bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes Involved in Control of Galactose Uptake in
Lactobacillus brevis
and Reconstitution of the Regulatory System in
Bacillus subtilis
Authors: Djordjevic Gordana M. , Tchieu Jason H. , Saier Milton H.
Published: July 2002

The heterofermentative lactic acid bacterium
Lactobacillus brevis
transports galactose and the nonmetabolizable galactose analogue thiomethyl-β-galactoside (TMG) by a permease-catalyzed sugar:H
+
symport mechanism. Addition of glucose to
L. brevis
cells loaded with [
14
C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H
+
symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in
L. brevis
, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H
+
symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned
galP
gene was integrated into the chromosome of
Bacillus subtilis
, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in
L. brevis
operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H
+
symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.
Microbial Origin of Plant-Type 2-Keto-3-Deoxy-
d
-
arabino
-Heptulosonate 7-Phosphate Synthases, Exemplified by the Chorismate- and Tryptophan-Regulated Enzyme from
Xanthomonas campestris
Authors: Gosset Guillermo , Bonner Carol A. , Jensen Roy A.
Published: July 2002

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-
d
-
arabino
-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic
Escherichia coli
paralogs are AroA
I
proteins, but many members of the
Bacteria
possess the AroA
II
class of enzyme, sometimes in combination with AroA
I
proteins. AroA
II
DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the
Xanthomonas campestris
AroA
II
protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis.
X. campestris
AroA
II
was cloned in
E. coli
by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA
II
protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector.
l
-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA
II
of higher plants,
X. campestris
AroA
II
was not hysteretically activated by dithiols. Compared to plant AroA
II
proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA
II
originated within the
Bacteria
domain, and it seems probable that higher-plant plastids acquired AroA
II
from a gram-negative bacterium via endosymbiosis. The
X. campestris
AroA
II
protein is suggested to exemplify a case of analog displacement whereby an ancestral
aroA
I
species was discarded, with the
aroA
II
replacement providing an alternative pattern of allosteric control. Three subgroups of AroA
II
proteins can be recognized: a large, central group containing the plant enzymes and that from
X. campestris
, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.
The PDZ Domain of the SpoIVB Serine Peptidase Facilitates Multiple Functions
Authors: Hoa Ngo T. , Brannigan James A. , Cutting Simon M.
Published: July 2002

During spore formation in
Bacillus subtilis
, the SpoIVB protein is a critical component of the ς
K
regulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor ς
K
in the outer mother cell chamber of the sporulating cell. This forms the basis of the ς
K
checkpoint and ensures accurate ς
K
-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for
trans
proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-ς
K
processing, and (iv) signaling of the second function of SpoIVB.
Iron-Dependent Transcription of the
frpB
Gene of
Helicobacter pylori
Is Controlled by the Fur Repressor Protein
Authors: Delany Isabel , Pacheco Ana Beatriz F. , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo
Published: July 2002

We have overexpressed and purified the
Helicobacter pylori
Fur protein and analyzed its interaction with the intergenic regions of divergent genes involved in iron uptake (
frpB
and
ceuE
) and oxygen radical detoxification (
katA
and
tsaA
). DNase I footprint analysis showed that Fur binds specifically to a high-affinity site overlapping the
P

frpB

promoter and to low-affinity sites located upstream from promoters within both the
frpB-katA
and
ceuE-tsaA
intergenic regions. Construction of an isogenic
fur
mutant indicated that Fur regulates transcription from the P

frpB

promoter in response to iron. In contrast, no effect by either Fur or iron was observed for the other promoters.
Identification and Characterization of Tn
4656
, a Novel Class II Transposon Carrying a Set of Toluene-Degrading Genes from TOL Plasmid pWW53
Authors: Tsuda Masataka , Genka Hiroyuki
Published: July 2002

It has been reported that the toluene-degrading (
xyl
) genes from
Pseudomonas putida
plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887–895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn
4656
, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the
res
site. The Tn
4656
TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn
1721
-related class II transposons, including another toluene transposon, Tn
4653
. Sequence analysis of the transposition-related genes and sites of Tn
4656
also supported the hypothesis that this transposon is closely related to the Tn
1721
-related transposons. The lower transposition frequency of Tn
4656
has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.
In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in
Bacillus subtilis
Authors: Quisel John D. , Burkholder William F. , Grossman Alan D.
Published: July 2002

The phosphorylated form of the response regulator Spo0A (Spo0A∼P) is required for the initiation of sporulation in
Bacillus subtilis
. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in
spo0A
allow initiation of sporulation in the absence of
spo0F
and
spo0B
, but the mechanisms by which these mutations allow bypass of
spo0F
and
spo0B
are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five
spo0A
mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A∼P-specific phosphatase, on sporulation of strains containing the
spo0A
mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some
spo0A
mutants.
Multiple Regulatory Mechanisms Act on the 5′ Untranslated Region of the S-Layer Gene from
Thermus thermophilus
HB8
Authors: Castán Pablo , de Pedro Miguel A. , Risco Cristina , Vallés Cristina , Fernández Luis A. , Schwarz Heinz , Berenguer José
Published: July 2002

The role of the 5′ untranslated region (5′UTR) of the S-layer gene from
Thermus thermophilus
was analyzed through the isolation of Δ5′UTR mutants. In these mutants the half-life of
splA
mRNA was strongly reduced and
slpA
transcription was no longer subjected to growth phase-dependent repression. Overproduction and detachment of the external envelopes of the mutants were observed in stationary phase.
S-Layer Variation in
Bacillus stearothermophilus
PV72 Is Based on DNA Rearrangements between the Chromosome and the Naturally Occurring Megaplasmids
Authors: Scholz Holger C. , Riedmann Eva , Witte Angela , Lubitz Werner , Kuen Beatrix
Published: July 2002

Bacillus stearothermophilus
PV72 expresses different S-layer genes (
sbsA
and
sbsB
) under different growth conditions. No stretches of significant sequence identity between
sbsA
and
sbsB
were detected. In order to investigate S-layer gene regulation in
B. stearothermophilus
PV72, we characterized the upstream regulatory region of
sbsA
and
sbsB
by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of
sbsB
in the
sbsA
-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene
sbsB
is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of
sbsB
was exclusively detected on the chromosome of PV72/p6. For
sbsB
expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to
sbsB
expression, the
sbsA
coding region was removed from the chromosome but could still be detected on the plasmid of the
sbsB
-expressing strain PV72/p2. The
sbsA
upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.
Anaerobic Initial Reaction of
n
-Alkanes in a Denitrifying Bacterium: Evidence for (1-Methylpentyl)succinate as Initial Product and for Involvement of an Organic Radical in
n
-Hexane Metabolism
Authors: Rabus Ralf , Wilkes Heinz , Behrends Astrid , Armstroff Antje , Fischer Thomas , Pierik Antonio J. , Widdel Friedrich
Published: July 2002

A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize
n
-alkanes anaerobically with nitrate as the electron acceptor to CO
2
formed (1-methylpentyl)succinate (MPS) during growth on
n
-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on
n
-hexanoate. Anaerobic growth with [1-
13
C]
n
-hexane or
d
14
-
n
-hexane led to a 1-methylpentyl side chain in MPS with one
13
C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from
n
-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in
n
-hexane-grown cells but not in
n
-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of
n
-hexane and that of toluene, even though
n
-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that
n
-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-
d
2
-fumarate was added to cultures growing on unlabeled
n
-hexane, 3-
d
1
-MPS rather than 2,3-
d
2
-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism.
Effects of Ribosomes and Intracellular Solutes on Activities and Stabilities of Elongation Factor 2 Proteins from Psychrotolerant and Thermophilic Methanogens
Authors: Thomas Torsten , Kumar Naresh , Cavicchioli Ricardo
Published: July 2002

Low-temperature-adapted archaea are abundant in the environment, yet little is known about the thermal adaptation of their proteins. We have previously compared elongation factor 2 (EF-2) proteins from Antarctic (
Methanococcoides burtonii
) and thermophilic (
Methanosarcina thermophila
) methanogens and found that the
M. burtonii
EF-2 had greater intrinsic activity at low temperatures and lower thermal stability at high temperatures (T. Thomas and R. Cavicchioli, J. Bacteriol. 182:1328–1332, 2000). While the gross thermal properties correlated with growth temperature, the activity and stability profiles of the EF-2 proteins did not precisely match the optimal growth temperature of each organism. This indicated that intracellular components may affect the thermal characteristics of the EF-2 proteins, and in this study we examined the effects of ribosomes and intracellular solutes. At a high growth temperature the thermophile produced high levels of potassium glutamate, which, when assayed in vitro with EF-2, retarded thermal unfolding and increased catalytic efficiency. In contrast, for the Antarctic methanogen adaptation to growth at a low temperature did not involve the accumulation of stabilizing organic solutes but appeared to result from an increased affinity of EF-2 for GTP and high levels of EF-2 in the cell relative to its low growth rate. Furthermore, ribosomes greatly stimulated GTPase activity and moderately stabilized both EF-2 proteins. These findings illustrate the different physiological strategies that have evolved in two phylogenetically related but thermally distinct methanogens to enable EF-2 to function satisfactorily.
Phase-Variable Expression of an Operon Encoding Extracellular Alkaline Protease, a Serine Protease Homolog, and Lipase in
Pseudomonas brassicacearum
Authors: Chabeaud Philippe , de Groot Arjan , Bitter Wilbert , Tommassen Jan , Heulin Thierry , Achouak Wafa
Published: July 2002

The rhizobacterium
Pseudomonas brassicacearum
forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional
lacZ
gene fusions revealed that the expression of the operon is under the control of phase variation.
FosB, a Cysteine-Dependent Fosfomycin Resistance Protein under the Control of ς
W
, an Extracytoplasmic-Function ς Factor in
Bacillus subtilis
Authors: Cao Min , Bernat Bryan A. , Wang Zhepeng , Armstrong Richard N. , Helmann John D.
Published: July 2002

We demonstrate that the
Bacillus subtilis fosB(yndN)
gene encodes a fosfomycin resistance protein. Expression of
fosB
requires ς
W
, and both
fosB
and
sigW
mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn
2+
-dependent glutathione transferases but with a preference for Mg
2+
and
l
-cysteine as cofactors.
Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked
Escherichia coli
Authors: Vázquez-Laslop Nora , Lee Hyunwoo , Hu Rong , Neyfakh Alex A.
Published: July 2002

Escherichia coli
cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises ∼10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated
E. coli
cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an
E. coli
homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.
NreB from
Achromobacter xylosoxidans
31A Is a Nickel-Induced Transporter Conferring Nickel Resistance
Authors: Grass Gregor , Fan Bin , Rosen Barry P. , Lemke Karin , Schlegel Hans-Günter , Rensing Christopher
Published: July 2002

There are two distinct nickel resistance loci on plasmid pTOM9 from
Achromobacter xylosoxidans
31A,
ncc
and
nre
. Expression of the
nreB
gene was specifically induced by nickel and conferred nickel resistance on both
A. xylosoxidans
31A and
Escherichia coli. E. coli
cells expressing
nreB
showed reduced accumulation of Ni
2+
, suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni
2+
resistance.
WbjA Adds Glucose To Complete the O-Antigen Trisaccharide Repeating Unit of the Lipopolysaccharide of
Pseudomonas aeruginosa
Serogroup O11
Authors: Dean Charles R. , Datta Anup , Carlson Russell W. , Goldberg Joanna B.
Published: July 2002

Lipopolysaccharide from a
wbjA
mutant, deficient in a putative glycosyltransferase from
Pseudomonas aeruginosa
serogroup O11, was compared to that from an O-antigen polymerase mutant. Results suggest that WbjA adds the terminal glucose to complete the serogroup O11 O-antigen unit and identifies the biological repeating unit as [-2)-β-D-glucose-(1-3)-α-L-
N
-acetylfucosamine-(1-3)-β-D-
N
-acetylfucosamine-(1].
The O-Antigen Gene Cluster of
Escherichia coli
O55:H7 and Identification of a New UDP-GlcNAc C4 Epimerase Gene
Authors: Wang Lei , Huskic Sandy , Cisterne Adam , Rothemund Deborah , Reeves Peter R.
Published: July 2002

Escherichia coli
O55 is an important antigen which is often associated with enteropathogenic
E. coli
clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a
gne
gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55
gne
product has only 20 and 26% identity to the
gne
genes of
Pseudomonas aeruginosa
and
E. coli
O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the
E. coli
O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the
galF
gene and located the other between the
hisG
and
amn
genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.
Identification and Physical Characterization of the HbpR Binding Sites of the
hbpC
and
hbpD
Promoters
Authors: Tropel David , Roelof van der Meer Jan
Published: July 2002

Pseudomonas azelaica
HBP1 can use 2-hydroxybiphenyl (2-HBP) and 2,2′-dihydroxybiphenyl as sole carbon and energy sources by means of the
hbp
regulon. This regulon is composed of three genes,
hbpCA
and
hbpD
, coding for enzymes of a
meta
-cleavage pathway and the
hbpR
gene, which codes for a XylR/DmpR-type transcription regulator. It was previously shown that HbpR activates transcription from two σ
54
-dependent promoters, P

hbpC

and P

hbpD

, in the presence of 2-HBP. In this study, by using gel mobility shift assays with a purified fusion protein containing calmodulin binding protein (CBP) and HbpR, we detected two binding regions for HbpR in P

hbpC

and one binding region in P

hbpD

. DNase I footprints of the proximal binding region of P

hbpC

and of the binding region in P

hbpD

showed that CBP-HbpR protected a region composed of two inverted repeat sequences which were homologous to the binding sites identified for XylR. Unlike the situation in the XylR/P

u

system, we observed simultaneous binding of CBP-HbpR on the two upstream activating sequences (UASs). Fragments with only one UAS did not show an interaction with HbpR, indicating that both pairs of UASs are needed for HbpR binding. The addition of both ATP and 2-HBP increased the DNA binding affinity of HbpR. These results showed for the first time that, for regulators of the XylR/DmpR type, the effector positively affects the recruitment of the regulatory protein on the enhancer DNA.
Repression of the
Staphylococcus aureus
Accessory Gene Regulator in Serum and In Vivo
Authors: Yarwood Jeremy M. , McCormick John K. , Paustian Michael L. , Kapur Vivek , Schlievert Patrick M.
Published: July 2002

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (
agr
locus) as well as multiple virulence-associated genes in
Staphylococcus aureus
. Gene expression was examined during growth of
S. aureus
in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the
agr
locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of
agr
and any staphylococcal virulence factors examined. Disruption of the
agr
locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of
agr
and virulence factors was also observed. These results suggest that
agr
activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override
agr
activity.
Induction of ResDE-Dependent Gene Expression in
Bacillus subtilis
in Response to Nitric Oxide and Nitrosative Stress
Authors: Nakano Michiko M.
Published: July 2002

Transcription of ResDE-controlled genes in
Bacillus subtilis
was induced by sodium nitroprusside and nitric oxide. This induction requires the sensor kinase ResE and the response regulator ResD. Among members of the ResDE regulon, only the flavohemoglobin gene was induced by nitrosative stress via both a ResDE-dependent mechanism and an unidentified ResDE-independent mechanism.
Metabolic Signals That Lead to Control of
CBB
Gene Expression in
Rhodobacter capsulatus
Authors: Tichi Mary A. , Tabita F. Robert
Published: July 2002

Various mutant strains were used to examine the regulation and metabolic control of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in
Rhodobacter capsulatus
. Previously, a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient strain (strain SBI/II) was found to show enhanced levels of
cbb
I
and
cbb
II
promoter activities during photoheterotrophic growth in the presence of dimethyl sulfoxide. With this strain as the starting point, additional mutations were made in genes encoding phosphoribulokinase and transketolase and in the gene encoding the LysR-type transcriptional activator, CbbR
II
. These strains revealed that a product generated by phosphoribulokinase was involved in control of CbbR-mediated
cbb
gene expression in SBI/II. Additionally, heterologous expression experiments indicated that
Rhodobacter sphaeroides
CbbR responded to the same metabolic signal in
R. capsulatus
SBI/II and mutant strain backgrounds.
Differential DNA Binding of Transcriptional Regulator PcaU from
Acinetobacter
sp. Strain ADP1
Authors: Popp Roland , Kohl Tobias , Patz Patricia , Trautwein Gaby , Gerischer Ulrike
Published: July 2002

Transcriptional regulator PcaU from
Acinetobacter
sp. strain ADP1 governs expression of genes for protocatechuate degradation (
pca
genes) as a repressor or an activator depending on the levels of the inducer protocatechuate and of its own gene. PcaU is a member of the IclR protein family. Here the DNA binding properties of the purified protein are described in terms of the location of the binding sites and the affinity to these sites. Native PcaU was purified after overexpression of the
pcaU
gene in
Escherichia coli
. It is a dimer in solution. The binding site in the
pcaU-pcaI
intergenic region is located between the two divergent promoters covering 45 bp, which includes three perfect 10-bp repetitions. A PcaU binding site downstream of
pcaU
is covered by PcaU across two palindromic sequence repetitions. The affinity of PcaU for the intergenic binding sites is 50-fold higher (dissociation constant [

K
d

], 0.16 nM) than the affinity for the site downstream of
pcaU
(

K
d

, 8 nM). The binding of PcaU was tested after modifications of the intergenic binding site. Removal of any external sequence repetition still allowed for specific binding of PcaU, but the affinity was significantly reduced, suggesting an important role for all three sequence repetitions in gene expression. The involvement of DNA bending in the regulatory process is suggested by the observed strong intrinsic curvature displayed by the
pcaU-pcaI
intergenic DNA.
Isolation of the Etiologic Agent of Human Granulocytic Ehrlichiosis from the White-Footed Mouse (
Peromyscus leucopus
)
Authors: Ravyn M. Dana , Kodner Carrie B. , Carter Sarah E. , Jarnefeld Janet L. , Johnson Russell C.
Published: July 2002

We examined white-footed mice (
Peromyscus leucopus
) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected
P. leucopus
from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.
Flow Cytometry Compared with Indirect Immunofluorescence for Rapid Detection of Dengue Virus Type 1 after Amplification in Tissue Culture
Authors: Kao Chuan-Liang , Wu Meng-Chan , Chiu Yen-Hui , Lin Jing-Lin , Wu Yin-Chang , Yueh Yi-Yung , Chen Li-Kuang , Shaio Men-Fang , King Chwan-Chuen
Published: July 2002

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.
Evaluation of BACTEC MGIT 960 and BACTEC 460TB Systems for Recovery of Mycobacteria from Clinical Specimens of a University Hospital with Low Incidence of Tuberculosis
Authors: Leitritz Lorenz , Schubert Sören , Bücherl Bettina , Masch Adelheid , Heesemann Jürgen , Roggenkamp Andreas
Published: July 2002

Clinical samples obtained over a period of 8 months (
n
= 2,624) were processed in parallel with the BACTEC 460TB system, with the MGIT 960 system, and in Löwenstein-Jensen (LJ) medium, resulting in the recovery of 127 mycobacteria. Recovery rates in combinations of the BACTEC 460TB or MGIT 960 system with LJ were, respectively, 94.7 and 94.7% for
Mycobacterium tuberculosis
complex (
n
= 57) and 91.4 and 70.0% for nontuberculous mycobacteria (
n
= 70). Contamination rates, elevated in the MGIT 960 system, were associated with patients (cystic fibrosis) and type of material but not with transport time. Detection time was reduced in the MGIT 960 system.
Heterogeneity and Compartmentalization of
Pneumocystis carinii
f. sp.
hominis
Genotypes in Autopsy Lungs
Authors: Helweg-Larsen Jannik , Lundgren Bettina , Lundgren Jens D.
Published: July 2002

The extent and importance of genotype heterogeneity of
Pneumocystis carinii
f. sp.
hominis
within lungs have not previously been investigated. Two hundred forty PCR clones obtained from respiratory specimens and lung segments from three patients with fatal
P. carinii
pneumonia were investigated to detect genetic diversity in the internal transcribed spacer (ITS) region of the nuclear rRNA operon, the mitochondrial large-subunit (mtLSU) rRNA gene, and the dihydropteroate synthase-encoding gene. For two of the three examined patients, a mixture of different mtLSU rRNA and ITS genotypes was observed. Not all genotypes present in the lungs at autopsy were detected in the diagnostic respiratory samples. Compartmentalization of specific ITS and mtLSU rRNA sequence types was observed in different lung segments. In conclusion, the interpretation of genotype data and in particular ITS sequence types in the assessment of epidemiological questions should be cautious since genotyping done on respiratory samples cannot a priori be assumed to represent all genotypes present within the lung.
Standardization of Broth Microdilution and Disk Diffusion Susceptibility Tests for
Actinobacillus pleuropneumoniae
and
Haemophilus somnus
: Quality Control Standards for Ceftiofur, Enrofloxacin, Florfenicol, Gentamicin, Penicillin, Tetracycline, Tilmicosin, and Trimethoprim-Sulfamethoxazole
Authors: McDermott P. F. , Barry A. L. , Jones R. N. , Stein G. E. , Thornsberry C. , Wu C. C. , Walker R. D.
Published: July 2002

Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens,
Actinobacillus pleuropneumoniae
and
Haemophilus somnus
, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are
A. pleuropneumoniae
ATCC 27090 and
H. somnus
ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.
Staphylococcus aureus
Isolated in Cases of Impetigo Produces Both Epidermolysin A or B and LukE-LukD in 78% of 131 Retrospective and Prospective Cases
Authors: Gravet A. , Couppié P. , Meunier O. , Clyti E. , Moreau B. , Pradinaud R. , Monteil H. , Prévost G.
Published: July 2002

Clinical symptoms of impetigo and staphylococcal scalded skin syndrome may not only be expressed as the splitting of cell layers within the epidermis but are often accompanied by some localized inflammation. Toxin patterns of
Staphylococcus aureus
isolates originating from patients with impetigo and also from those with other primary and secondary skin infections in a retrospective isolate collection in France and a prospective isolate collection in French Guiana revealed a significant association (75% of the cases studied) of impetigo with production of at least one of the epidermolysins A and B and the bicomponent leucotoxin LukE-LukD (
P
< 0.001). However, most of the isolates were able to produce one of the nonubiquitous enterotoxins. Pulsed-field gel electrophoresis (PFGE) of genomic DNA hydrolyzed with
Sma
I showed a polymorphism of the two groups of isolates despite the fact that endemic clones were suspected in French Guiana and France. The combination of toxin patterns with PFGE fingerprinting may provide further discrimination among isolates defined in a given cluster or a given pulsotype and account for a specific virulence. The new association of toxins with a clinical syndrome may reveal principles of the pathological process.
Sterilization of
Mycobacterium tuberculosis
Erdman Samples by Antimicrobial Fixation in a Biosafety Level 3 Laboratory
Authors: Schwebach J. Reid , Jacobs William R. , Casadevall Arturo
Published: July 2002

Incomplete sterilization of
Mycobacterium tuberculosis
Erdman cultures followed 1 h of incubation in low concentrations of glutaraldehyde (0.5 and 1.0%) or azide. In contrast, 2.5% glutaraldehyde, paraformaldehyde (2 or 4%), Vesphine IIse or 5% formalin sterilized these samples after 1 h. These results suggest caution in removing fixed
M. tuberculosis
samples from biosafety level 3.
Use of Immunoglobulin G Antibody Avidity for Differentiation of Primary Human Herpesvirus 6 and 7 Infections
Authors: Ward K. N. , Turner D. J. , Parada X. Couto , Thiruchelvam A. D.
Published: July 2002

A human herpesvirus 7 (HHV-7) indirect immunofluorescence antibody avidity test was developed and used with an existing human herpesvirus 6 (HHV-6) antibody avidity test to detect and distinguish low-avidity antibodies to HHV-6 and HHV-7 and hence the respective primary infections. With sera from 269 British children aged 0 to 179 weeks, the tests showed that most (10 of 98 serum samples [13%]) HHV-6 low-avidity antibody was found in the first year of life, whereas for HHV-7, most (18 of 101 serum samples [20%]) HHV-7 low-avidity antibody was found in the second year of life. Five children had low-avidity antibodies to both viruses. Of nine Japanese children with previously serologically proven primary HHV-6 or HHV-7 infections, eight had low-avidity antibody only to the relevant virus, but one child had low-avidity antibodies to HHV-6 and HHV-7. The avidity tests were applied to five British children and further proof of viral infection was sought by the detection of specific DNA in serum or plasma, and saliva or cerebrospinal fluid. In two children who had low-avidity antibody to HHV-7 but who were seronegative for HHV-6, only HHV-7 was found. Both viruses were detected in one child with low-avidity HHV-7 antibody and high-avidity HHV-6 antibody. In two children with low-avidity antibodies to both viruses, HHV-6 and HHV-7 DNAs were found, confirming dual primary infections and excluding antibody cross-reactivity.
Outbreak of
Pichia anomala
Infection in the Pediatric Service of a Tertiary-Care Center in Northern India
Authors: Chakrabarti A. , Singh K. , Narang A. , Singhi S. , Batra R. , Rao K. L. N. , Ray P. , Gopalan S. , Das S. , Gupta V. , Gupta A. K. , Bose S. M. , McNeil M. M.
Published: July 2002

An outbreak of nosocomial fungemia due to the unusual yeast,
Pichia anomala
occurred in the pediatric wards of our hospital over a period of 23 months (April 1996 to February 1998). A total of 379 neonates and children (4.2% admissions) were infected. The probable index case was admitted to the pediatric emergency ward, with subsequent transmission to the premature nursery, pediatric intensive care units, and other children wards. Carriage on the hands of health care personnel was likely to be responsible for dissemination of the fungus. The outbreak could only be controlled after a health education campaign to improve hand-washing practices was instituted and after nystatin-fluconazole prophylaxis to all premature neonates and high-risk infants was introduced. In a case-control study, we identified a lower gestational age, a very low birth weight (<1,500 g), and a longer duration of hospital stay as significant risk factors associated with
P. anomala
fungemia in premature neonates. We conducted a culture prevalence survey of 50 consecutive premature neonates and found that 28% were colonized with
P. anomala
at a skin or mucosal site on the date of delivery and that 20% of these neonates subsequently developed
P. anomala
fungemia. We performed multilocus enzyme electrophoresis on 40
P. anomala
outbreak isolates (including patient and health care workers' hand isolates), and the results suggested that these isolates were identical. Our study highlights the importance of
P. anomala
as an emerging nosocomial fungal pathogen.
Helicobacter
sp. Flexispira Bacteremia in an Immunocompetent Young Adult
Authors: Iten Anne , Graf Susanne , Egger Martin , Täuber Martin , Graf Joerg
Published: July 2002

A young immunocompetent patient was admitted for a febrile illness with malaise, arthralgias, painful leg swelling, and polyserositis. Shortly prior to becoming ill, the patient had traveled to the Northern African desert. The symptoms disappeared during treatment with antibiotics (doxycycline and ceftriaxone) but recurred twice after stopping therapy. A motile gram-negative fusiform rod was isolated from a blood culture taken on the first admission. Analysis of the 16S rRNA gene of the blood culture isolate revealed close similarity with
Helicobacter
sp. flexispira taxon 8 (99.9% identity), a species that was previously reported as “
Flexispira rappini
.” This is the first reported case of a recurrent
Helicobacter
sp. flexispira bacteremia in an adult, immunocompetent patient.
Genotyping
Neisseria gonorrhoeae
Using Fluorescent Amplified Fragment Length Polymorphism Analysis
Authors: Palmer Helen M. , Arnold Catherine
Published: July 2002

An evaluation of fluorescent amplified fragment length polymorphism (FAFLP) for typing
Neisseria gonorrhoeae
using 52 isolates revealed that its discriminatory power approached that of Opa-typing in identifying epidemiologically linked isolates. Automated, accurate sizing of FAFLP amplified fragments permits objective data analysis and storage, making it an attractive method for large surveillance projects.
Characterization of IS
6110
Restriction Fragment Length Polymorphism Patterns and Mechanisms of Antimicrobial Resistance for Multidrug-Resistant Isolates of
Mycobacterium tuberculosis
from a Major Reference Hospital in Assiut, Egypt
Authors: Abbadi Said , Rashed Heba G. , Morlock Glenn P. , Woodley Charles L. , El Shanawy O. , Cooksey Robert C.
Published: July 2002

We evaluated 25
Mycobacterium tuberculosis
isolates from patients at a major Egyptian reference hospital in Assiut, Egypt, who had been treated for at least 1 year for tuberculosis. Typing patterns (IS
6110
) were diverse, and multidrug resistance was found among 11 (44%) of the isolates. Mutations associated with antimicrobial drug resistance were found in
rpoB,
katG, rpsL,
and
embB
in the resistant isolates.
Comparison of Restriction Fragment Length Polymorphism, Microsatellite Length Polymorphism, and Random Amplification of Polymorphic DNA Analyses for Fingerprinting
Aspergillus fumigatus
Isolates
Authors: Bart-Delabesse Emmanuelle , Sarfati Jacqueline , Debeaupuis Jean-Paul , van Leeuwen Willem , van Belkum Alex , Bretagne Stephane , Latge Jean-Paul
Published: July 2002

Aspergillus fumigatus
fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.
Evaluation of the VITEK 2 System for the Identification and Susceptibility Testing of Three Species of Nonfermenting Gram-Negative Rods Frequently Isolated from Clinical Samples
Authors: Joyanes Providencia , del Carmen Conejo Marı́a , Martı́nez-Martı́nez Luis , Perea Evelio J.
Published: July 2002

VITEK 2 is a new automatic system for the identification and susceptibility testing of the most clinically important bacteria. In the present study 198 clinical isolates, including
Pseudomonas aeruginosa
(
n
= 146),
Acinetobacter baumannii
(
n
= 25), and
Stenotrophomonas maltophilia
(
n
= 27) were evaluated. Reference susceptibility testing of cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin, tobramycin, levofloxacin (only for
P. aeruginosa
), co-trimoxazole (only for
S. maltophilia
), and ampicillin-sulbactam and tetracycline (only for
A. baumannii
) was performed by microdilution (NCCLS guidelines). The VITEK 2 system correctly identified 91.6, 100, and 76% of
P. aeruginosa
,
S. maltophilia
, and
A. baumannii
isolates, respectively, within 3 h. The respective percentages of essential agreement (to within 1 twofold dilution) for
P. aeruginosa
and
A. baumannii
were 89.0 and 88.0% (cefepime), 91.1 and 100% (cefotaxime), 95.2 and 96.0% (ceftazidime), 98.6 and 100% (ciprofloxacin), 88.4 and 100% (gentamicin), 87.0 and 92.0% (imipenem), 85.0 and 88.0% (meropenem), 84.2 and 96.0% (piperacillin), and 97.3 and 80% (tobramycin). The essential agreement for levofloxacin against
P. aeruginosa
was 86.3%. The percentages of essential agreement for ampicillin-sulbactam and tetracycline against
A. baumannii
were 88.0 and 100%, respectively. Very major errors for
P. aeruginosa
(resistant by the reference method, susceptible with the VITEK 2 system [resistant to susceptible]) were noted for cefepime (0.7%), cefotaxime (0.7%), gentamicin (0.7%), imipenem (1.4%), levofloxacin (2.7%), and piperacillin (2.7%) and, for one strain of
A. baumannii,
for imipenem. Major errors (susceptible to resistant) were noted only for
P. aeruginosa
and cefepime (2.0%), ceftazidime (0.7%), and piperacillin (3.4%). Minor errors ranged from 0.0% for piperacillin to 22.6% for cefotaxime against
P. aeruginosa
and from 0.0% for piperacillin and ciprofloxacin to 20.0% for cefepime against
A. baumannii
. The VITEK 2 system provided co-trimoxazole MICs only for
S. maltophilia
; no very major or major errors were obtained for co-trimoxazole against this species. It is concluded that the VITEK 2 system allows the rapid identification of
S. maltophilia
and most
P. aeruginosa
and
A. baumannii
isolates. The VITEK 2 system can perform reliable susceptibility testing of many of the antimicrobial agents used against
P. aeruginosa
and
A. baumannii
. It would be desirable if new versions of the VITEK 2 software were able to determine MICs and the corresponding clinical categories of agents active against
S. maltophilia
.