bluuebunny/crossref_metadata_2025 huggingface dataset:
Noncatalytic Docking Domains of Cellulosomes of Anaerobic Fungi
Authors: Steenbakkers Peter J. M. , Li Xin-Liang , Ximenes Eduardo A. , Arts Jorik G. , Chen Huizhong , Ljungdahl Lars G. , Op den Camp Huub J. M.
Published: July 2002

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus
Piromyces
sp. strain E2 and the polycentric fungus
Orpinomyces
sp. strain PC-2. Through PCR using cDNA from
Orpinomyces
sp. and genomic DNA from
Piromyces
sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an
Orpinomyces
sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus
Orpinomyces
sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.
LuxArray, a High-Density, Genomewide Transcription Analysis of
Escherichia coli
Using Bioluminescent Reporter Strains
Authors: Van Dyk Tina K. , DeRose Ellen J. , Gonye Gregory E.
Published: July 2002

A sequenced collection of plasmid-borne random fusions of
Escherichia coli
DNA to a
Photorhabdus luminescens luxCDABE
reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions. This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in
E. coli
. High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression. The cellular response to nalidixic acid perturbation was analyzed using this format. As expected, fusions to promoters of LexA-controlled SOS-responsive genes
dinG
,
dinB
,
uvrA
, and
ydjM
were found to be upregulated in the presence of nalidixic acid. In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated. The responses of two of these, fusions to
oraA
and
yigN
, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon. The responses of the other four were neither induced by mitomycin C nor dependent on
lexA
function. Thus, the promoters of
ycgH, intG
,
rihC
, and a putative operon consisting of
lpxA, lpxB, rnhB,
and
dnaE
were not generally DNA damage responsive and represent a more specific response to nalidixic acid. These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses.
Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of
Haemophilus ducreyi
: Restoration of Full-Length LOS Restores Pyocin Sensitivity
Authors: Filiatrault Melanie J. , Munson Robert S. , Campagnari Anthony A.
Published: July 2002

DNA sequence and Southern blot analyses were used to determine the genetic defect of a
Haemophilus ducreyi
pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire
waaQ
gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of
H. influenzae
, the gene encoding an argininosuccinate synthase homolog, and a change in the 3′ sequence of the
lgtF
gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.
Temporal Regulation of Genes Encoding the Flagellar Proximal Rod in
Caulobacter crescentus
Authors: Boyd Charles H. , Gober James W.
Published: July 2002

The gram-negative bacterium
Caulobacter crescentus
has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid
trans
-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. The
flgBC
-
fliE
genes were identified as a result of the
C. crescentus
genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with ς
70
is responsible for regulating transcription of other early flagellar genes in
C. crescentus
. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and
flgB
deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing
fliP
, which encodes a member of the flagellum-specific protein export apparatus.
The TOL Plasmid pWW0
xylN
Gene Product from
Pseudomonas putida
Is Involved in
m-
Xylene Uptake
Authors: Kasai Yuki , Inoue Jun , Harayama Shigeaki
Published: July 2002

The upper operon of the TOL plasmid pWW0 of
Pseudomonas putida
encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon,
xylN
, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in
Escherichia coli
. To analyze the role of the
xylN
gene product,
xylN
on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of
P. putida
harboring the wild-type and
xylN
mutant TOL plasmids were characterized. The growth of
P. putida
harboring the wild-type TOL plasmid was inhibited by a high concentration of
m
-xylene, while that of
P. putida
harboring the
xylN
mutant TOL plasmid was not. The apparent
K

s

value for the oxidation of
m
-xylene in intact cells of the
xylN
mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the
xylN
gene product is a porin involved in the transport of
m
-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.
Regulation of Gene Expression in Response to Oxygen in
Rhizobium etli
: Role of FnrN in
fixNOQP
Expression and in Symbiotic Nitrogen Fixation
Authors: Lopez Oswaldo , Morera Claudia , Miranda-Rios Juan , Girard Lourdes , Romero David , Soberón Mario
Published: July 2002

Previously, we reported finding duplicated
fixNOQP
operons in
Rhizobium etli
CFN42. One of these duplicated operons is located in the symbiotic plasmid (
fixNOQP
d), while the other is located in a cryptic plasmid (
fixNOQP
f). Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both
fixNOQP
duplicated operons, we found that a mutation in
fixL
eliminates
fixNOQP
f expression but has only a moderate effect on expression of
fixNOQP
d. This suggests that there are differential regulatory controls. Interestingly, only the
fixNOQP
d operon was essential for symbiotic nitrogen fixation (L. Girard, S. Brom, A. Dávalos, O. Lopez, M. Soberón, and D. Romero, Mol. Plant-Microbe Interact. 13:1283–1292, 2000). Searching for potential candidates responsible for the differential expression, we characterized two
fnrN
homologs (encoding transcriptional activators of the cyclic AMP receptor protein [CRP]-Fnr family) in
R. etli
CFN42. One of these genes (
fnrN
d) is located on the symbiotic plasmid, while the other (
fnrN
chr) is located on the chromosome. Analysis of the expression of the
fnrN
genes using transcriptional fusions with
lacZ
showed that the two
fnrN
genes are differentially regulated, since only
fnrN
d is expressed in microaerobic cultures of the wild-type strain while
fnrN
chr is negatively controlled by FixL. Mutagenesis of the two
fnrN
genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the
fixNOQP
d operon. Participation of these genes is also seen during the symbiotic process, in which mutations in
fnrN
d and
fnrN
chr, either singly or in combination, lead to reductions in nitrogen fixation. Therefore,
R. etli
employs a regulatory circuit for induction of the
fixNOQP
d operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory circuit may be important for ensuring optimal production of the
cbb
3
, terminal oxidase during symbiosis.
Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in
Bacillus subtilis
Authors: Ould Ali Naima , Bignon Joelle , Rapoport Georges , Debarbouille Michel
Published: July 2002

Bacillus subtilis
grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The
acoABCL
operon encodes the E1α, E1β, E2, and E3 subunits of the acetoin dehydrogenase complex in
B. subtilis
. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The
acoR
gene is located downstream from the
acoABCL
operon and encodes a positive regulator which stimulates the transcription of the operon. The product of
acoR
has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a −12, −24 promoter, and transcription is abolished in
acoR
and
sigL
mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an
acoR
′-′
lacZ
strain of
B. subtilis
showed that the expression of
acoR
is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of
acoR
is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of
acoR
was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of
acoR
is mediated by the binding of CcpA to the promoter region of
acoR
.
Genetic Isolation of Meningococci of the Electrophoretic Type 37 Complex
Authors: Claus H. , Stoevesandt J. , Frosch M. , Vogel U.
Published: July 2002

Neisseria meningitidis
(the meningococcus) is a naturally competent bacterial species in which intra- and interspecific horizontal gene transfer is a major source of genetic diversity. In strains of the electrophoretic type 37 (ET-37) complex and of the A4 cluster, we identified genomic DNA coding for a novel restriction-modification system and for the tail of a previously unidentified prophage. Furthermore, a novel 7.2-kb DNA segment restricted to clones of the ET-37 complex and the A4 cluster was isolated and shown to occur both as a plasmid (pJS-B) and as a chromosomal integration. Neither the genomic loci nor pJS-B was present in ET-5 complex, lineage 3, or serogroup A meningococci. The differential distribution of the DNA segments described herein, as well as of
opcA, porB, nmeAI, nmeBI
, and
nmeDI
described previously, supports the concept of genetic isolation of hypervirulent lineages responsible for most cases of serogroup C disease worldwide.
Transcriptional Regulation of the
orf19
Gene and the
tir-cesT-eae
Operon of Enteropathogenic
Escherichia coli
Authors: Sánchez-SanMartı́n Claudia , Bustamante Vı́ctor H. , Calva Edmundo , Puente José Luis
Published: July 2002

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic
Escherichia coli
(EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The
tir
gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by
orf19
and downstream by the
cesT
and
eae
genes. With the use of a series of
cat
transcriptional fusions and primer extension analysis, we confirmed that
tir, cesT
, and
eae
form the
LEE5
operon, which is under the control of a promoter located upstream from
tir
, and found that the
orf19
gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the
tir
and the
orf19
promoters and that a sequence motif located between positions −204 and −157 was needed for the Ler-dependent activation of the
tir
operon. Sequence elements located between positions −204 and −97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the
tir
promoter was fully active even in the absence of Ler, indicating that
tir
expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25°C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of
orf19
, but not of the
tir
operon, became Ler independent in an
hns
mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene.
Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from
Xanthobacter autotrophicus
Strain Py2 and
Rhodobacter capsulatus
Strain B10
Authors: Sluis Miriam K. , Larsen Rachel A. , Krum Jonathan G. , Anderson Ruth , Metcalf William W. , Ensign Scott A.
Published: July 2002

Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO
2
to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium
Rhodobacter capsulatus
was purified to homogeneity and compared to that of
Xanthobacter autotrophicus
strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (α
2
β
2
γ
2
multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH
3
COCH
3
+ CO
2
+ ATP→CH
3
COCH
2
COO
−
+ H
+
+ AMP + 2P
i
), and kinetic properties (

K
m

for acetone, 8 μM;
k
cat
= 45 min
−1
). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of
X. autotrophicus
and sequence analysis of the
R. capsulatus
genome and were found to be clustered in similar operons consisting of the genes
acxA
(β subunit),
acxB
(α subunit), and
acxC
(γ subunit). Transposon mutagenesis of
X. autotrophicus
revealed a requirement of σ
54
and a σ
54
-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential σ
54
-dependent promoter 122 bp upstream of
X. autotrophicus acxABC
was identified. An AcxR gene homolog was identified 127 bp upstream of
acxA
in
R. capsulatus
, but this activator lacked key features of σ
54
-dependent activators, and the associated
acxABC
lacked an apparent σ
54
-dependent promoter, suggesting that σ
54
is not required for expression of
acxABC
in
R. capsulatus.
These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.
Characterization and Regulation of the
gbuA
Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of
Pseudomonas aeruginosa
PAO1
Authors: Nakada Yuji , Itoh Yoshifumi
Published: July 2002

The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO
2
and urea. Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway. We investigated the structure and regulation of the gene for GBase (designated
gbuA
) of
Pseudomonas aeruginosa
PAO1 and characterized the
gbuA
product. The
gbuA
and the adjacent
gbuR
genes were cloned by functional complementation of a
gbuA9005
mutant of strain PAO1 defective in 4-GB utilization. The deduced amino acid sequence of GbuA (319 amino acids;
M
r
34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases. Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with

K
m

and
K
cat
values of 49 mM and 1,012 s
−1,
respectively. The divergent
gbuR
gene, which shared the intergenic promoter region of 206 bp with
gbuA
, encoded a putative regulatory protein (297 amino acids;
M
r
33,385) homologous to the LysR family of proteins. Insertional inactivation of
gbuR
by a gentamicin resistance cassette caused a defect in 4-GB utilization. GBase and
gbuA
′::′
lacZ
fusion assays demonstrated that this
gbuR
mutation abolishes the inducible expression of
gbuA
by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene. Northern blotting located an inducible promoter for
gbuA
in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of
gbuA
. The
gbuRA
genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene
kauB
at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway.
Identification of Target Genes Regulated by the Two-Component System HP166-HP165 of
Helicobacter pylori
Authors: Dietz Patricia , Gerlach Gabriele , Beier Dagmar
Published: July 2002

Two-component systems are signal transduction systems which enable bacteria to regulate cellular functions in response to changing environmental conditions. In most cases regulation is accomplished on the transcriptional level by a response regulator protein, which, according to the phosphorylation state of its receiver domain, displays different affinities for its target promoters. Here we describe identification of genes regulated by the two-component system HP166-HP165 of
Helicobacter pylori
and characterization of the corresponding target promoters. We demonstrated that expression of the HP166-HP165 two-component system is negatively autoregulated under conditions favoring autophosphorylation of the histidine kinase. Furthermore, we found that response regulator HP166 activates transcription of genes encoding a protein family with an unknown function present in
H. pylori
26695, as well as an operon composed of five
H. pylori-
specific genes. While open reading frame HP166 is an essential gene, the target genes of the response regulator are not required for growth under in vitro culture conditions.
Luteolin and GroESL Modulate In Vitro Activity of NodD
Authors: Yeh Kuo-Chen , Peck Melicent C. , Long Sharon R.
Published: July 2002

In the early stages of symbiosis between the soil bacterium
Sinorhizobium meliloti
and its leguminous host plant, alfalfa, bacterial nodulation (
nod
) genes are controlled by NodD1, NodD2, and NodD3, members of the LysR family of transcriptional regulators, in response to flavonoid and other inducers released by alfalfa. To gain an understanding of the biochemical aspects of this action, epitope-tagged recombinant NodD1 and NodD3 were overexpressed in
Escherichia coli
. The DNA binding properties of the purified recombinant NodD proteins were indistinguishable from those of NodD isolated from
S. meliloti
. In addition, the
E. coli
GroEL chaperonin copurified with the recombinant NodD proteins. In this study, we showed that NodD proteins are in vitro substrates of the GroESL chaperonin system and that their DNA binding activity is modulated by GroESL. This confirmed the earlier genetic implication that the GroESL chaperonin system is essential for the function of these regulators. Increased DNA binding activity by NodD1 in the presence of luteolin confirmed that NodD1 is involved in recognizing the plant signal during the early stages of symbiosis.
Nonenzymatic Turnover of anErwinia carotovoraQuorum-Sensing Signaling Molecule
Authors: Byers Joseph T. , Lucas Claire , Salmond George P. C. , Welch Martin
Published: July 2002

The production of virulence factors and carbapenem antibiotic in the phytopathogenErwinia carotovorais under the control of quorum sensing. The quorum-sensing signaling molecule,N-(3-oxohexanoyl)-l-homoserine lactone (OHHL), accumulates in log-phase culture supernatants ofE. carotovorabut diminishes in concentration during the stationary phase. In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur. Rather, it was caused by degradation of the molecule. The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing. The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to ∼8.5. OHHL became unstable over a narrow pH range (pH 7 to 8). Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30°C) by buffering the samples at pH 6.8. These results may provide a rationale for the observation that an early response of plants which are under attack byErwiniais to activate a proton pump which alkalizes the site of infection to a pH of >8.2.
act
Operon Control of Developmental Gene Expression in
Myxococcus xanthus
Authors: Gronewold Thomas M. A. , Kaiser Dale
Published: July 2002

Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the
csgA
gene, which encodes the C-signal, is directed by four genes of the
act
operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the
act
genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in
actA
,
actB
,
actC
, or
actD
mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null
act
mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The
actA
and
actB
null mutations substantially decreased the expression of all these reporters. The other
act
null mutations caused either premature expression to wild-type levels (
actC
) or delayed expression (
actD
), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.
Identification of Two
myo-
Inositol Transporter Genes of
Bacillus subtilis
Authors: Yoshida Ken-Ichi , Yamamoto Yoshiyuki , Omae Kaoru , Yamamoto Mami , Fujita Yasutaro
Published: July 2002

Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the
Bacillus subtilis
genome project, we found that a mutant with inactivation of
iolT
(
ydjK
) exhibited a growth defect on
myo
-inositol as the sole carbon source. The putative product of
iolT
exhibits significant similarity with many bacterial sugar transporters in the databases. In
B. subtilis
, the
iolABCDEFGHIJ
and
iolRS
operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the
iol
genes,
iolF
was predicted to encode an inositol transporter. Inactivation of
iolF
alone did not cause such an obvious growth defect on inositol as the
iolT
inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single
iolT
inactivation. Determination of inositol uptake by the mutants revealed that
iolT
inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the

K
m

and
V
max
values for the IolT and IolF inositol transporters, indicated that
iolT
and
iolF
encode major and minor inositol transporters, respectively. Northern and primer extension analyses of
iolT
transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by ςsgr;
A
RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the
iolT
promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the
iol
divergon.
Rubredoxins Involved in Alkane Oxidation
Authors: van Beilen Jan B. , Neuenschwander Martin , Smits Theo H. M. , Roth Christian , Balada Stefanie B. , Witholt Bernard
Published: July 2002

Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on
n-
alkanes (Alk-Rds). Complementation tests in an
Escherichia coli
recombinant containing all
Pseudomonas putida
GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds. All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in
n-
octane hydroxylation. Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria. Common to most Rds are the two iron-binding CXXCG motifs. All Alk-Rds possess four negatively charged residues that are not conserved in other Rds. The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif. In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids. Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.
Strains of
Escherichia coli
O157:H7 Differ Primarily by Insertions or Deletions, Not Single-Nucleotide Polymorphisms
Authors: Kudva Indira T. , Evans Peter S. , Perna Nicole T. , Barrett Timothy J. , Ausubel Frederick M. , Blattner Frederick R. , Calderwood Stephen B.
Published: July 2002

Escherichia coli
O157:H7 (O157) strains demonstrate varied pulsed-field gel electrophoresis patterns following
Xba
I digestion, which enable epidemiological surveillance of this important human pathogen. The genetic events underlying PFGE differences between strains, however, are not defined. We investigated the mechanisms for strain variation in O157 by recovering and examining nucleotide sequences flanking each of the
Xba
I restriction enzyme sites in the genome. Our analysis demonstrated that differences between O157 strains were due to discrete insertions or deletions that contained the
Xba
I sites polymorphic between strains rather than single-nucleotide polymorphisms in the
Xba
I sites themselves. These insertions and deletions were found to be uniquely localized within the regions of the genome that are specific to O157 compared to
E. coli
K-12 (O islands), suggesting that strain-to-strain variation occurs in these O islands. These results may be utilized to devise novel strain-typing tools for this pathogen.
Chemiosmotic Energy Conservation with Na
+
as the Coupling Ion during Hydrogen-Dependent Caffeate Reduction by
Acetobacterium woodii
Authors: Imkamp Frank , Müller Volker
Published: July 2002

Cell suspensions of
Acetobacterium woodii
prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na
+
dependent with a

K
m

for Na
+
of 0.38 mM; Li
+
could substitute for Na
+
. The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na
+
extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na
+
dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na
+
gradient in ATP synthesis. The ATPase inhibitor
N
,
N
′-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na
+
as the coupling ion during hydrogen-dependent caffeate reduction by
A
.
woodii
.
Evolutionary Analysis by Whole-Genome Comparisons
Authors: Bansal Arvind K. , Meyer Terrance E.
Published: July 2002

A total of 37 complete genome sequences of bacteria, archaea, and eukaryotes were compared. The percentage of orthologous genes of each species contained within any of the other 36 genomes was established. In addition, the mean identity of the orthologs was calculated. Several conclusions result: (i) a greater absolute number of orthologs of a given species is found in larger species than in smaller ones; (ii) a greater percentage of the orthologous genes of smaller genomes is contained in other species than is the case for larger genomes, which corresponds to a larger proportion of essential genes; (iii) before species can be specifically related to one another in terms of gene content, it is first necessary to correct for the size of the genome; (iv) eukaryotes have a significantly smaller percentage of bacterial orthologs after correction for genome size, which is consistent with their placement in a separate domain; (v) the archaebacteria are specifically related to one another but are not significantly different in gene content from the bacteria as a whole; (vi) determination of the mean identity of all orthologs (involving hundreds of gene comparisons per genome pair) reduces the impact of errors in misidentification of orthologs and to misalignments, and thus it is far more reliable than single gene comparisons; (vii) however, there is a maximum amount of change in protein sequences of 37% mean identity, which limits the use of percentage sequence identity to the lower taxa, a result which should also be true for single gene comparisons of both proteins and rRNA; (viii) most of the species that appear to be specifically related based upon gene content also appear to be specifically related based upon the mean identity of orthologs; (ix) the genes of a majority of species considered in this study have diverged too much to allow the construction of all-encompassing evolutionary trees. However, we have shown that eight species of gram-negative bacteria, six species of gram-positive bacteria, and eight species of archaebacteria are specifically related in terms of gene content, mean identity of orthologs, or both.
Firm but Slippery Attachment of
Deinococcus geothermalis
Authors: Kolari M. , Schmidt U. , Kuismanen E. , Salkinoja-Salonen M. S.
Published: July 2002

Bacterial biofilms impair the operation of many industrial processes.
Deinococcus geothermalis
is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of
D. geothermalis
E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the
D. geothermalis
E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living
D. geothermalis
E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment.
D. geothermalis
E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of
Burkholderia cepacia
F28L1 or the well-characterized biofilm former
Staphylococcus epidermidis
O-47.
Deinococcus radiodurans
strain DSM 20539
T
also formed tenacious biofilms. This paper shows that
D. geothermalis
has firm but laterally slippery attachment not reported before for a nonmotile species.
Improved Detection of Amphotericin B-Resistant Isolates of
Candida lusitaniae
by Etest
Authors: Peyron Florence , Favel Anne , Michel-Nguyen Annie , Gilly Magali , Regli Patrick , Bolmström Anne
Published: July 2002

Both intrinsic and acquired resistance to amphotericin B have been documented for
Candida lusitaniae
. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of
C. lusitaniae
. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0.032 to 16 μg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at ≥0.38 μg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 μg/ml), and there were six putatively resistant isolates for which MICs were >1 μg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 μg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of
C. lusitaniae
.
Evaluation of Pulsed-Field Gel Electrophoresis in Epidemiological Investigations of Meningococcal Disease Outbreaks Caused by
Neisseria meningitidis
Serogroup C
Authors: Popovic Tanja , Schmink Susanna , Rosenstein Nancy A. , Ajello Gloria W. , Reeves Michael W. , Plikaytis Brian , Hunter Susan B. , Ribot Efrain M. , Boxrud David , Tondella Maria L. , Kim Chung , Noble Corie , Mothershed Elizabeth , Besser John , Perkins Bradley A.
Published: July 2002

Since 1990, the frequency of
Neisseria meningitidis
serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (
Cla
I), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39
Nhe
I PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.
International Multicenter Evaluation of Latex Agglutination Tests for Identification of
Staphylococcus aureus
Authors: van Griethuysen Arjanne , Bes Michèle , Etienne Jerome , Zbinden Reinhard , Kluytmans Jan
Published: July 2002

A newly marketed rapid agglutination kit for the identification of
Staphylococcus aureus
, Slidex Staph Plus (bioMérieux), was compared to Staphaurex Plus (Murex Diagnostics) and Pastorex Staph-Plus (Sanofi Diagnostics Pasteur). The study took place in three clinical microbiology laboratories in three different European countries. A total of 892 staphylococcal isolates, including 278 methicillin-sensitive
S. aureus
(MSSA) isolates, 171 methicillin-resistant
S. aureus
(MRSA) isolates, and 443 coagulase-negative staphylococcal isolates, were analyzed. The sensitivities (MSSA/MRSA) and specificities, respectively, were 98.2% (98.9%/97.1%) and 98.9% for Slidex Staph Plus, 98.2% (98.2%/98.2%) and 96.2% for Staphaurex Plus, and 98.7% (98.6%/98.8%) and 95.7% for Pastorex Staph Plus. The specificity of the Slidex Staph Plus kit was statistically significantly higher than the specificities of Staphaurex Plus and Pastorex Staph-Plus. The Slidex Staph Plus is a very reliable test for the identification of
S. aureus
.
Value of Throat Swab in Diagnosis of Melioidosis
Authors: Wuthiekanun Vanaporn , Suputtamongkol Yupin , Simpson A. J. H. , Kanaphun Panida , White N. J.
Published: July 2002

Throat swab (TS) cultures were performed for 1,011 patients with melioidosis and 3,524 healthy subjects or patients with other diseases. The specificity of TS culture for the diagnosis of melioidosis was 100%, and the overall sensitivity was 36% (24% for sputum-negative patients and 79% for sputum-positive patients). Direct plating of the TS specimen on Ashdown's medium was rapid (colonies were usually evident within 24 h) but only 63% sensitive compared to the results of primary culture in a selective broth. A throat swab should be cultured in all cases of suspected melioidosis.
Emergence of G9 P[6] Human Rotaviruses in Argentina: Phylogenetic Relationships among G9 Strains
Authors: Bok Karin , Palacios Gustavo , Sijvarger Karina , Matson David , Gomez Jorge
Published: July 2002

Because rotavirus diarrhea can be reduced through vaccination and because current vaccine candidates provide protection against only the most common G antigenic types (G1 to G4), detection of uncommon G types is one of the main goals of rotavirus surveillance. After a 2-year nationwide rotavirus surveillance study in Argentina concluded, surveillance was continued and an increase of G9 prevalence in several Argentine cities was detected. During this period G9 strains predominated in the south, and a gradient of decreasing G9 prevalence was observed from south to north (41 to 0%). Sequence analysis of gene 9, encoding the G antigen, showed that Argentine strains cluster with most G9 isolates from other countries, showing less than 2% nucleotide divergence among them, but are distinctive from them in that they present some unique amino acid changes. Our results agree with reports of increased G9 prevalence in other parts of the world, suggesting the need to incorporate G9 into candidate rotavirus vaccines.
Isolation and Characterization of Polymorphic DNA from
Entamoeba histolytica
Authors: Zaki Mehreen , Clark C. Graham
Published: July 2002

An important gap in our understanding of the epidemiology of amebiasis is what determines the outcome of
Entamoeba histolytica
infections. To investigate the possible existence of invasive and noninvasive strains as one factor, the ability to differentiate individual isolates of
E. histolytica
is necessary. Two new loci containing internal repeats, locus 1-2 and locus 5-6, have been isolated. Each contains a single repeat block with two types of related direct repeats arranged in tandem. Southern blot analysis suggests that both loci are multicopy and may themselves be arranged in tandem arrays. Three other previously reported, internally repetitive loci containing at least two repeat blocks each with one or more related repeat units were also investigated. PCR was used to study polymorphism at each of these loci, which was detected to various degrees in each case. Variation was seen in the total number of bands obtained per isolate and their sizes. Nucleotide sequence comparison of loci 1-2 and 5-6 in five axenic isolates revealed differences in the number of repeat units, which correlated with the observed PCR product size variation, and in repeat sequence. Use of multiple loci collectively allowed differentiation of a majority of the 13 isolates studied, and we believe that these loci have the potential to be used as polymorphic molecular markers for investigating the epidemiology of
E. histolytica
and the potential existence of genetically distinct invasive and noninvasive strains.
Detection and Culture of
Bartonella quintana, Serratia marcescens
, and
Acinetobacter
spp. from Decontaminated Human Body Lice
Authors: La Scola Bernard , Fournier Pierre-Edouard , Brouqui Philippe , Raoult Didier
Published: July 2002

As part of a survey for trench fever among homeless people in Marseilles, France, we attempted isolation of
Bartonella quintana
from body lice. A decontamination protocol of immersion in 70% ethanol with 0.2% iodine was devised and was tested with a laboratory colony of body lice. Lice which had been experimentally contaminated with either
Escherichia coli, Staphylococcus epidermidis
, or
Acinetobacter
spp. were successfully decontaminated, and this process did not prevent the culture of
B. quintana
from these lice. One hundred sixty-one lice obtained from homeless patients were studied by the protocol.
B. quintana
was isolated on axenic medium from 15 of 161 body lice and was detected in 41 of 161 lice by PCR.
Acinetobacter
spp. and
Serratia marcescens
were also isolated from body lice. The sensitivities of PCR and culture of
B. quintana
were 98 and 36%, respectively. These procedures may be useful for epidemiologic studies of trench fever and for the recovery of strains for characterization and comparison.
Development of Molecular Methods for Identification of
Schizophyllum commune
from Clinical Samples
Authors: Buzina Walter , Lang-Loidolt Doris , Braun Hannes , Freudenschuss Kurt , Stammberger Heinz
Published: July 2002

In the last 50 years, to our knowledge, only 16 cases of diseases caused by
Schizophyllum commune
in humans have been reported. Within only 6 months, we found four isolates of this basidiomycetous fungus, obtained from patients suffering from chronic sinusitis. The cultures of the isolated fungi showed neither clamp connections nor fruiting bodies (basidiocarps), which are distinctive features for
S. commune
, but fast-growing cottony white mycelium only. This was harvested, and DNA was extracted. The internal transcribed spacer region of the ribosomal DNA (rDNA) was amplified with fungus-specific primers, and the PCR products were sequenced. Two strains of
S. commune
, collected from branches of a European hornbeam (
Carpinus betulus
) and a tree of heaven (
Ailanthus altissima
), respectively; four specimens from the herbarium of the Institute of Botany, Karl-Franzens-University Graz; and two strains from internationally known culture collections (CBS 340.81 [ATCC 44201] and CBS 405.96) were investigated in the same way. The sequence data of all strains were compared and showed homology of over 99% in this 660-bp-long fragment of rDNA. With these results, a map of restriction enzyme cutting sites and a primer set specific for
S. commune
were created for reliable identification of this human pathogenic fungus.
Comparative Evaluation of Penicillin, Ampicillin, and Imipenem MICs and Susceptibility Breakpoints for Vancomycin-Susceptible and Vancomycin-Resistant
Enterococcus faecalis
and
Enterococcus faecium
Authors: Weinstein Melvin P.
Published: July 2002

Although imipenem has in vitro activity against
Enterococcus faecalis
and Food and Drug Administration-approved indications for treatment of infections caused by this microorganism, there are no NCCLS guidelines for susceptibility testing of imipenem versus enterococci. Therefore, the in vitro activities of penicillin, ampicillin, imipenem, and vancomycin against 201 blood isolates of
E. faecalis
and 24 blood isolates of
Enterococcus faecium
were compared. The susceptibility of isolates to penicillin or ampicillin accurately predicted the in vitro activity of imipenem. Since the susceptibility of enterococci to imipenem can be predicted by the results obtained by testing of penicillin or ampicillin, testing of imipenem by clinical laboratories probably is not necessary.