bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization ofVibrio choleraeO1 Antigen as the Bacteriophage K139 Receptor and Identification of IS1004Insertions Aborting O1 Antigen Biosynthesis
Authors: Nesper Jutta , Kapfhammer Dagmar , Klose Karl E. , Merkert Hilde , Reidl Joachim
Published: July 2002

Bacteriophage K139 was recently characterized as a temperate phage of O1Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfbregion). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004into therfbgene cluster. Treating onewbeW::IS1004serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.
Purine Catabolism in
Escherichia coli
and Function of Xanthine Dehydrogenase in Purine Salvage
Authors: Xi Hualin , Schneider Barbara L. , Reitzer Larry
Published: July 2002

Escherichia coli
is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential ς
54
-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the ς
54
-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced
14
CO
2
from minimal medium containing [
14
C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated
14
CO
2
production from [
14
C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to
xdhA
. The
xdhA
mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.
Regulation of
Vibrio cholerae
Genes Required for Acid Tolerance by a Member of the “ToxR-Like” Family of Transcriptional Regulators
Authors: Merrell D. Scott , Camilli Andrew
Published: July 2002

The ability of the intestinal pathogen
Vibrio cholerae
to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential component of the ATR is CadA-mediated lysine decarboxylation. CadA is encoded by the acid- and infection-induced gene
cadA
. Herein,
cadA
is shown to be the second gene in an operon with
cadB
, encoding a lysine/cadaverine antiporter.
cadC
, which is 5′ of
cadB
, encodes an acid-responsive, positive transcriptional regulator of
cadBA
. Unlike in
Escherichia coli
,
V. cholerae cadB
and
cadA
are also transcribed monocistronically. Of note, bicistronic
cadBA
is transcribed at low constitutive levels in an acid- and CadC-independent manner. CadC represents a new member of the “ToxR-like” family of transcriptional regulators in
V. cholerae
and, in addition, exhibits extensive amino acid and functional similarity to
E. coli
CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.
Cloning and Characterization of Secretory Tyrosine Phosphatases of
Mycobacterium tuberculosis
Authors: Koul Anil , Choidas Axel , Treder Martin , Tyagi Anil K. , Drlica Karl , Singh Yogendra , Ullrich Axel
Published: July 2002

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of
Mycobacterium tuberculosis
H
37
Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione
S
-transferase fusion proteins in
Escherichia coli
. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while
mptpA
is present in slow-growing mycobacterial species as well as fast-growing saprophytes,
mptpB
was restricted to members of the
M. tuberculosis
complex. These phosphatases were present in both whole-cell lysates and culture filtrates of
M. tuberculosis
, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of
mptpB
makes it a good candidate for a virulence gene of
M. tuberculosis
.
The Feedback Response of
Escherichia coli
rRNA Synthesis Is Not Identical to the Mechanism of Growth Rate-Dependent Control
Authors: Voulgaris Justina , Pokholok Dmitry , Holmes W. Mike , Squires Craig , Squires Catherine L.
Published: July 2002

Growth rate-independent
rrn
P1 promoter mutants were tested for their ability to respond to changes in
rrn
gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the
rrn
gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.
Inorganic Polyphosphate in
Vibrio cholerae
: Genetic, Biochemical, and Physiologic Features
Authors: Ogawa Nobuo , Tzeng Chi-Meng , Fraley Cresson D. , Kornberg Arthur
Published: July 2002

Vibrio cholerae
O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the
ppk
gene, which has been cloned from
V. cholerae
, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of
Escherichia coli
, which it resembles biochemically. As in
E. coli
,
ppk
is part of an operon with
ppx
, the gene that encodes exopolyphosphatase (PPX). However, unlike in
E. coli
, PPX activity was not detected in cell extracts of wild-type
V. cholerae
. The
ppk
null mutant of
V. cholerae
has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.
WhiD and WhiB, Homologous Proteins Required for Different Stages of Sporulation in
Streptomyces coelicolor
A3(2)
Authors: Molle Virginie , Palframan Wendy J. , Findlay Kim C. , Buttner Mark J.
Published: July 2002

The
whiD
locus, which is required for the differentiation of
Streptomyces coelicolor
aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77–96, 1996). Subcloning and sequencing showed that
whiD
encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in
S. coelicolor
. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed
whiD
null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The
whiD
null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores.
whiD
null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These “minicompartments” appeared to be devoid of chromosomal DNA. Two
whiD
promoters,
whiDp1
and
whiDp2
, were identified, and their activities were analyzed during development of wild-type
S. coelicolor
on solid medium. Both promoters were developmentally regulated;
whiDp1
and
whiDp2
transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.
Regulation of rRNA Transcription Is Remarkably Robust: FIS Compensates for Altered Nucleoside Triphosphate Sensing by Mutant RNA Polymerases at
Escherichia coli rrn
P1 Promoters
Authors: Bartlett Michael S. , Gaal Tamas , Ross Wilma , Gourse Richard L.
Published: July 2002

We recently identified
Escherichia coli
RNA polymerase (RNAP) mutants (RNAP β′ Δ215–220 and β RH454) that form extremely unstable complexes with rRNA P1 (
rrn
P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of
rrn
P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the
rrn
transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent
K
ATP
for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of
rrn
P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.
Regions of RNase E Important for 5′-End-Dependent RNA Cleavage and Autoregulated Synthesis
Authors: Jiang Xunqing , Diwa Alexis , Belasco Joel G.
Published: July 2002

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in
Escherichia coli
. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5′ end. Here we show that the preference of
E. coli
RNase E for 5′-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related
E. coli
ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from
Synechocystis
, indicating that the 5′-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5′-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in
E. coli
. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining
E. coli
cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.
Carbon Flux Distribution and Kinetics of Cellulose Fermentation in Steady-State Continuous Cultures of
Clostridium cellulolyticum
on a Chemically Defined Medium
Authors: Desvaux Mickaël , Guedon Emmanuel , Petitdemange Henri
Published: July 2002

The metabolic characteristics of
Clostridium cellulolyticum
, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with
C. cellulolyticum
(i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (μ) is low, and (iii) there is a decrease of the NADH/NAD
+
ratio and
q
NADH produced
/
q
NADH used
ratio as the dilution rate (
D
) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as μ increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262–3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with
D
. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble β-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with
D
. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with
D
, reaching 27.3 and 16.8%, respectively, at 0.083 h
−1
. Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)
−1
] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)
−1
]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h
−1
. Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower μ, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results,
C. cellulolyticum
appeared well adapted and even restricted to a cellulolytic lifestyle.
The LuxM Homologue VanM from
Vibrio anguillarum
Directs the Synthesis of
N
-(3-Hydroxyhexanoyl)homoserine Lactone and
N
-Hexanoylhomoserine Lactone
Authors: Milton Debra L. , Chalker Victoria J. , Kirke David , Hardman Andrea , Cámara Miguel , Williams Paul
Published: July 2002

Vibrio anguillarum
, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (
vanRI
) and to produce
N
-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a
vanI
null mutant still activated
N
-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in
V. anguillarum
. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as
N
-hexanoylhomoserine lactone (C6-HSL) and
N
-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase
V. anguillarum
spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore,
vanM
, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the
luxL
and
luxM
genes, which are required for the production of
N
-(3-hydroxybutanoyl)homoserine lactone in
Vibrio harveyi
. However, resequencing of the
V. harveyi luxL/luxM
junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed
luxM
). Downstream of
vanM
, we identified a homologue of
luxN
(
vanN
) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in
V. harveyi
. A mutation in
vanM
abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the
vanM
mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via
vanRI
. However, a
vanN
mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that
V. anguillarum
possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both
V. fischeri
(the LuxRI homologues VanRI) and
V. harveyi
(the LuxMN homologues, VanMN).
Sodium Ion-Driven Serine/Threonine Transport in
Porphyromonas gingivalis
Authors: Dashper S. G. , Brownfield L. , Slakeski N. , Zilm P. S. , Rogers A. H. , Reynolds E. C.
Published: July 2002

Porphyromonas gingivalis
is an asaccharolytic, gram-negative bacterium that relies on the fermentation of amino acids for metabolic energy. When grown in continuous culture in complex medium containing 4 mM (each) free serine, threonine, and arginine,
P. gingivalis
assimilated mainly glutamate/glutamine, serine, threonine, aspartate/asparagine, and leucine in free and/or peptide form. Serine and threonine were assimilated in approximately equal amounts in free and peptide form. We characterized serine transport in this bacterium by measuring uptake of the radiolabeled amino acid in washed cells of
P. gingivalis
energized with a tetrapeptide not containing serine. Serine was transported by a single system with an affinity constant for transport (

K
t

) of 24 μM that was competitively inhibited by threonine. Serine transport was dependent on sodium ion concentration in the suspending buffer, and the addition of the ionophore gramicidin caused the inhibition of serine uptake. Together these data indicate that serine transport was sodium ion-motive force driven. A
P. gingivalis
gene potentially encoding a serine transporter was identified by sequence similarity to an
Escherichia coli
serine transporter (SstT). This
P. gingivalis
gene, designated
sstT,
was inactivated by insertion of a
Bacteroides
tetQ
gene, producing the mutant W50ST. The mutant was unable to transport serine, confirming the presence of a single serine transporter in this bacterium under these growth conditions. The transport of serine by
P. gingivalis
was dependent on the presence of free cysteine in the suspension buffer. Other reducing agents were unable to stimulate serine uptake. These data show that
P. gingivalis
assimilates free serine and threonine from culture media via a cysteine-activated, sodium ion-motive force-driven serine/threonine transporter.
Competence Modulation by the NADH Oxidase of
Streptococcus pneumoniae
Involves Signal Transduction
Authors: Echenique José R. , Trombe Marie C.
Published: July 2002

Oxygen controls competence development in
Streptococcus pneumoniae
. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by
comC
and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of
ciaRH
and
comCDE
. Thus, in
S. pneumoniae,
Nox, with O
2
as its secondary substrate, is part of the O
2
-signaling pathway.
HcwA, an Autolysin, Is Required for Heterocyst Maturation in
Anabaena
sp. Strain PCC 7120
Authors: Zhu Jinsong , Jäger Karin , Black Todd , Zarka Kelly , Koksharova Olga , Wolk C. Peter
Published: July 2002

In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N
2
. Synthesis of the heterocyst envelope polysaccharide is dependent on the gene
hepA
in
Anabaena
sp. strain PCC 7120. In search of genes that are involved in the regulation of
hepA
, we transposon mutagenized strain DR1069, which bears a chromosomal
hepA
::
luxAB
fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of
hepA
in response to nitrogen deprivation. In HNL3, transposon Tn
5-
1058 is inserted within gene
hcwA
, a constitutively expressed open reading frame whose predicted product resembles
N
-acetylmuramoyl-
l
-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction of
hepA
in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type
hcwA
. Moreover, HcwA expressed in
Escherichia coli
exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.
Binding Site of Macrolide Antibiotics on the Ribosome: New Resistance Mutation Identifies a Specific Interaction of Ketolides with rRNA
Authors: Garza-Ramos Georgina , Xiong Liqun , Zhong Ping , Mankin Alexander
Published: July 2002

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA renderedE. colicells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.
Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface
Authors: Etz Hildegard , Minh Duc Bui , Schellack Carola , Nagy Eszter , Meinke Andreas
Published: July 2002

Four outer membrane proteins of
Escherichia coli
were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of
E. coli
as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B
12
and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and λ, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of
E. coli
for diverse applications.
Characterization of a Novel Transferrin Receptor in Bovine Strains of
Pasteurella multocida
Authors: Ogunnariwo Julius A. , Schryvers Anthony B.
Published: July 2002

Analysis of bovine respiratory isolates of
Pasteurella multocida
demonstrated that six of nine strains tested were capable of growth dependent upon bovine transferrin and of specifically binding ruminant transferrins. A single 82-kDa protein was affinity isolated from the
P. multocida
strains with immobilized bovine transferrin. In contrast to what has been observed in other species, binding of this protein to immobilized transferrin was specifically blocked by the N-lobe subfragment of bovine transferrin. A single gene encoding the 82-kDa protein was flanked by a leucyl-tRNA synthetase gene and an IS
1060
element, in contrast to other species where genes encoding the two receptor proteins (TbpB and TbpA) are found in an operonic arrangement. A similar gene arrangement was observed in all of the receptor-positive strains, in spite of the observation that they belonged to different genomic groups. Analysis of the deduced amino acid sequence of the receptor protein indicated that it is a member of the TonB-dependent outer membrane receptor family, and although it is related to transferrin and lactoferrin receptor proteins (TbpAs and LbpAs) from other species, it differs substantially from other members of this group. Amino acid alignments suggest that the reduced size (20 kDa smaller) of the
P. multocida
TbpA is primarily due to the absence of larger predicted external loops. Collectively these results suggest that
P. multocida
has a single, novel receptor protein (TbpA) that is capable of efficiently mediating iron acquisition from bovine transferrin without the involvement of a second receptor protein (TbpB).
Complete Genomic Sequence of the Lytic Bacteriophage φYeO3-12 of
Yersinia enterocolitica
Serotype O:3
Authors: Pajunen Maria I. , Kiljunen Saija J. , Söderholm M. E.-Lotta , Skurnik Mikael
Published: July 2002

φYeO3-12 is a T3-related lytic bacteriophage of
Yersinia enterocolitica
serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the φYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted φYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, φYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on
Shigella sonnei
D
2
371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114–5120, 2000). These findings indicate that φYeO3-12 is a T3-like phage that has adapted to
Y. enterocolitica
O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported.
Degradation of Aromatics and Chloroaromatics byPseudomonassp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase
Authors: Kaschabek Stefan R. , Kuhn Bernd , Müller Dagmar , Schmidt Eberhard , Reineke Walter
Published: July 2002

The degradation of 3-oxoadipate inPseudomonassp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 ± 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4.Kmvalues were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-Oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 ± 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8.Kmvalues were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.
Rescue of Social Motility Lost during Evolution of
Myxococcus xanthus
in an Asocial Environment
Authors: Velicer Gregory J. , Lenski Richard E. , Kroos Lee
Published: July 2002

Replicate populations of the social bacterium
Myxococcus xanthus
underwent extensive evolutionary adaptation to an asocial selective environment (liquid batch culture). All 12 populations showed partial or complete loss of their social (S) motility function after 1,000 generations of evolution. Mutations in the
pil
gene cluster (responsible for type IV pilus biogenesis and function) were found to be at least partially responsible for the loss of S motility in the majority of evolved lines. Restoration (partial or complete) of S motility in the evolved lines by genetic complementation with wild-type
pil
genes positively affected their fruiting body development and sporulation while negatively affecting their competitive fitness in the asocial regime. This genetic tradeoff indicates that mutations in the
pil
region were adaptive in the asocial selective environment. This finding was confirmed by experiments showing that defined deletions of
pil
gene regions conferred a competitive advantage under asocial conditions. Moreover, an amino acid substitution in an evolved genotype was located in a region predicted by genetic complementation analysis to bear an adaptive mutation.
Pseudomonas aeruginosaVirulence Analyzed in aDictyostelium discoideumHost System
Authors: Cosson Pierre , Zulianello Laurence , Join-Lambert Olivier , Faurisson François , Gebbie Leigh , Benghezal Mohammed , van Delden Christian , Curty Lasta Kocjancic , Köhler Thilo
Published: July 2002

Pseudomonas aeruginosais an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors.P. aeruginosainfections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoebaDictyostelium discoideumcan be used as a simple model system to analyze the virulence ofP. aeruginosastrains. The virulent wild-type strain PAO1 was shown to inhibit growth ofD. discoideum. Isogenic mutants deficient in thelasquorum-sensing system were almost as inhibitory as the wild type, whilerhlquorum-sensing mutants permitted growth ofDictyosteliumcells. Therefore, in this model system, factors controlled by therhlquorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis ofD. discoideumcells. By using this simple model system, we predicted that certain antibiotic-resistant mutants ofP. aeruginosashould show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus,D. discoideumcould be used as a simple nonmammalian host system to assess pathogenicity ofP. aeruginosa.
Competence without a Competence Pheromone in a Natural Isolate ofStreptococcus infantis
Authors: Ween Ola , Teigen Svanhild , Gaustad Peter , Kilian Mogens , Håvarstein Leiv Sigve
Published: July 2002

Many streptococcal species belonging to the mitis and anginosus phylogenetic groups are known to be naturally competent for genetic transformation. Induction of the competent state in these bacteria is regulated by a quorum-sensing mechanism consisting of a secreted peptide pheromone encoded bycomCand a two-component regulatory system encoded bycomDE. Here we report that a natural isolate of a mitis group streptococcus (Atu-4) is competent for genetic transformation even though it has lost the gene encoding the competence pheromone. In contrast to other strains, induction of competence in Atu-4 is not regulated by cell density, since highly diluted cultures of this strain are still competent. Interestingly, competence in the Atu-4 strain is lost if the gene encoding the response regulator ComE is disrupted, demonstrating that this component of the quorum-sensing apparatus is still needed for competence development. These results indicate that mutations in ComD or ComE have resulted in a gain-of-function phenotype that allows competence without a competence pheromone. A highly similar strain lackingcomCwas isolated independently from another individual, suggesting that strains with this phenotype are able to survive in nature in competition with wild-type strains.
Natural Transformation and DNA Uptake Signal Sequences in
Actinobacillus actinomycetemcomitans
Authors: Wang Ying , Goodman Steve D. , Redfield Rosemary J. , Chen Casey
Published: July 2002

Actinobacillus actinomycetemcomitans
is a member of the family
Pasteurellaceae
and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation,
A. actinomycetemcomitans
has yet to be fully characterized. Here we show that the competence of
A. actinomycetemcomitans
is remarkably similar to that of
Haemophilus influenzae
. In addition to having a similar frequency of transformation as
H. influenzae
,
A. actinomycetemcomitans
competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in
H. influenzae
, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative
A. actinomycetemcomitans
DNA uptake signal sequence (USS) virtually identical to the USS of
H. influenzae
. Moreover, we provide evidence that this sequence functions in the same capacity as that from
H. influenzae
; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated
A. actinomycetemcomitans
.
Identification of theClostridium perfringensGenes Involved in the Adaptive Response to Oxidative Stress
Authors: Briolat V. , Reysset G.
Published: July 2002

Clostridium perfringensis a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. AlthoughC. perfringensis strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response,C. perfringensstrain 13 mutants were generated by Tn916insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to theydaDandycdFgenes ofBacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress inC. perfringensand are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the β-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.
Development and Evaluation of a Fluorogenic 5′ Nuclease Assay To Detect and Differentiate between Ebola Virus Subtypes Zaire and Sudan
Authors: Gibb Tammy R. , Norwood David A. , Woollen Neal , Henchal Erik A.
Published: July 2002

The ability to rapidly recognize Ebola virus infections is critical to quickly limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of Ebola virus infection and to distinguish it from other diseases that can cause similar clinical symptoms. A one-tube reverse transcription-PCR assay for the identification of Ebola virus subtype Zaire (Ebola Zaire) and Ebola virus subtype Sudan (Ebola Sudan) was developed and evaluated by using the ABI PRISM 7700 sequence detection system. This assay uses one common primer set and two differentially labeled fluorescent probes to simultaneously detect and differentiate these two subtypes of Ebola virus. The sensitivity of the primer set was comparable to that of previously designed primer sets, as determined by limit-of-detection experiments. This assay is unique in its ability to simultaneously detect and differentiate Ebola Zaire and Ebola Sudan. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a very useful diagnostic tool for the control and management of future outbreaks.
Susceptibility of
Arcobacter butzleri, Arcobacter cryaerophilus
, and
Arcobacter skirrowii
to Antimicrobial Agents Used in Selective Media
Authors: Houf Kurt , Devriese Luc A. , zutter Lieven De , Van Hoof Jan , Vandamme Peter
Published: July 2002

Several antimicrobial agents used in selective media for the isolation of
Arcobacter
were found to be inhibitory to strains belonging to this genus. All three species tested were susceptible to colistin and rifampin at concentrations used in selective media.
Arcobacter skirrowii
was the most susceptible species. 5-Fluorouracil, novobiocin, trimethoprim, and teicoplanin or vancomycin were found to be without any inhibitory effect on the strains tested at concentrations described for the isolation of
Arcobacter
species.
Endocarditis Caused by Culture-Negative Organisms Visible by Brown and Brenn Staining: Utility of PCR and DNA Sequencing for Diagnosis
Authors: Wilck Marissa B. , Wu Yanyun , Howe John G. , Crouch Jill Y. , Edberg Stephen C.
Published: July 2002

Two cases of culture-negative endocarditis with cocci seen in valve vegetations are presented. The organisms were identified by molecular analysis using broad-range PCR primers complementary to the 16S rRNA gene, sequencing, and database search using BLAST software. The results and utility of this method are discussed.
Rapid Identification of
Campylobacter
spp. by Melting Peak Analysis of Biprobes in Real-Time PCR
Authors: Logan J. M. J. , Edwards K. J. , Saunders N. A. , Stanley J.
Published: July 2002

We describe rapid PCR-biprobe identification of
Campylobacter
spp.. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all
Campylobacter
species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of
Campylobacter, Helicobacter
, and
Arcobacter
spp. and 59 Penner serotype reference strains of
Campylobacter jejuni
and
C. coli
. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (
C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis
, and
C. fetus
), as well as
C. helveticus
and
C. lanienae
. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR–enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.
Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR
Authors: Couto Isabel , Pereira Sandro , Miragaia Maria , Sanches Ilda Santos , de Lencastre Hermı́nia
Published: July 2002

The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates ofStaphylococcus epidermidis, 157 ofS. haemolyticus, 79 ofS. aureus, 21 ofS. hominis, 14 ofS. saprophyticus, 8 ofS. warneri, 6 ofS. simulans, 2 ofS. lugdunensis, and 1 each ofS. caprae,S. carnosus, andS. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the groupS. saprophyticus,S. cohnii,S. gallinarum,S. xylosus,S. lentus,S. equorum, andS. chromogenes, the pairS. schleiferiandS. vitulus, and the pairS. piscifermentansandS. carnosus. Four species,S. aureus,S. caprae,S. haemolyticus, andS. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.
Survival of Some Medically Important Fungi on Hospital Fabrics and Plastics
Authors: Neely Alice N. , Orloff Mary M.
Published: July 2002

Tests of the survival of
Candida
spp.,
Aspergillus
spp., a
Fusarium
sp.
,
a
Mucor
sp.
,
and a
Paecilomyces
sp. on hospital fabrics and plastics indicated that viability was variable, with most fungi surviving at least 1 day but many living for weeks. These findings reinforce the need for appropriate disinfection and conscientious contact control precautions.
Serotype Identification of Group B Streptococci by PCR and Sequencing
Authors: Kong Fanrong , Gowan Sonia , Martin Diana , James Gregory , Gilbert Gwendolyn L.
Published: July 2002

Group B streptococcus (GBS;
Streptococcus agalactiae
) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (
cps
) gene clusters to further define partial
cps
gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.