overexpression of the
KlPDR5
gene confers resistance to oligomycin, antimycin, econazole, and ketoconazole, whereas cells with a disrupted allele of
KlPDR5
are hypersensitive to the drugs and have a decreased capacity to carry out efflux of the anionic fluorescent dye rhodamine 123. It was found that a chromosomal disruption of
KlPDR5
abolishes the drug-resistant phenotype associated with
sit4
mutations and that a synergistic hyperresistance to the drugs can be created by overexpressing
KlPDR5
in
sit4
mutants. These data strongly indicate that the multidrug-resistant phenotype of
sit4
mutants is mediated by negatively modulating the activity of KlPdr5p. As the transcriptional level of
KlPDR5
and the steady-state level of KlPdr5p are not significantly affected by mutations in
SIT4
, the regulation by Sit4p appears to be a posttranslational process.
Three Efflux Pumps Are Required To Provide Efficient Tolerance to Toluene in
Pseudomonas putida
DOT-T1E
Authors: Rojas Antonia , Duque Estrella , Mosqueda Gilberto , Golden Geir , Hurtado Ana , Ramos Juan L. , Segura Ana 
Published: July 2002

ABSTRACT

In
Pseudomonas putida
DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The
ttgGHI
genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single
ttgH
mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene,
m
-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.
OhrR Is a Repressor of
ohrA
, a Key Organic Hydroperoxide Resistance Determinant in
Bacillus subtilis
Authors: Fuangthong Mayuree , Atichartpongkul Sopapan , Mongkolsuk Skorn , Helmann John D. 
Published: July 2002

ABSTRACT

Bacillus subtilis
displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and ς
B
regulons. We investigated the function of two
B. subtilis
homologs of the
Xanthomonas campestris
organic hydroperoxide resistance (
ohr
) gene. Mutational analyses indicate that both
ohrA
and
ohrB
contribute to organic peroxide resistance in
B. subtilis
, with the OhrA protein playing the more important role in growing cells. Expression of
ohrA
, but not
ohrB
, is strongly and specifically induced by organic peroxides. Regulation of
ohrA
requires the convergently transcribed gene,
ohrR
, which encodes a member of the MarR family of transcriptional repressors. In an
ohrR
mutant,
ohrA
expression is constitutive, whereas expression of the neighboring
ohrB
gene is unaffected. Selection for mutant strains that are derepressed for
ohrA
transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus,
B. subtilis
contains at least three regulons (ς
B
, PerR, and OhrR) that contribute to peroxide stress responses.
Reduced Function of a Phenylacetate-Oxidizing Cytochrome P450 Caused Strong Genetic Improvement in Early Phylogeny of Penicillin-Producing Strains
Authors: Rodrı́guez-Sáiz M. , Barredo J. L. , Moreno M. A. , Fernández-Cañón J. M. , Peñalva M. A. , Dı́ez B. 
Published: July 2002

ABSTRACT

The single-copy
pahA
gene from
Penicillium chrysogenum
encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the
Aspergillus nidulans
homologue PhacA. Expression level of
pahA
displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by
pahA
genes from penicillin-overproducing strains of
P. chrysogenum
compared to the activity conferred by
phacA
of
A. nidulans
. Sequencing and expression of wild-type
pahA
from
P. chrysogenum
NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49–133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.
Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of
Escherichia coli
Authors: Yang Xiaoming , Ishiguro Edward E. 
Published: July 2002

ABSTRACT

Amino acid-deprived
rplK
(previously known as
relC
) mutants of
Escherichia coli
cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The
rplK
gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of
rplK
encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.
The First Step in Polyethylene Glycol Degradation by Sphingomonads Proceeds via a Flavoprotein Alcohol Dehydrogenase Containing Flavin Adenine Dinucleotide
Authors: Sugimoto Manabu , Tanabe Miwa , Hataya Misako , Enokibara Shogo , Duine Johannis A. , Kawai Fusako 
Published: July 2002

ABSTRACT

Several
Sphingomonas
spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from
Sphingomonas terrae
revealed that the enzyme is membrane bound. The gene encoding this enzyme (
pegA
) was cloned, sequenced, and expressed in
Escherichia coli
. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from
Pseudomonas oleovorans
(46%); choline dehydrogenase from
E. coli
(40%);
l
-sorbose dehydrogenase from
Gluconobacter oxydans
(38%); and 4-nitrobenzyl alcohol dehydrogenase from a
Pseudomonas
species (35%).
Role of Alginate and Its O Acetylation in Formation of
Pseudomonas aeruginosa
Microcolonies and Biofilms
Authors: Nivens David E. , Ohman Dennis E. , Williams Jessica , Franklin Michael J. 
Published: July 2002

ABSTRACT

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of
Pseudomonas aeruginosa
on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm
−1
. All strains produced biofilms that absorbed IR radiation near 1,650 cm
−1
(amide I), 1,550 cm
−1
(amide II), 1,240 cm
−1
(PO stretching, C—O—C stretching, and/or amide III vibrations), 1,100 to 1,000 cm
−1
(C—OH and P—O stretching) 1,450 cm
−1
, and 1,400 cm
−1
. The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm
−1
(C—OH stretching of alginate) and 1,250 cm
−1
(C—O stretching of the
O
-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 μm, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 μm in depth. In contrast, FRD1 produced microcolonies that were approximately 40 μm in depth. An
algJ
mutant strain that produced alginate lacking
O
-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the
algJ
mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for
P. aeruginosa
biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in
P. aeruginosa
biofilm architecture.