bluuebunny/crossref_metadata_2025 huggingface dataset:
The Nucleotide Sequence of Shiga Toxin (Stx) 2e-Encoding Phage φP27 Is Not Related to Other Stx Phage Genomes, but the Modular Genetic Structure Is Conserved
Authors: Recktenwald Jürgen , Schmidt Herbert
Published: July 2002

In this study we determined the complete nucleotide sequence of Shiga toxin 2e-encoding bacteriophage φP27, isolated from the Shiga toxin-producing
Escherichia coli
patient isolate 2771/97. φP27 is integrated as a prophage in the chromosomal
yecE
gene. This integration generates identity segments of
attL
and
attR
sites with lengths of 11 nucleotides. The integrated prophage genome has a size of 42,575 bp. We identified 58 open reading frames (ORFs), each with a length of >150 nucleotides. The deduced proteins of 44 ORFs showed significant homologies to other proteins present in sequence databases, whereas 14 putative proteins did not. For 29 proteins, we could deduce a putative function. Most of these are related to the basic phage propagation cycle. The φP27 genome represents a mosaic composed of genetic elements which are obviously derived from related and unrelated phages. We identified five short linker sequences of 22 to 151 bp in the φP27 sequence which have also been detected in a couple of other lambdoid phages. These linkers are located between functional modules in the phage genome and are thought to play a role in genetic recombination. Although the overall DNA sequence of φP27 is not highly related to other known phages, the data obtained demonstrate a typical lambdoid genome structure.
NK T Cells Are a Source of Early Interleukin-4 following Infection with Third-Stage Larvae of the Filarial NematodeBrugia pahangi
Authors: Balmer Paul , Devaney Eileen
Published: July 2002

Infection of C57BL/6 mice with the third-stage larvae ofBrugia pahangiresults in a rapid expansion of NK1.1+T cells in the spleen and draining lymph nodes. NK T cells produced interleukin-4 in the spleen within 24 h of infection, and these cells were CD4−.
LACK-Specific CD4
+
T Cells That Induce Gamma Interferon Production in Patients with Localized Cutaneous Leishmaniasis during an Early Stage of Infection
Authors: Bourreau Eliane , Prévot Ghislaine , Gardon Jacques , Pradinaud Roger , Hasagewa Hitoshi , Milon Geneviève , Launois Pascal
Published: July 2002

The profile of cytokines induced by soluble leishmania antigen (SLA) and the
Leishmania
homologue of the mammalian receptor for activated C kinase (LACK), a candidate vaccine against leishmaniasis, and the cellular source of the cytokines produced in response to these antigens were analyzed in patients infected with
Leishmania guyanensis
. Gamma interferon (IFN-γ) and interleukin-10 (IL-10) were produced in response to LACK. Although LACK-specific CD4
+
cells producing IFN-γ were isolated only during the early phase of infection (less than 30 days following the onset of infection), cells producing IL-10 in response to LACK were detected in all patients. CD4
+
T cells producing IFN-γ and IL-13 were produced in response to SLA in all patients. SLA- and LACK-specific T cells are effector memory cells, as they are CD45RA
−
CCR7
−
CD4
+
T cells. CD4
+
T cells producing IFN-γ are CD62L
−
, and CD4
+
T cells producing IL-10 are CD62L
+
, indicating that these cells have different tissue-homing capacities. These findings show that SLA and LACK induce both type 1 (IFN-γ) and type 2 (IL-10 or IL-13) cell responses.
The
Pseudomonas
Quinolone Signal Regulates
rhl
Quorum Sensing in
Pseudomonas aeruginosa
Authors: McKnight Susan L. , Iglewski Barbara H. , Pesci Everett C.
Published: July 2002

The opportunistic pathogen
Pseudomonas aeruginosa
uses intercellular signals to control the density-dependent expression of many virulence factors. The
las
and
rhl
quorum-sensing systems function, respectively, through the autoinducers
N
-(3-oxododecanoyl)-
l
-homoserine lactone and
N
-butyryl-
l
-homoserine lactone (C
4
-HSL), which are known to positively regulate the transcription of the elastase-encoding gene,
lasB
. Recently, we reported that a second type of intercellular signal is involved in
lasB
induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the
Pseudomonas
quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the
las
and
rhl
quorum-sensing systems, respectively. In order to define the role of PQS in the
P. aeruginosa
quorum-sensing cascade,
lacZ
gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes
lasR
,
lasI
,
rhlR
, and
rhlI
. We found that in
P. aeruginosa
, PQS caused a major induction of
rhlI′-lacZ
and had lesser effects on the transcription of
lasR′-lacZ
and
rhlR′-lacZ
. We also observed that the transcription of both
rhlI′-lacZ
and
lasB′-lacZ
was cooperatively effected by C
4
-HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the
las
and
rhl
quorum-sensing systems and that this signal is not involved in sensing cell density.
Effects of Limited Aeration and of the ArcAB System on Intermediary Pyruvate Catabolism in
Escherichia coli
Authors: Alexeeva Svetlana , de Kort Bart , Sawers Gary , Hellingwerf Klaas J. , de Mattos M. Joost Teixeira
Published: July 2002

The capacity of
Escherichia coli
to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome
bd
and cytochrome
bo
oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome
bd
oxidase-dependent respiration. The synthesis of PFL and cytochrome
bd
oxidase was found to be maximal in the lower microaerobic range but not in a ΔArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis.
WhiA, a Protein of Unknown Function Conserved among Gram-Positive Bacteria, Is Essential for Sporulation in
Streptomyces coelicolor
A3(2)
Authors: Aínsa J. A. , Ryding N. J. , Hartley N. , Findlay K. C. , Bruton C. J. , Chater K. F.
Published: July 2002

The
whiA
sporulation gene of
Streptomyces coelicolor
A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of
whiA
mutants. DNA sequencing of the wild-type allele and five
whiA
mutations verified that
whiA
is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in
S. coelicolor
, the
whiA
-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that
whiA
is essential for sporulation.
whiA
is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional
whiA
gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the
whiA
promoter does not depend on the sporulation-specific ς factor encoded by
whiG
.
Role and Mechanism of Action of C ·
Pvu
II, a Regulatory Protein Conserved among Restriction-Modification Systems
Authors: Vijesurier Roy M. , Carlock Leon , Blumenthal Robert M. , Dunbar Joan C.
Published: July 2002

The
Pvu
II restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The
Pvu
II system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (
pvuIIC
) between the divergently oriented genes for the restriction endonuclease (
pvuIIR
) and modification methyltransferase (
pvuIIM
), with
pvuIIC
in the same orientation as and partially overlapping
pvuIIR
. The product of
pvuIIC
, C ·
Pvu
II, was found to act in
trans
and to be required for expression of
pvuIIR
. In this study we demonstrate that premature expression of
pvuIIC
prevents establishment of the
Pvu
II genes, consistent with the model that requiring C ·
Pvu
II for
pvuIIR
expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing
pvuIIC
and
pvuIIM
transcripts overlap by over 60 nucleotides at their 5′ ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C ·
Pvu
II, demonstrating that it is a sequence-specific DNA-binding protein that binds to the
pvuIIC
promoter and stimulates transcription of both
pvuIIC
and
pvuIIR
into a polycistronic mRNA. The apparent location of C ·
Pvu
II binding, overlapping the −10 promoter hexamer and the
pvuIICR
transcriptional starting points, is highly unusual for transcriptional activators.
Cloning and Characterization of the
str
Operon and Elongation Factor Tu Expression in
Bacillus stearothermophilus
Authors: Krásný Libor , Vacík Tomáš , Fučík Vladimír , Jonák Jiří
Published: July 2002

The complete primary structure of the
str
operon of
Bacillus stearothermophilus
was determined. It was established that the operon is a five-gene transcriptional unit: 5′-
ybxF
(unknown function; homology to eukaryotic ribosomal protein L30)-
rpsL
(S12)-
rpsG
(S7)-
fus
(elongation factor G [EF-G])-
tuf
(elongation factor Tu [EF-Tu])-3′. The main operon promoter (
str
p) was mapped upstream of
ybxF
, and its strength was compared with the strength of the
tuf
-specific promoter (
tuf
p) located in the
fus-tuf
intergenic region. The strength of the
tuf
p region to initiate transcription is about 20-fold higher than that of the
str
p region, as determined in chloramphenicol acetyltransferase assays. Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting
cis
elements, identified upstream of
str
p (an inhibitory region) and
tuf
p (a stimulatory A/T-rich block). Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the
str
operon genes, as monitored by the expression of EF-Tu and EF-G.
Cooperative Action of the Catabolite Activator Protein and AraC In Vitro at the
araFGH
Promoter
Authors: Johnson Casonya M. , Schleif Robert F.
Published: July 2002

Full activation of transcription of the
araFGH
promoter,

p
FGH

, requires both the catabolite activator protein (CAP) and AraC protein. At

p
FGH

, the binding site for CAP is centered at position −41.5, an essential binding site for AraC is centered at position −79.5, and a second, nonessential binding site is centered at position −154.5. In this work, we used the minimal promoter region required for in vivo activation of

p
FGH

to examine the roles of CAP and AraC in stimulating formation of open complexes at

p
FGH

. Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-

p
FGH

complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true

p
FGH

promoter.
Genetic Characterization and Evolutionary Implications of a
car
Gene Cluster in the Carbazole Degrader
Pseudomonas
sp. Strain CA10
Authors: Nojiri Hideaki , Sekiguchi Hiroyo , Maeda Kana , Urata Masaaki , Nakai Sei-Ichiro , Yoshida Takako , Habe Hiroshi , Omori Toshio
Published: July 2002

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the
carAaAaBaBbCAc
(ORF7)
Ad
genes of carbazole-degrading
Pseudomonas
sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the
car
gene cluster was consequently revealed to consist of 10 genes (
carAaAaBaBbCAcAdDFE
) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between
carD
and
carFE
, makes it quite likely that the
carFE
genes were recruited from another locus. In the 21-kb region upstream from
carAa
, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated
antABC
. Four homologous insertion sequences, IS
5car1
to IS
5car4
, were identified in the neighboring regions of
car
and
ant
genes. IS
5car2
and IS
5car3
constituted the putative composite transposon containing
antABC
. One-ended transposition of IS
5car2
together with the 5′ portion of
antA
into the region immediately upstream of
carAa
had resulted in the formation of IS
5car1
and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the
car
gene cluster has been constructed. In addition, it was also revealed that the
car
and
ant
gene clusters are located on the megaplasmid pCAR1.
Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi
Authors: Navas Jesús , González-Zorn Bruno , Ladrón Néstor , Garrido Patricia , Vázquez-Boland José A.
Published: July 2002

The virulence mechanisms of the facultative intracellular parasiteRhodococcus equiremain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, thechoEmonocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified inBrevibacterium sterolicumandStreptomycesspp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded byMycobacterium tuberculosisandMycobacterium leprae. Genetic tools for use withR. equiare poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (theaacC4apramycin resistance gene fromSalmonella), and homologous recombination. ThechoEallele was disrupted by insertion of theaacC4gene, cloned in pUC19 and introduced by electroporation inR. equi. choErecombinants were isolated at frequencies between 10−2and 10−3. Twelve percent of the recombinants were double-crossoverchoEmutants. ThechoEmutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression ofchoEfrom pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor forR. equi. The highly efficient targeted mutagenesis procedure that we used to generatechoEisogenic mutants will be a valuable tool for the molecular analysis ofR. equivirulence.
Methanococcus jannaschiiGeneratesl-Proline by Cyclization ofl-Ornithine
Authors: Graupner Marion , White Robert H.
Published: July 2002

Cell extracts ofMethanococcus jannaschiihave been shown to readily convertl-ornithine tol-proline. This cyclization reaction proceeds with the loss of only the C-2 nitrogen, as has been documented for ornithine cyclodeaminase (EC4.3.1.12). Since no gene homologous to that coding for ornithine cyclodeaminase is present in the genome ofM. jannaschii, these results indicate that proline biosynthesis inM. jannaschiiis accomplished by a previously unrecognized enzyme.
Role of NtcB in Activation of Nitrate Assimilation Genes in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Aichi Makiko , Takatani Nobuyuki , Omata Tatsuo
Published: July 2002

In
Synechocystis
sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units,
nrtABCD
-
narB
and
nirA
, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in
Synechococcus
sp. strain PCC 7942. The
slr0395
gene, which encodes a protein 47% identical to
Synechococcus
NtcB, was identified as the
Synechocystis ntcB
gene, on the basis of the inability of an
slr0395
mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While
Synechococcus
NtcB strictly requires nitrite for its action,
Synechocystis
NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the
Synechococcus ntcB
mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the
Synechocystis ntcB
mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in
Synechocystis
. Activities of the nitrate assimilation enzymes in the
Synechocystis ntcB
mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.
The Global Posttranscriptional Regulator RsmA Modulates Production of Virulence Determinants and
N-
Acylhomoserine Lactones in
Pseudomonas aeruginosa
Authors: Pessi Gabriella , Williams Faye , Hindle Zoë , Heurlier Karin , Holden Matthew T. G. , Cámara Miguel , Haas Dieter , Williams Paul
Published: July 2002

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a
Pseudomonas aeruginosa
gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of
rsmA
resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of
rsmA
in
P. aeruginosa
had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on
N
-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of
N
-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and
N
-butanoylhomoserine lactone (C4-HSL) produced by the
rsmA
mutant and the
rsmA
-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using
lasI
and
rhlI
translational fusions. These data also highlighted the temporal expression control of the
lasI
gene, which was induced much earlier and to a higher level during the exponential growth phase in an
rsmA
mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control,
hcn
translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (
hcnABC
). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the
hcnA
ribosome-binding site. This suggests that, in
P. aeruginosa,
RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.
Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria
Authors: Leveau Johan H. J. , Lindow Steven E.
Published: July 2002

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-
gfp
fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P

A1/O4/O3

in response to increasing concentrations of the inducer IPTG (isopropyl-
β
-
d
-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P

A1/O4/O3

. Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in
Erwinia herbicola
than in
Escherichia coli
. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-
gfp
fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.
Noncoupled NADH:Ubiquinone Oxidoreductase of
Azotobacter vinelandii
Is Required for Diazotrophic Growth at High Oxygen Concentrations
Authors: Bertsova Yulia V. , Bogachev Alexander V. , Skulachev Vladimir P.
Published: July 2002

The gene encoding the noncoupled NADH:ubiquinone oxidoreductase (NDH II) from
Azotobacter vinelandii
was cloned, sequenced, and used to construct an NDH II-deficient mutant strain. Compared to the wild type, this strain showed a marked decrease in respiratory activity. It was unable to grow diazotrophically at high aeration, while it was fully capable of growth at low aeration or in the presence of NH
4
+
. This result suggests that the role of NDH II is as a vital component of the respiratory protection mechanism of the nitrogenase complex in
A. vinelandii
. It was also found that the oxidation of NADPH in the
A. vinelandii
respiratory chain is catalyzed solely by NDH II.
The LexA Protein from
Deinococcus radiodurans
Is Not Involved in RecA Induction following γ Irradiation
Authors: Narumi Issay , Satoh Katsuya , Kikuchi Masahiro , Funayama Tomoo , Yanagisawa Tadashi , Kobayashi Yasuhiko , Watanabe Hiroshi , Yamamoto Kazuo
Published: July 2002

The involvement of LexA in induction of RecA was investigated in
Deinococcus radiodurans
. As in the wild-type strain, an increase in RecA protein synthesis following γ irradiation was detected in a
lexA
disruptant, indicating that LexA is not involved in the induction of RecA in
D. radiodurans
.
Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from
Ralstonia eutropha
Strain Bo
Authors: Zarnt Grit , Schräder Thomas , Andreesen Jan R.
Published: July 2002

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from
Ralstonia eutropha
strain Bo was investigated for its catalytic properties. The apparent
k
cat
/

K
m

and
K
i
values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with
n
-pentanol exhibiting a
k
cat
/

K
m

value of 788 × 10
4
M
−1
s
−1
. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from
R. eutropha
strain Bo (
tfaA
) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.
The
ner
Gene of
Photorhabdus
: Effects on Primary-Form-Specific Phenotypes and Outer Membrane Protein Composition
Authors: O'Neill Keith H. , Roche Declan M. , Clarke David J. , Dowds Barbara C. A.
Published: July 2002

The nematode-bacterium complex of
Heterorhabditis-Photorhabdus
is pathogenic to insect larvae. The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes. The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries. Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as
ner
, a regulatory gene in gram-negative bacteria and their phages. Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form. These and other pieces of evidence indicate that the endogenous
ner
gene is not responsible for the secondary phenotype, but that
ner
, when overexpressed, can repress expression of primary phenotypes at stationary phase. Inactivation of the endogenous
ner
gene in the primary form affected the outer membrane protein profile. A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the
ner
primary strain.
Atypical Processing in Domain III of 23S rRNA of
Rhizobium leguminosarum
ATCC 10004
T
at a Position Homologous to an rRNA Fragmentation Site in Protozoa
Authors: Klein Franziska , Samorski Regina , Klug Gabriele , Evguenieva-Hackenberg Elena
Published: July 2002

For still unknown reasons, the 23S rRNA of many α-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The α-proteobacterium
Rhizobium leguminosarum
ATCC 10004
T
possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5′ end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.
The Products of the
spoVA
Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of
Bacillus subtilis
Authors: Tovar-Rojo Federico , Chander Monica , Setlow Barbara , Setlow Peter
Published: July 2002

Bacillus subtilis
cells with mutations in the
spoVA
operon do not complete sporulation. However, a
spoVA
strain with mutations that remove all three of the spore’s functional nutrient germinant receptors (termed the
ger3
mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation.
ger3 spoVA
and
sleB spoVA
spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or
ger3
spores. These properties of
ger3 spoVA
and
sleB spoVA
spores are identical to those of
ger3 spoVF
and
sleB spoVF
spores that lack DPA due to deletion of the
spoVF
operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in
ger3 spoVF
spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into
ger3 spoVA
spores. These data indicate that one or more products of the
spoVA
operon are involved in DPA transport into the developing forespore during sporulation.
Identification of a Gene Cluster in
Klebsiella pneumoniae
Which Includes
citX
, a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group
Authors: Schneider Karin , Kästner Christopher N. , Meyer Margareta , Wessel Mirja , Dimroth Peter , Bott Michael
Published: July 2002

The biosynthesis of the 2′-(5"-phosphoribosyl)-3′-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2′-(5"-triphosphoribosyl)-3′-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In
Escherichia coli
, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster,
citCDEFXG
. In the homologous
citCDEFG
operon of
Klebsiella pneumoniae
,
citX
is missing. A search for
K. pneumoniae citX
led to the identification of a second genome region involved in citrate fermentation which comprised the
citWX
genes and the divergent
citYZ
genes. The
citX
gene was confirmed to encode holo-citrate lyase synthase, whereas
citW
was shown to encode a citrate carrier, the third one identified in this species. The
citYZ
genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed ≥40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in
K. pneumoniae
. A
citZ
insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.
Detection of Antibodies to a Pathogenic Mycoplasma in American Alligators (Alligator mississippiensis), Broad-Nosed Caimans (Caiman latirostris), and Siamese Crocodiles (Crocodylus siamensis)
Authors: Brown D. R. , Schumacher I. M. , Nogueira M. F. , Richey L. J. , Zacher L. A. , Schoeb T. R. , Vliet K. A. , Bennett R. A. , Jacobson E. R. , Brown M. B.
Published: July 2002

An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995.Mycoplasma alligatorissp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled forM. alligatorisas the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response toM. alligatorisexposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P< 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P< 0.05) in the relatively closely related broad-nosed caimanCaiman latirostrisand the relatively distantly related Siamese crocodileCrocodylus siamensisfollowing experimental inoculation withM. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogenM. alligatorisamong captive, repatriated, and wild crocodilian species.
Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies toNeospora caninumin Cattle
Authors: Baszler Timothy V. , Adams Scott , Vander-Schalie John , Mathison Bruce A. , Kostovic Miladin
Published: July 2002

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody toNeospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing nativeN. caninumantigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a “gold standard” set of 184 cow sera defined by fetal histopathology andN. caninumimmunohistochemistry and by maternalN. caninumindirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknownN. caninumstatus. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknownN. caninumstatus revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modifiedN. caninumcELISA provided a simple, rapid, and versatile method to accurately identifyN. caninuminfection status in cattle using a single cutoff value.
Comparison of Methods for Identification of
Mycobacterium abscessus
and
M. chelonae
Isolates
Authors: Yakrus Mitchell A. , Hernandez S. Moises , Floyd Margaret M. , Sikes David , Butler W. Ray , Metchock Beverly
Published: July 2002

Mycobacterium abscessus
and
Mycobacterium chelonae
are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either
M. abscessus
or
M. chelonae
that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be
M. abscessus
and 29 were identified as
M. chelonae
. Eight isolates originally submitted as
M. chelonae
were identified as
M. abscessus
, and one isolate submitted as
M. abscessus
was found to be
M. chelonae
. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of
M. abscessus
exhibited resistance to tobramycin (MIC of 8 to ≥16 μg/ml), while all isolates of
M. chelonae
were susceptible to this drug (MIC of ≤4 μg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of
M. abscessus
and
M. chelonae
.
Identification of
Mycobacterium avium
subsp.
paratuberculosis
in Biopsy Specimens from Patients with Crohn's Disease Identified by In Situ Hybridization
Authors: Sechi Leonardo A. , Manuela Mura , Francesco Tanda , Amelia Lissia , Antonello Solinas , Giovanni Fadda , Stefania Zanetti
Published: July 2002

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient
Mycobacterium avium
subsp.
paratuberculosis
in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS
900
as a probe.
Molecular and Phenotypic Characterization of Potentially New
Shigella dysenteriae
Serotype
Authors: Coimbra Roney S. , Lenormand Pascal , Grimont Francine , Bouvet Philippe , Matsushita Shigeru , Grimont Patrick A. D.
Published: July 2002

From September 1997 to November 1998, the French National Center for
Salmonella
and
Shigella
received 22
Shigella
isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established
Shigella
serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new
Shigella dysenteriae
serotype (represented by strain 96–204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96–204, showed biochemical reactions typical of
S. dysenteriae
and gave positive results in a PCR assay for detection of the plasmid
ipaH
gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to
S. dysenteriae
serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other
Shigella
or
Escherichia coli
serotypes.
Comparison of Quantitative and Qualitative PCR Assays for Cytomegalovirus DNA in Plasma
Authors: Caliendo Angela M. , Schuurman Rob , Yen-Lieberman Belinda , Spector Stephen A. , Andersen Janet , Manjiry R. , Crumpacker Clyde , Lurain Nell S. , Erice Alejo
Published: July 2002

We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.
Precore Stop Mutant in HBeAg-Positive Patients with Chronic Hepatitis B: Clinical Characteristics and Correlation with the Course of HBeAg-to-Anti-HBe Seroconversion
Authors: Chu Chia-Ming , Yeh Chau-Ting , Lee Ching-Song , Sheen I-Shyan , Liaw Yun-Fan
Published: July 2002

This study aimed to investigate the ratios of precore stop mutant (codon 28; TGG to TAG) to total viremia in 53 HBeAg-positive patients with chronic hepatitis B by amplification-created restriction site assays along the course of HBeAg-to-anti-HBe seroconversion. At baseline, 11% had exclusive wild-type hepatitis B virus (HBV), 15% had exclusively precore mutant, and 74% had mixed viral strains. Precore mutant ratios correlated little with age, sex, or HBV DNA levels (allP> 0.1), but correlated modestly with alanine aminotransferase (ALT) levels (P= 0.05). The intervals from presentation to anti-HBe seroconversion correlated significantly with ALT and precore mutant ratios in univariate analysis but with only precore mutant ratios in multivariate analysis (P= 0.003). Precore mutant ratios at baseline were significantly higher (P< 0.001) in six patients with persistent high viremia and ALT elevation after anti-HBe seroconversion (group 1) than in 47 with remission (group 2). All group 1 patients had exclusive precore mutant after anti-HBe seroconversion, as did only 14 (30%) of the group 2 patients (P= 0.003). Among group 2 patients, precore mutant ratios at baseline or after anti-HBe seroconversion showed no significant difference between 34 patients with sustained remission and 13 with relapse. Cirrhosis developed in 50% (5 of 10) of patients with precore mutant ratios >50% at baseline but only in 12% (5 of 43) of those with precore mutant ratios of <50% at baseline (P< 0.05). In conclusion, precore mutant of variable ratios was frequently detected in HBeAg-positive patients with chronic hepatitis B. Precore mutant ratios tended to correlate with ALT levels and anti-HBe seroconversion, but high precore mutant ratios were associated with persistent hepatitis after anti-HBe seroconversion and increased risk of cirrhosis.
Comparative Evaluation of the VERSANT HCV RNA 3.0, QUANTIPLEX HCV RNA 2.0, and COBAS AMPLICOR HCV MONITOR Version 2.0 Assays for Quantification of Hepatitis C Virus RNA in Serum
Authors: Germer Jeffrey J. , Heimgartner Paul J. , Ilstrup Duane M. , Harmsen W. Scott , Jenkins Greg D. , Patel Robin
Published: July 2002

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (
P
= 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (
P
≤ 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (
P
≤ 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.
Application of a 5′ Nuclease Assay for Detection ofLawsonia intracellularisin Fecal Samples from Pigs
Authors: Lindecrona R. H. , Jensen T. K. , Andersen P. H. , Møller K.
Published: July 2002

A 5′ nuclease assay was developed to detectLawsonia intracellularisin porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5′ nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of immunohistochemistry (IM) on ileal sections of the same animals. There was 91% agreement between the results of IM and the 5′ nuclease assay. In the 5′ nuclease assay, 111 (54%) of the pigs tested positive forL. intracellularisinfection, with a mean cycle threshold (Ct) value of 27.2, whereas 98 (48%) of the pigs tested positive by IM. On average, the Ct and ΔRn values for the positive samples were 27.2 (standard deviation [SD], 3.7) and 1.6 (SD, 0.7), respectively. A Ct value of 27.2 corresponds to a fecal excretion of approximately 107L. intracellulariscells per g of feces. Furthermore, a total of 40 fecal samples derived from a herd known to be free from infection withL. intracellularisall tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1L. intracellulariscell per PCR tube. In conclusion, the 5′ nuclease assay that has been developed represents an applicable fast method for detection ofL. intracellularisin fecal samples, with a sensitivity and specificity comparable to those of IM.