bluuebunny/crossref_metadata_2025 huggingface dataset:
Hybrid Protein between Ribosomal Protein S16 and RimM of
Escherichia coli
Retains the Ribosome Maturation Function of Both Proteins
Authors: Lövgren J. Mattias , Wikström P. Mikael
Published: July 2002

The RimM protein in
Escherichia coli
is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of
E. coli
approximately threefold. Several faster-growing derivatives of the
rimM
amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the
rimM
mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the
rimM
mRNA. Three other independently isolated suppressor mutations created a fusion between
rpsP
, encoding the ribosomal protein S16, and
rimM
on the chromosome as a result of mutations in the
rpsP
stop codon preceding
rimM
. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.
Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the
fha
Promoter of
Bordetella pertussis
Authors: Boucher Philip E. , Yang Mei-Shin , Schmidt Deanna M. , Stibitz Scott
Published: July 2002

The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in
Bordetella pertussis
. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (
fha
) and pertussis toxin (
ptx
). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the
fha
promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Evaluation of Etest Method for Determining Caspofungin (MK-0991) Susceptibilities of 726 Clinical Isolates of
Candida
Species
Authors: Pfaller M. A. , Messer S. A. , Mills K. , Bolmström A. , Jones R. N.
Published: July 2002

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 isolates of
Candida
spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. MICs were determined by Etest for all 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35°C. The
Candida
isolates included
Candida albicans
(
n
= 486),
Candida glabrata
(
n
= 96),
Candida tropicalis
(
n
= 51),
Candida parapsilosis
(
n
= 47),
Candida krusei
(
n
= 11),
Candida lusitaniae
(
n
= 2), and
Candida guilliermondii
(
n
= 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for
C. parapsilosis
to 100% for
C. krusei
,
C. lusitaniae
, and
C. guilliermondii
. When CAS was used, agreement ranged from 0% for
C. lusitaniae
to 100% for
C. glabrata
. With AM3, agreement ranged from 0% for
C. lusitaniae
to 100% for
C. guilliermondii
. All three media supported growth of each of the
Candida
species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of
Candida
species.
Isolations of
Leclercia adecarboxylata
from a Patient with a Chronically Inflamed Gallbladder and from a Patient with Sepsis without Focus
Authors: de Baere Thierry , Wauters Georges , Huylenbroeck Anne , Claeys Geert , Peleman Renaat , Verschraegen Gerda , Allemeersch Daniel , Vaneechoutte Mario
Published: July 2002

Leclercia adecarboxylata
was isolated from a patient with a chronically inflamed gallbladder, together with
Enterococcus
sp. The organism was considered clinically significant and was susceptible to all antibiotics tested. Another strain of
L. adecarboxylata
was cultured from blood, together with
Escherichia hermannii
and
E. faecalis
, from a patient with sepsis.
Identification of
Actinobacillus actinomycetemcomitans
Serotypes by Multiplex PCR
Authors: Suzuki Nao , Nakano Yoshio , Yoshida Yasuo , Ikeda Daisuke , Koga Toshihiko
Published: July 2002

Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify
Actinobacillus actinomycetemcomitans
serotypes a to e using the multiplex PCR. This method may be useful for serotype-specific genotyping rapidly and directly from clinical samples containing various organisms.
Genomic Diversity of Group A Rotavirus Strains Infecting Humans in Eastern India
Authors: Das Soma , Sen A. , Uma G. , Varghese V. , Chaudhuri S. , Bhattacharya S. K. , Krishnan T. , Dutta P. , Dutta D. , Bhattacharya M. K. , Mitra U. , Kobayashi N. , Naik T. N.
Published: July 2002

Between 1998 and 2000, a total of 266 samples were found positive for group A rotaviruses by RNA electrophoresis. Samples were collected from patients admitted to two leading hospitals in Calcutta. Serotyping could be done only with 22% of the positive samples, leaving 78% untypeable. The G (VP7 genotypes) and P (VP4 genotypes) types were determined for 159 samples by reverse transcription and multiplex PCR. The predominant genotype was G1P[8] (20%), followed by G2P[4] (15%) and G4P[8] (6%). A number of uncommon genotypes, G1P[4] (4%), G2P[8] (2.5%), G2P[6] (0.6%), G4P[4] (2.5%), and G4P[6] (1.25%), were also detected during this study period. Twenty two percent of specimens showed mixed infections, 38 (24%) of the total samples remained untypeable for either VP7 or VP4, while only 4 (2.5%) of the samples were untypeable for both genes. Eleven specimens collected from Manipur were also genotyped and revealed a very high degree of genomic reassortment.
Testing of Susceptibility of
Mycobacterium tuberculosis
to Pyrazinamide with the Nonradiometric BACTEC MGIT 960 System
Authors: Pfyffer Gaby E. , Palicova Frantiska , Rüsch-Gerdes Sabine
Published: July 2002

The reliability of the novel BACTEC MGIT 960 pyrazinamide (PZA) kit (Becton Dickinson Microbiology Systems, Sparks, Md.) was assessed for testing of susceptibility of
Mycobacterium tuberculosis
to PZA. Results generated by the BACTEC MGIT 960 system (Becton Dickinson) were compared with those obtained with the BACTEC 460TB system. Extensive proficiency testing (phase I) and reproducibility testing (phase II) as well as susceptibility testing of blinded strains of
M. tuberculosis
from the Centers for Disease Control and Prevention (phase III) were performed prior to testing 58 strains isolated from clinical specimens (phase IV). After resolution of discrepant results obtained by the two BACTEC methods by two other laboratories which acted as independent arbiters (phase V), overall agreement of the BACTEC MGIT 960 system with the BACTEC 460TB system for PZA testing of phase IV strains was 96.6%. Between the two systems there was no statistically significant difference in time until results were obtained, i.e., 6.8 days (BACTEC MGIT 960) versus 5.4 days (BACTEC 460TB), the latter not counting the time required for a subculture with a growth index of 200, however. The new BACTEC MGIT PZA susceptibility testing procedure works equally well for inocula prepared from liquid (MGIT) and solid (Löwenstein-Jensen) cultures. PZA MGIT medium in plastic tubes yielded results equivalent to medium dispensed in glass tubes.
Detection, Subgroup Specificity, and Genotype Diversity of Rotavirus Strains in Children with Acute Diarrhea in Paraguay
Authors: Coluchi Norma , Munford Veridiana , Manzur Julio , Vazquez Cynthia , Escobar Mabel , Weber Ernesto , Mármol Perla , Rácz Maria Lucia
Published: July 2002

Of a total of 220 stool specimens from children with acute diarrhea, mostly under the age of 3 years, collected in Paraguay between January 1999 and March 2000, 70 (31.8%) were found positive for rotaviruses (RV). Positive samples were characterized by electropherotyping and subgrouping. Sixty-one (87.1%) were classified as group A, subgroup II; one (1.4%) was classified as group A, subgroup I; six (8.6%) were group A, non-I non-II; and two (2.9%) were not tested. RV strains were G and P genotyped by reverse transcription-PCR. The following G types were detected: G4 (34.3%), G1 (21.4%), G2 (1.4%), and G9 (5.7%). Mixtures of human and animal genotypes were detected in 15 (21.4%) samples, and 11 samples (15.7%) were nontypeable. The following P types were detected: P[8] (48.6%), P[4] (1.4%), and P[1] (1.4%). A mixed type was found in 10% of samples, and an unexpectedly high percentage (38.6%) of nontypeable samples was found. The common human G- and P-type combinations P[8], G4 (15.7%) and P[8], G1 (14.2%) were detected. Mixed human and animal genotypes were observed as the following combinations: G4 + G5, G4 + G5 + G10, and G1 + G10 for G types and P[8]-P[1] for P types. The emerging G9 genotype was detected in four samples. These results show for the first time the diversity of RV circulating among children in Paraguay and contribute to the knowledge of this pathogen required to devise strategies to prevent diarrheal illness in this country. The finding of mixed genotypes may indicate interspecies transmission of RV between humans and animals.
Evaluation of the DipStreak, a New Device with an Original Streaking Mechanism for Detection, Counting, and Presumptive Identification of Urinary Tract Pathogens
Authors: Scarparo Claudio , Piccoli Paola , Ricordi Paolo , Scagnelli Mariuccia
Published: July 2002

DipStreak is a new urine culture device with two types of agar attached back-to-back on a plastic paddle. It combines dip-slide technology and an original streaking inoculation mechanism, allowing for bacterial counting and colony isolation. The performance of the DipStreak device with two different medium formulations, CHROMagar and MacConkey media in study A and UriSelect 3 and MacConkey media in study B, was evaluated and compared to that of the reference streak method by using plates of cystine-lactose-electrolyte-deficient (CLED) agar, tryptic soy agar with 5% sheep blood, and UriSelect 3 medium. In study A, 2,000 urine specimens were processed and 511 cultures were found positive. The DipStreak device and the UriSelect 3 and CLED medium plates gave the same detection rate, 99.7%. For the direct identification of
Escherichia coli
,
Proteus mirabilis
, and
Enterococcus
sp. isolates, the DipStreak device and the UriSelect 3 medium plate showed overall sensitivities of 97 and 93.4%, respectively. In study B, 3,000 urine specimens were processed and 714 cultures were found positive. The DipStreak device and the UriSelect 3 and CLED medium plates gave detection rates of 99.4, 99.9, and 99.2%, respectively. For the direct identification of
E. coli
,
P. mirabilis
, and
Enterococcus
sp. isolates, the DipStreak device and the UriSelect 3 medium plate showed overall sensitivities of 88 and 94.4%, respectively. In conclusion, the DipStreak device with both medium formulations represents an attractive and excellent screening method for the reliable detection, counting, and presumptive identification of urinary tract pathogens. It enables bedside urine inoculation and provides a valid means of transporting the sample back to the laboratory, decreasing drastically the rate of false-positive results due to bacterial overgrowth and reducing associated costs.
Expression of the Bundle-Forming Pilus by Enteropathogenic
Escherichia coli
Strains of Heterologous Serotypes
Authors: Gismero-Ordoñez Juana , Dall'Agnol Monique , Trabulsi Luiz R. , Girón Jorge A.
Published: July 2002

The production of the bundle-forming pilus subunit BfpA was investigated in 44 enteropathogenic
Escherichia coli
(EPEC) diarrheal isolates after growth on several conventional bacteriological media. In all, the use of brucella agar supplemented with 4.5 mM sodium bisulfite resulted in a higher detection of BfpA-producing EPEC.
Pneumonia Due toBordetella bronchisepticain a Cystic Fibrosis Patient: 16S rRNA Sequencing for Diagnosis Confirmation
Authors: Wallet Frédéric , Perez Thierry , Armand Sylvie , Wallaert Benoit , Courcol René J.
Published: July 2002

Bordetella bronchisepticawas identified as an unusual etiologic agent of pulmonary recurrent exacerbations and pneumonia in a cystic fibrosis (CF) patient by utilizing a 16S rRNA molecular kit in our hospital's clinical laboratory. This method appears to be a useful approach for identifying new emerging CF pathogens when discrepancies exist between phenotypical tests.
Characterization of Two Divergent Lineages of Macaque Rhadinoviruses Related to Kaposi's Sarcoma-Associated Herpesvirus
Authors: Schultz Emily R. , Rankin George W. , Blanc Marie-Pierre , Raden Brian W. , Tsai Che-Chung , Rose Timothy M.
Published: July 2002

We have cloned and characterized the entire DNA polymerase gene and flanking regions from Kaposi's sarcoma-associated herpesvirus (KSHV) and two closely related macaque homologs of KSHV, retroperitoneal fibromatosis-associated herpesvirus-
Macaca nemestrina
(RFHVMn) and -
Macaca mulatta
(RFHVMm). We have also identified and partially characterized the corresponding genomic region of another KSHV-like herpesvirus, provisionally named “
M. nemestrina
rhadinovirus type 2 (MneRV-2),” with close similarity to rhesus rhadinovirus (RRV). A sequence comparison of these four macaque viruses and two KSHV-like gammaherpesviruses recently identified in African green monkeys, Chlorocebus rhadinovirus types 1 and 2 (ChRV-1 and ChRV-2) reveals the presence of two distinct lineages of KSHV-like rhadinoviruses in Old World primates. The first rhadinovirus lineage consists of KSHV and its closely related homologs RFHVMn, RFHVMm, and ChRV-1, while the second more distantly related lineage consists of RRV, MneRV-2, and ChRV-2. Our findings raise the possibility of the existence of another human KSHV-like herpesvirus belonging to the second rhadinovirus lineage.
Functional Analysis of theenvOpen Reading Frame in Human Endogenous Retrovirus IDDMK1,222 Encoding Superantigen Activity
Authors: Lapatschek Matthias , Dürr Susanne , Löwer Roswitha , Magin Christine , Wagner Hermann , Miethke Thomas
Published: July 2002

Mice harbor a family of endogenous retroviruses, the mouse mammary tumor viruses (MMTV), which encode superantigens. These superantigens are responsible for the deletion of T cells expressing certain Vβ chains of the T-cell receptor in the thymus. Human T cells are able to recognize MMTV-encoded superantigens presented by human major histocompatibility complex class II-positive cells. Owing to this and to the similarity of the human and murine immune systems, it was speculated that human endogenous retroviruses might also code for superantigens. Recently, it was reported that a proviral clone (IDDMK1,222) of the human endogenous retrovirus family HTDV/HERV-K encodes a superantigen. The putative superantigen gene was located within theenvregion of the virus. Stimulated by these findings, we amplified by PCR and cloned into eucaryotic expression vectors open reading frames (ORFs) which were identical or very similar to IDDMK1,222. When we transfected these vectors into A20 cells, a murine B-cell lymphoma, we were able to demonstrate mRNA expression and protein production. However, we did not find any evidence that the ORF stimulated human or murine T cells in a Vβ-specific fashion, the most prominent feature of superantigens.
The Murine Gammaherpesvirus 68 v-Cyclin Is a Critical Regulator of Reactivation from Latency
Authors: van Dyk Linda F. , Virgin Herbert W. , Speck Samuel H.
Published: July 2002

Gamma-2 herpesviruses encode a homolog of mammalian D-type cyclins. The v-cyclin encoded by murine gammaherpesvirus 68 (γHV68) induces cell cycle progression and is an oncogene (L. F. van Dyk, J. L. Hess, J. D. Katz, M. Jacoby, S. H. Speck, and H. W. Virgin IV, J. Virol. 73:5110–5122, 1999). However, the role of the pro-proliferative v-cyclins in gamma-2 herpesvirus pathogenesis is not known. Here we report the generation and characterization of a γHV68 v-cyclin mutant (v-cyclin.LacZ) that is unable to express a functional v-cyclin protein. Notably, although the γHV68 v-cyclin is expressed from an early-late lytic transcript, v-cyclin.LacZ replicated normally in fibroblasts in vitro and during acute infection in the spleen, liver, and lungs in vivo. Moreover, v-cyclin.LacZ exhibited wild-type (wt) virulence in mice with severe combined immunodeficiency. In addition, in a model of γHV68-induced chronic disease in mice lacking the gamma interferon receptor (IFNγR−/−), v-cyclin.LacZ virus was similar to wt γHV68 in terms of the incidence of mortality and vasculitis. Further analysis revealed that the frequencies of splenocytes and peritoneal cells harboring the latent γHV68 genome in normal and B-cell-deficient mice infected with wt γHV68 or v-cyclin.LacZ were very similar. However, v-cyclin.LacZ was significantly compromised in its capacity to reactivate from latency. This phenotype was conclusively mapped to the v-cyclin gene by (i) generating a marker rescue virus (v-cyclin.MR) from the v-cyclin.LacZ mutant, which restored the frequency of cells in which virus reactivated from latency to the levels observed with wt γHV68; and (ii) generating a second v-cyclin mutant virus containing a translation stop codon within the v-cyclin gene (v-cyclin.stop), which was compromised in reactivation from latency. These studies demonstrate that despite expression as a lytic cycle gene, the pro-proliferative γHV68 v-cyclin is not required for γHV68 replication either in vitro or during acute infection in vivo but rather is a critical determinant of reactivation from latency.
Identification of Mouse Hepatitis Virus Papain-Like Proteinase 2 Activity
Authors: Kanjanahaluethai Amornrat , Baker Susan C.
Published: July 2002

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5′-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in
trans
. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.
Human Immunodeficiency Virus Type 1- and Cytomegalovirus-Specific Cytotoxic T Lymphocytes Can Persist at High Frequency for Prolonged Periods in the Absence of Circulating Peripheral CD4+T Cells
Authors: Spiegel Hans M. L. , Ogg Graham S. , DeFalcon Elizabeth , Sheehy Megan E. , Monard Simon , Haslett Patrick A. J. , Gillespie Geraldine , Donahoe Sean M. , Pollack Henry , Borkowsky William , McMichael Andrew J. , Nixon Douglas F.
Published: July 2002

CD4+T cells are thought to be critical in the maintenance of virus-specific CD8+cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4+T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8+T cells when the peripheral CD4+T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8+-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8+T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4+T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.
Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors
Authors: Schneider Urs , Bullough Frances , Vongpunsawad Sompong , Russell Stephen J. , Cattaneo Roberto
Published: July 2002

We sought proof of principle that one of the safest human vaccines, measles virus Edmonston B (MV-Edm), can be genetically modified to allow entry via cell surface molecules other than its receptor CD46. Hybrid proteins consisting of the epidermal growth factor (EGF) or the insulin-like growth factor 1 (IGF1) linked to the extracellular (carboxyl) terminus of the MV-Edm attachment protein hemagglutinin (H) were produced. The standard H protein gene was replaced by one coding for H/EGF or H/IGF1 in cDNA copies of the MV genome. Recombinant viruses were rescued and replicated to titers approaching those of the parental strain. MV displaying EGF or IGF1 efficiently entered CD46-negative rodent cells expressing the human EGF or the IGF1 receptor, respectively, and the EGF virus caused extensive syncytium formation and cell death. Taking advantage of a factor Xa protease recognition site engineered in the hybrid H proteins, the displayed domain was cleaved off from virus particles, and specific entry in rodent cells was abrogated. These studies prove that MV can be engineered to selectively eliminate cells expressing a targeted receptor and provide insights into the mechanism of MV entry.