bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipase and Its Modulator from
Pseudomonas
sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator
Authors: Kim Eun Kyung , Jang Won Hee , Ko Jung Ho , Kang Jong Seok , Noh Moon Jong , Yoo Ook Joon 
Published: July 2002

ABSTRACT

A lipase gene,
lipK
, and a lipase modulator gene,
limK
, of
Pseudomonas
sp. strain KFCC 10818 have been cloned, sequenced, and expressed in
Escherichia coli
. The
limK
gene is located immediately downstream of the
lipK
gene. Enzymatically active lipase was produced only in the presence of the
limK
gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the
Pseudomonas
subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known
Pseudomonas
Lifs, suggesting that
Pseudomonas
sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2
Pseudomonas
species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro
112
residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.
Peroxisomal Catalase in the Methylotrophic Yeast
Candida boidinii
: Transport Efficiency and Metabolic Significance
Authors: Horiguchi Hirofumi , Yurimoto Hiroya , Goh Toh-Kheng , Nakagawa Tomoyuki , Kato Nobuo , Sakai Yasuyoshi 
Published: July 2002

ABSTRACT

In this study we cloned
CTA1
, the gene encoding peroxisomal catalase, from the methylotrophic yeast
Candida boidinii
and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which
CTA1
was deleted (
cta1
Δ strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and
d
-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and
d
-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and
d
-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pΔnkf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H
2
O
2
, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.
Cell Density Modulates Acid Adaptation in
Streptococcus mutans
: Implications for Survival in Biofilms
Authors: Li Yung-Hua , Hanna Michael N. , Svensäter Gunnel , Ellen Richard P. , Cvitkovitch Dennis G. 
Published: July 2002

ABSTRACT

Streptococcus mutans
normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of
S. mutans
to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of
S. mutans
growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by
S. mutans
. This study was initiated to examine the acid tolerance response (ATR) of
S. mutans
biofilm cells and to determine the effect of cell density on the induction of acid adaptation.
S. mutans
BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that
S. mutans
BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in
S. mutans
log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in
S. mutans
. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the
comC
, -
D
, or -
E
genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the
comC
mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in
S. mutans
, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.
Ammonia Switch-Off of Nitrogen Fixation in the Methanogenic Archaeon
Methanococcus maripaludis
: Mechanistic Features and Requirement for the Novel GlnB Homologues, NifI
1
and NifI
2
Authors: Kessler Peter S. , Daniel Catherine , Leigh John A. 
Published: July 2002

ABSTRACT

Ammonia switch-off is the immediate inactivation of nitrogen fixation that occurs when a superior nitrogen source is encountered. In certain bacteria switch-off occurs by reversible covalent ADP-ribosylation of the dinitrogenase reductase protein, NifH. Ammonia switch-off occurs in diazotrophic species of the methanogenic
Archaea
as well. We showed previously that in
Methanococcus maripaludis
switch-off requires at least one of two novel homologues of
glnB
, a family of genes whose products play a central role in nitrogen sensing and regulation in bacteria. The novel
glnB
homologues have recently been named
nifI
1
and
nifI
2
. Here we use in-frame deletions and genetic complementation analysis in
M. maripaludis
to show that the
nifI
1
and
nifI
2
genes are both required for switch-off. We could not detect ADP-ribosylation or any other covalent modification of dinitrogenase reductase during switch-off, suggesting that the mechanism differs from the well-studied bacterial system. Furthermore, switch-off did not affect
nif
gene transcription,
nifH
mRNA stability, or NifH protein stability. Nitrogenase activity resumed within a short time after ammonia was removed from a switched-off culture, suggesting that whatever the mechanism, it is reversible. We demonstrate the physiological importance of switch-off by showing that it allows growth to accelerate substantially when a diazotrophic culture is switched to ammonia.
Chlorocatechols Substituted at Positions 4 and 5 Are Substrates of the Broad-Spectrum Chlorocatechol 1,2-Dioxygenase of
Pseudomonas chlororaphis
RW71
Authors: Potrawfke Thomas , Armengaud Jean , Wittich Rolf-Michael 
Published: July 2002

ABSTRACT

The nucleotide sequence of a 10,528-bp region comprising the chlorocatechol pathway gene cluster
tetRtetCDEF
of the 1,2,3,4-tetrachlorobenzene via the tetrachlorocatechol-mineralizing bacterium
Pseudomonas chlororaphis
RW71 (T. Potrawfke, K. N. Timmis, and R.-M. Wittich, Appl. Environ. Microbiol. 64:3798–3806, 1998) was analyzed. The chlorocatechol 1,2-dioxygenase gene
tetC
was cloned and overexpressed in
Escherichia coli
. The recombinant gene product was purified, and the α,α-homodimeric TetC was characterized. Electron paramagnetic resonance measurements confirmed the presence of a high-spin-state Fe(III) atom per monomer in the holoprotein. The productive transformation by purified TetC of chlorocatechols bearing chlorine atoms in positions 4 and 5 provided strong evidence for a significantly broadened substrate spectrum of this dioxygenase compared with other chlorocatechol dioxygenases. The conversion of 4,5-dichloro- or tetrachlorocatechol, in the presence of catechol, displayed strong competitive inhibition of catechol turnover. 3-Chlorocatechol, however, was simultaneously transformed, with a rate similar to that of the 4,5-halogenated catechols, indicating similar specificity constants. These novel characteristics of TetC thus differ significantly from results obtained from hitherto analyzed catechol 1,2-dioxygenases and chlorocatechol 1,2-dioxygenases.
Functional Expression in
Escherichia coli
of Low-Affinity and High-Affinity Na
+
(Li
+
)/H
+
Antiporters of
Synechocystis
Authors: Inaba Masami , Sakamoto Atsushi , Murata Norio 
Published: July 2002

ABSTRACT

Synechocystis
sp. strain PCC 6803 has five genes for putative Na
+
/H
+
antiporters (designated
nhaS1
,
nhaS2
,
nhaS3
,
nhaS4
, and
nhaS5
). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na
+
/H
+
antiporter of
Pseudomonas aeruginosa
, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na
+
/H
+
antiporter of
Enterococcus hirae
. We successfully induced the expression of
nhaS1
,
nhaS3
, and
nhaS4
under control of an Na
+
-dependent promoter in
Escherichia coli
TO114, a strain that is deficient in Na
+
/H
+
antiport activity. Inverted membrane vesicles prepared from TO114
nhaS1
and TO114
nhaS3
cells exhibited Na
+
(Li
+
)/H
+
antiport activity. Kinetic analysis of this activity revealed that
nhaS1
encodes a low-affinity Na
+
/H
+
antiporter with a

K
m

of 7.7 mM for Na
+
ions and a

K
m

of 2.5 mM for Li
+
ions, while
nhaS3
encodes a high-affinity Na
+
/H
+
antiporter with a

K
m

of 0.7 mM for Na
+
ions and a

K
m

of 0.01 mM for Li
+
ions. Transformation of
E. coli
TO114 with the
nhaS1
and
nhaS3
genes increased cellular tolerance to high concentrations of Na
+
and Li
+
ions, as well as to depletion of K
+
ions during cell growth. To our knowledge, this is the first functional characterization of Na
+
/H
+
antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114
nhaS4
cells did not have Na
+
/H
+
antiport activity, and the cells themselves were as sensitive to Na
+
and Li
+
ions as the original TO114 cells. However, the TO114
nhaS4
cells were tolerant to depletion of K
+
ions. Taking into account these results and the growth characteristics of
Synechocystis
mutants in which
nhaS
genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in
Synechocystis
.
Targeted Inactivation of the
mecB
Gene, Encoding Cystathionine-γ-Lyase, Shows that the Reverse Transsulfuration Pathway Is Required for High-Level Cephalosporin Biosynthesis in
Acremonium chrysogenum
C10 but Not for Methionine Induction of the Cephalosporin Genes
Authors: Liu Gang , Casqueiro Javier , Bañuelos Oscar , Cardoza Rosa E. , Gutiérrez Santiago , Martı́n Juan F. 
Published: July 2002

ABSTRACT

Targeted gene disruption efficiency in
Acremonium chrysogenum
was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the
mecB
gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive
mecB
gene by Southern blot analysis and no cystathionine-γ-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain,
A. chrysogenum
C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain
A. chrysogenum
C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in
A. chrysogenum
derives from the autotrophic (SH
2
) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene
pcbC
were identical in the parental strain and the
mecB
mutants, indicating that the induction effect is not mediated by cystathionine-γ-lyase.
Genetic Interactions between the
Escherichia coli umuDC
Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase
Authors: Sutton Mark D. , Farrow Mary F. , Burton Briana M. , Walker Graham C. 
Published: July 2002

ABSTRACT

The
Escherichia coli umuDC
gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the
umuDC
gene products are regulated by RecA–single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD′ (which enables TLS). In addition, even modest overexpression of the
umuDC
gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD
2
C. We have previously reported that overexpression of the ɛ proofreading subunit of DNA polymerase III suppresses
umuDC
-mediated cold sensitivity, suggesting that interaction of ɛ with UmuD
2
C is important for the DNA damage checkpoint control function of the
umuDC
gene products. Here, we report that overexpression of the β processivity clamp of the
E. coli
replicative DNA polymerase (encoded by the
dnaN
gene) not only exacerbates the cold sensitivity conferred by elevated levels of the
umuDC
gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the
umuD
′
C
gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant β proteins possibly deficient for physical interactions with the
umuDC
gene products, we selected for novel
dnaN
alleles unable to confer a cold-sensitive growth phenotype upon a
umuD
′
C
-overexpressing strain. In all, we identified 75
dnaN
alleles, 62 of which either reduced the expression of β or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of
dnaN
. Each of the
dnaN
missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight
dnaN
alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the
umuDC
gene products, suggesting that the interactions between UmuD′ and β are a subset of those between UmuD and β. Taken together, these findings suggest that interaction of β with UmuD
2
C is important for the DNA damage checkpoint function of the
umuDC
gene products. Four possible models for how interactions of UmuD
2
C with the ɛ and the β subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.
Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering
Authors: Suenaga Hikaru , Watanabe Takahito , Sato Mika , Ngadiman  , Furukawa Kensuke 
Published: July 2002

ABSTRACT

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs). Based on crystallographic analyses of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure
6:
571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of
Pseudomonas pseudoalcaligenes
KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in
Escherichia coli
. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2′,5′-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2′-trichlorobiphenyl and 2,5,4′-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2′,3′-dihydroxy biphenyl with concomitant dechlorination.
Rapid Identification and Antibiotic Susceptibility Testing of
Salmonella enterica
Serovar Typhi Isolated from Blood: Implications for Therapy
Authors: Saha Samir K. , Darmstadt Gary L. , Baqui Abdullah H. , Hanif M. , Ruhulamin M. , Santosham Mathuram , Nagatake T. , Black Robert E. 
Published: July 2002

ABSTRACT

The turnaround time (TAT) for
Salmonella enterica
serovar Typhi identification and reporting of the antibiotic susceptibility profile was determined for 391 cases of typhoid fever, using the lysis direct plating or lysis centrifugation method of blood culture along with rapid antimicrobial susceptibility testing. The TAT was more rapid (TAT for 90% of the patients [TAT
90
] = 30 h; TAT
100
≤ 67 h) than was possible with conventional methodologies and was equivalent to that reported previously using more advanced, costly technologies that are largely unavailable in developing countries. Antibiotic susceptibility profiles, determined by the rapid antimicrobial susceptibility testing method, of randomly selected 60
S. enterica
serovar Typhi isolates were identical to those determined by overnight conventional testing. Preliminary assessment of the impact of the reduced TAT on physician practices revealed that initial empirical therapy was prescribed at the time of presentation in most cases (87 of 108 [81%]) despite awareness that the final report would be available on the following day. Patients treated empirically with first-line antibiotics and shown subsequently to be infected with a multidrug-resistant strain benefited most (8 cases), since therapy was changed appropriately on the following day. In an additional 21 cases, therapy with an appropriate antibiotic was initiated after culture results were available. Thus, in approximately one-fourth (29 of 108 [27%]) of the cases, a change in management to an agent active for treatment of the isolate was made after receipt of the test results. However, in no case was therapy changed from a second-line to a first-line agent, even if the isolate was reported on the day after presentation to be sensitive to first-line therapy (33 cases). Ways in which to utilize rapid-TAT result reporting in order to positively influence physicians' prescribing in Bangladesh are the subject of ongoing research.
Clinical Evaluation of Anti-Tuberculous Glycolipid Immunoglobulin G Antibody Assay for Rapid Serodiagnosis of Pulmonary Tuberculosis
Authors: Maekura Ryoji , Okuda Yoshinari , Nakagawa Masaru , Hiraga Touru , Yokota Souichirou , Ito Masami , Yano Ikuya , Kohno Hiroaki , Wada Masako , Abe Chiyoji , Toyoda Takeo , Kishimoto Toshio , Ogura Takeshi 
Published: July 2002

ABSTRACT

Previously we reported the development of a highly sensitive enzyme-linked immunosorbent assay specific for anti-tuberculous glycolipid (anti-TBGL) for the rapid serodiagnosis of tuberculosis. In this study, the usefulness of an anti-TBGL antibody assay kit for rapid serodiagnosis was evaluated in a controlled multicenter study. Antibody titers in sera from 318 patients with active pulmonary tuberculosis (216 positive for
Mycobacterium tuberculosis
in smear and/or culture tests and 102 smear and culture negative and clinically diagnosed), 58 patients with old tuberculosis, 177 patients with other respiratory diseases, 156 patients with nonrespiratory diseases, and 454 healthy subjects were examined. Sera from 256 younger healthy subjects from among the 454 healthy subjects were examined as a control. When the cutoff point of anti-TBGL antibody titer was determined as 2.0 U/ml, the sensitivity for active tuberculosis patients was 81.1% and the specificity was 95.7%. Sensitivity in patients with smear-negative and culture-negative active pulmonary tuberculosis was 73.5%. Even in patients with noncavitary minimally advanced lesions, the positivity rate (60.0%) and the antibody titer (4.6 ± 9.4 U/ml) were significantly higher than those in the healthy group. These results indicate that this assay using anti-TBGL antibody is useful for the rapid serodiagnosis of active pulmonary tuberculosis.
Evaluation of BacT/Alert 3D Liquid Culture System for Recovery of Mycobacteria from Clinical Specimens Using Sodium Dodecyl (Lauryl) Sulfate-NaOH Decontamination
Authors: Carricajo A. , Fonsale N. , Vautrin A. C. , Aubert G. 
Published: July 2002

ABSTRACT

A total of 52 mycobacterial isolates were recovered from 1,197 clinical specimens decontaminated by a sodium dodecyl (lauryl) sulfate (SDS)-NaOH protocol. Of these, 94% were recovered with the BacT/Alert 3D system (Organon Teknika, Durham, N.C.) and 79% were recovered on Löwenstein-Jensen (LJ) medium. Mean times to detection of organisms of the
Mycobacterium tuberculosis
complex (
n
= 47) were 22.8 days with LJ medium and 16.2 days with the system. The BacT/Alert 3D system is a rapid and efficient detection system which can be used with an SDS-NaOH decontamination procedure.
Trends in Antifungal Susceptibility among Swedish
Candida
Species Bloodstream Isolates from 1994 to 1998: Comparison of the E-test and the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A Reference Method
Authors: Chryssanthou Erja 
Published: July 2002

ABSTRACT

A comparative evaluation of the NCCLS macrodilution method, the E-test, and the Sensititre YeastOne Colorimetric Antifungal Panel for the susceptibility testing of fluconazole, itraconazole, amphotericin B, and flucytosine was conducted with 233 blood isolates of
Candida
species collected between 1994 and 1998 in Sweden. Antifungal susceptibility profiles of
Candida albicans
and non-
C.
albicans Candida
species remained essentially unchanged within the 5-year study period. The overall agreement rates for the E-test and the NCCLS MICs and for the YeastOne and the NCCLS MICs were ≥86 and ≥87%, respectively, within ±1 dilution for fluconazole, amphotericin B, and flucytosine, and ≥66 and ≥57%, respectively, for itraconazole. The E-test and the YeastOne panels are equivalent, and both are convenient methods for routine use.