bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Targeted Mutagenesis in
Borrelia burgdorferi
Authors: Bono James L. , Elias Abdallah F. , Kupko John J. , Stevenson Brian , Tilly Kit , Rosa Patricia
Published: July 2002

Genetic studies in
Borrelia burgdorferi
have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is

gyrB
r

, a mutated form of the chromosomal
gyrB
gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A
1
. The utility of the coumermycin-resistant

gyrB
r

gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous
gyrB
gene. A kanamycin resistance gene (
kan
) was introduced into
B. burgdorferi
, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen.
B. burgdorferi
transformants with the
kan
gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the
kan
gene expressed from either the
B. burgdorferi flaB
or
flgB
promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in
B. burgdorferi
and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.
Divergence in Fitness and Evolution of Drug Resistance in Experimental Populations of
Candida albicans
Authors: Cowen Leah E. , Kohn Linda M. , Anderson James B.
Published: July 2002

The dissemination and persistence of drug-resistant organisms in nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage compared to less resistant genotypes in the presence of drug, resistance may incur a cost; resistant genotypes may be at a disadvantage in the absence of drug. We measured the fitness of replicate experimental populations of the pathogenic yeast
Candida albicans
founded from a single progenitor cell in a previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol. 182:1515–1522, 2000) and evolved in the presence, and in the absence, of the antifungal agent fluconazole. Fitness was measured both in the presence and in the absence of fluconazole by placing each evolved population in direct competition with the drug-sensitive ancestor and measuring the reproductive output of each competitor in the mixture. Populations evolved in the presence of drug diverged in fitness. Any significant cost of resistance, indicated by reduced fitness in the absence of drug, was eliminated with further evolution. Populations evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation, suggesting the importance of interactions between mixed genotypes in competitions.
Osmoregulated Periplasmic Glucans of
Erwinia chrysanthemi
Authors: Cogez Virginie , Talaga Philippe , Lemoine Jerome , Bohin Jean-Pierre
Published: July 2002

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of
Erwinia chrysanthemi
. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000–0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and
1
H and
13
C nuclear magnetic resonance analyses. OPGs produced by
E. chrysanthemi
are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of β-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by β-1,6 linkages in a random way. This glucan backbone may be substituted by
O
-acetyl and
O
-succinyl ester-linked residues.
ComE, a Competence Protein from
Neisseria gonorrhoeae
with DNA-Binding Activity
Authors: Chen Inês , Gotschlich Emil C.
Published: July 2002

Neisseria gonorrhoeae
is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in
N. gonorrhoeae
, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene
comE
was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent
Bacillus subtilis
. The gene
comE
is present in four identical copies in the genomes of both
N. gonorrhoeae
and
Neisseria meningitidis
, located downstream of each of the rRNA operons. Single-copy deletion of
comE
in
N. gonorrhoeae
did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 × 10
4
-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of
comE
has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of
N. gonorrhoeae
and
N. meningitidis
. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.
NtcA-Dependent Expression of the
devBCA
Operon, Encoding a Heterocyst-Specific ATP-Binding Cassette Transporter in
Anabaena
spp
Authors: Fiedler Gabriele , Muro-Pastor Alicia M. , Flores Enrique , Maldener Iris
Published: July 2002

The
devBCA
operon, encoding subunits of an ATP-binding cassette exporter, is essential for differentiation of N
2
-fixing heterocysts in
Anabaena
spp. Nitrogen deficiency-dependent transcription of the operon and the use of its transcriptional start point, located 762 (
Anabaena variabilis
strain ATCC 29413-FD) or 704 (
Anabaena
sp. strain PCC 7120) bp upstream of the translation start site, were found to require the global nitrogen transcriptional regulator NtcA. Furthermore, NtcA was shown to bind in vitro to the promoter of
devBCA
.
Global versus Local Regulatory Roles for Lrp-Related Proteins:
Haemophilus influenzae
as a Case Study
Authors: Friedberg Devorah , Midkiff Michael , Calvo Joseph M.
Published: July 2002

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in
Escherichia coli
, affecting expression of dozens of operons. Numerous
lrp
-related genes have been identified in different bacteria and archaea, including
asnC
, an
E. coli
gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with
E. coli
Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from
Haemophilus influenzae
that shares 75% sequence identity with
E. coli
Lrp (highest sequence identity among 42 sequences compared). A strain of
H. influenzae
having an
lrfB
null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with
lrp
+
and
lrp E. coli
strains by others showed 20 differences). The abundance of LrfB in
H. influenzae
, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per
E. coli
cell). LrfB expressed in
E. coli
replaced Lrp as a repressor of the
lrp
gene but acted only to a limited extent as an activator of the
ilvIH
operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.
Cytotoxic and Genotoxic Consequences of Heat Stress Are Dependent on the Presence of Oxygen in
Saccharomyces cerevisiae
Authors: Davidson John F. , Schiestl Robert H.
Published: July 2002

Lethal heat stress generates oxidative stress in
Saccharomyces cerevisiae
, and anaerobic cells are several orders of magnitude more resistant than aerobic cells to a 50°C heat shock. Here we characterize the oxidative effects of this heat stress. The thermoprotective effect in anaerobic cells was not due to expression of
HSP104
or any other heat shock gene, raising the possibility that the toxicity of lethal heat shock is due mainly to oxidative stress. Aerobic but not anaerobic heat stress caused elevated frequencies of forward mutations and interchromosomal DNA recombination. Oxidative DNA repair glycosylase-deficient strains under aerobic conditions showed a powerful induction of forward mutation frequencies compared to wild-type cells, which was completely abolished under anaerobiosis. We also investigated potential causes for this oxygen-dependent heat shock-induced genetic instability. Levels of sulfhydryl groups, dominated mainly by the high levels of the antioxidant glutathione (reduced form) and levels of vitamin E, decreased after aerobic heat stress but not after anaerobic heat stress. Aerobic heat stress also led to an increase in mitochondrial membrane disruption of several hundredfold, which was 100-fold reduced under anaerobic conditions.
Release of Compact Nucleoids with Characteristic Shapes from
Escherichia coli
Authors: Zimmerman Steven B. , Murphy Lizabeth D.
Published: July 2002

The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from
Escherichia coli
upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189–3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.
Molecular and Functional Analyses of the Gene (
eshA
) Encoding the 52-Kilodalton Protein of
Streptomyces coelicolor
A3(2) Required for Antibiotic Production
Authors: Kawamoto Shinichi , Watanabe Masakatsu , Saito Natsumi , Hesketh Andrew , Vachalova Katerina , Matsubara Keiko , Ochi Kozo
Published: July 2002

Analysis of proteins recovered in the S100 precipitate fraction of
Streptomyces griseus
after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (
eshA
) which codes for this protein was cloned from
S. griseus
, and then its homologue was cloned from
Streptomyces coelicolor
A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in
Mycobacterium leprae,
a major antigenic protein to humans. The
eshA
gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The
eshA
gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the
relA
mutation, which blocks ppGpp synthesis.
Novel Motility Mutants of
Synechocystis
Strain PCC 6803 Generated by In Vitro Transposon Mutagenesis
Authors: Bhaya Devaki , Takahashi Akiko , Shahi Payam , Grossman Arthur R.
Published: July 2002

We screened for transposon-generated mutants of
Synechocystis
sp. strain PCC 6803 that exhibited aberrant phototactic movement. Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes. These novel genes and their possible roles in motility are discussed.
Identification of Essential Amino Acids in theAzorhizobium caulinodansFucosyltransferase NodZ
Authors: Chazalet Valerie , Uehara Kazuyoshi , Geremia Roberto A. , Breton Christelle
Published: July 2002

ThenodZgene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an α1-6 linkage to the reducingN-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form inEscherichia coliand was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a “Rossmann-type” nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases.
Potential Symbiosis-Specific Genes Uncovered by Sequencing a 410-Kilobase DNA Region of the
Bradyrhizobium japonicum
Chromosome
Authors: Göttfert Michael , Röthlisberger Sandra , Kündig Christoph , Beck Christoph , Marty Roger , Hennecke Hauke
Published: July 2002

The physical and genetic map of the
Bradyrhizobium japonicum
chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G+C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from
nodM
, all nodulation genes that were known to exist in
B. japonicum
. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by −24/−12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.
Functional Characterization of a Novel Xylanase from a Corn Strain of
Erwinia chrysanthemi
Authors: Hurlbert Jason C. , Preston James F.
Published: July 2002

A β-1,4-xylan hydrolase (xylanase A) produced by
Erwinia chrysanthemi
D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35°C, with a secondary structure under those conditions that consists of approximately 10 to 15% α-helices. The enzyme was still active at temperatures higher than 40°C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45°C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-
O
-methyl-
d
-glucuronic acid and appears to require two 4-
O
-methyl-
d
-glucuronic acid residues for substrate recognition and/or cleavage of a β-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-
O
-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process.
Determination ofWolbachiaGenome Size by Pulsed-Field Gel Electrophoresis
Authors: Sun Ling V. , Foster Jeremy M. , Tzertzinis George , Ono Midori , Bandi Claudio , Slatko Barton E. , O'Neill Scott L.
Published: July 2002

Genome sizes of six differentWolbachiastrains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. EnzymesSmaI,ApaI,AscI, andFseI cleaved the studiedWolbachiastrains at a small number of sites and were used for the determination of the genome sizes ofwMelPop,wMel, andwMelCS (each 1.36 Mb),wRi (1.66 Mb),wBma (1.1 Mb), andwDim (0.95 Mb). TheWolbachiagenomes studied were all much smaller than the genomes of free-living bacteria such asEscherichia coli(4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability amongWolbachiastrains, especially between the more parasitic A groupWolbachiainfections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that theWolbachiagenome is circular.
Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase from
Sphingomonas xenophaga
BN6 by Random Mutagenesis
Authors: Riegert Ulrich , Bürger Sibylle , Stolz Andreas
Published: July 2002

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from
Sphingomonas xenophaga
strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction,
bphC1
was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.
Topology of OxlT, the Oxalate Transporter of
Oxalobacter formigenes
, Determined by Site-Directed Fluorescence Labeling
Authors: Ye Liwen , Jia Zhenzhen , Jung Thomas , Maloney Peter C.
Published: July 2002

The topology of OxlT, the oxalate:formate exchange protein of
Oxalobacter formigenes
, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His
9
-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni
2+
-linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.
Targeting of
D
MinC/MinD and
D
MinC/DicB Complexes to Septal Rings in
Escherichia coli
Suggests a Multistep Mechanism for MinC-Mediated Destruction of Nascent FtsZ Rings
Authors: Johnson Jay E. , Lackner Laura L. , de Boer Piet A. J.
Published: July 2002

The MinC protein is an important determinant of septal ring positioning in
Escherichia coli
. The N-terminal domain (
Z
MinC) suppresses septal ring formation by interfering with FtsZ polymerization, whereas the C-terminal domain (
D
MinC) is required for dimerization as well as for interaction with the MinD protein. MinD oscillates between the membrane of both cell halves in a MinE-dependent fashion. MinC oscillates along with MinD such that the time-integrated concentration of
Z
MinC at the membrane is minimal, and hence the stability of FtsZ polymers is maximal, at the cell center. MinC is cytoplasmic and fails to block FtsZ assembly in the absence of MinD, indicating that recruitment of MinC by MinD to the membrane enhances
Z
MinC function. Here, we present evidence that the binding of
D
MinC to MinD endows the MinC/MinD complex with a more specific affinity for a septal ring-associated target in vivo. Thus, MinD does not merely attract MinC to the membrane but also aids MinC in specifically binding to, or in close proximity to, the substrate of its
Z
MinC domain. MinC-mediated division inhibition can also be activated in a MinD-independent fashion by the DicB protein of cryptic prophage Kim. DicB shows little homology to MinD, and how it stimulates MinC function has been unclear. Similar to the results obtained with MinD, we find that DicB interacts directly with
D
MinC, that the
D
MinC/DicB complex has a high affinity for some septal ring target(s), and that MinC/DicB interferes with the assembly and/or integrity of FtsZ rings in vivo. The results suggest a multistep mechanism for the activation of MinC-mediated division inhibition by either MinD or DicB and further expand the number of properties that can be ascribed to the Min proteins.
Cloning and Analysis of the Telomere and Terminal Inverted Repeat of the Linear Chromosome of
Streptomyces griseus
Authors: Goshi Kohei , Uchida Tetsuya , Lezhava Alexander , Yamasaki Masayuki , Hiratsu Keiichiro , Shinkawa Hidenori , Kinashi Haruyasu
Published: July 2002

Cloning and sequencing of the telomere of
Streptomyces griseus
revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in
Streptomyces
telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.
Genetic Locus Encoding Functions Involved in Biosynthesis and Outer Membrane Localization of Xanthomonadin in
Xanthomonas oryzae
pv. oryzae
Authors: Goel Ajay Kumar , Rajagopal Lakshmi , Nagesh Narayana , Sonti Ramesh V.
Published: July 2002

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus
Xanthomonas
. We have characterized a genetic locus (
pig
) from
Xanthomonas oryzae
pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the
pig
locus is required for outer membrane localization of xanthomonadin in
X. oryzae
pv. oryzae. We also report the identification of a novel 145-bp palindromic
Xanthomonas
repetitive intergenic consensus element that is present in two places in the
pig
locus. We estimate that more than 100 copies of this element might be present in the genome of
X. oryzae
pv. oryzae and other xanthomonads.
The
Rhizobium etli cyaC
Product: Characterization of a Novel Adenylate Cyclase Class
Authors: Téllez-Sosa Juan , Soberón Nora , Vega-Segura Alicia , Torres-Márquez María E. , Cevallos Miguel A.
Published: July 2002

Adenylate cyclases (ACs) catalyze the formation of 3′,5′-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene,
cyaC
, was isolated from
Rhizobium etli
by phenotypic complementation of an
Escherichia coli cya
mutant. The functionality of the
cyaC
gene was corroborated by its ability to restore cAMP accumulation in an
E. coli cya
mutant. Further, overexpression of a
malE
::
cyaC
fusion protein allowed the detection of significant AC activity levels in cell extracts of an
E. coli cya
mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to
cyaC
have been identified in the genomes of the related bacterial species
Mesorhizobium loti
,
Sinorhizobium meliloti
, and
Agrobacterium tumefaciens
. The
cyaC
gene is cotranscribed with a gene similar to
ohr
of
Xanthomonas campestris
and is expressed only in the presence of organic hydroperoxides. The physiological performance of an
R. etli cyaC
mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis.
Two Opines Control Conjugal Transfer of anAgrobacteriumPlasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator GenetraR
Authors: Oger Philippe , Farrand Stephen K.
Published: July 2002

Conjugal transfer of Ti plasmids fromAgrobacteriumspp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of thetraregulon. Opines control transfer because thetraRgene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenicAgrobacterium radiobacterisolate K84. In this study we showed that this plasmid contains two copies oftraR, and each is associated with a different opine-regulated operon. One copy,traRnoc, is the last gene of thenoxoperon and was induced by nopaline but not by agrocinopines A and B. MutatingtraRnocabolished induction of transfer by nopaline but not by the agrocinopines. A mutation inocd, an upstream gene of thenoxoperon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy,traRacc, is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in bothtraRgenes was not induced for transfer by either opine. However, expression of atraRgene intransto this plasmid resulted in opine-independent transfer. The association oftraRnocwithnoxis unique, but the operon containingtraRaccis related to thearcoperons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies oftraR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.
Rapid Detection of Methicillin Resistance in Coagulase-Negative Staphylococci by a Penicillin-Binding Protein 2a-Specific Latex Agglutination Test
Authors: Horstkotte Matthias A. , Knobloch Johannes K.-M. , Rohde Holger , Mack Dietrich
Published: July 2002

The detection of PBP 2a by the MRSA-Screen latex agglutination test with 201 clinical coagulase-negative staphylococci had an initial sensitivity of 98% and a high degree of specificity for
Staphylococcus epidermidis
strains compared to PCR for
mecA
. Determination of oxacillin MICs evaluated according to the new breakpoint (0.5 μg/ml) of the National Committee for Clinical Laboratory Standards exhibited an extremely low specificity for this population.
Identification and Characterization of Phage Variants of a Strain of Epidemic Methicillin-Resistant
Staphylococcus aureus
(EMRSA-15)
Authors: O'Neill G. L. , Murchan S. , Gil-Setas A. , Aucken H. M.
Published: July 2002

EMRSA-15 is one of the most important strains of epidemic methicillin-resistant
Staphylococcus aureus
(EMRSA) found in the United Kingdom. It was originally characterized by weak lysis with phage 75 and production of enterotoxin C but not urease. Two variant strains of EMRSA-15 which show a broader phage pattern than the progenitor strain have emerged. A total of 153 recent clinical isolates representing classical EMRSA-15 (55 isolates) or these phage variants (98 isolates) were compared by
Sma
I macrorestriction profiles in pulsed-field gel electrophoresis (PFGE) as well as by urease and enterotoxin C production. Eight of the 98 isolates were shown to be other unrelated strains by both PFGE and their production of urease, a misidentification rate of 8% by phage typing. Seventy-one EMRSA-15 isolates were enterotoxin C negative, and the majority of these were sensitive to phage 81. Examination of PFGE profiles and Southern blotting studies suggest that the enterotoxin C gene locus is encoded on a potentially mobile DNA segment of ca. 15 kb. After elimination of the eight non-EMRSA-15 isolates, the remaining 145 were characterized by PFGE, yielding 22 profiles. All profiles were within five band differences of at least one other profile. Classical EMRSA-15 isolates showed nine PFGE profiles, with the majority of isolates (68%) in profile B1. Six of these nine PFGE profiles were unique to the classical EMRSA-15 isolates. Among the phage variants of EMRSA-15, 16 profiles were seen, but the majority of isolates (83%) fell into 1 of 4 profiles (B2, B3, B4, and B7) which correlated well with phage patterns. The most divergent PFGE profiles among the EMRSA-15 isolates had as many as 12 band differences from one another, suggesting that in examining isolates belonging to such a temporally and geographically disseminated epidemic strain, the range of PFGE profiles must be regarded as a continuum and analyzed by relating the profiles back to the most common or progenitor profile.
Candida
Species Exhibit Differential In Vitro Hemolytic Activities
Authors: Luo Gang , Samaranayake Lakshman P. , Yau Joyce Y. Y.
Published: July 2002

A total of 80
Candida
isolates representing 14 species were examined for their respective responses to an in vitro hemolytic test. A modification of a previously described plate assay system where the yeasts are incubated on glucose (3%)-enriched sheep blood agar in a carbon dioxide (5%)-rich environment for 48 h was used to evaluate the hemolytic activity. A group of eight
Candida
species which included
Candida albicans
(15 isolates),
C. dubliniensis
(2),
C. kefyr
(2),
C. krusei
(4),
C. zeylanoides
(1),
C. glabrata
(34),
C. tropicalis
(5), and
C. lusitaniae
(2) demonstrated both alpha and beta hemolysis at 48 h postinoculation. Only alpha hemolysis was detectable in four
Candida
species, viz.,
C. famata
(3),
C. guilliermondii
(4),
C. rugosa
(1), and
C. utilis
(1), while
C. parapsilosis
(5) and
C. pelliculosa
(1) failed to demonstrate any hemolytic activity after incubation for 48 h or longer. This is the first study to demonstrate the variable expression profiles of hemolysins by different
Candida
species.
Immunofluorescent Detection of Intraerythrocytic
Bartonella henselae
in Naturally Infected Cats
Authors: Rolain J. M. , La Scola B. , Liang Z. , Davoust B. , Raoult D.
Published: July 2002

To determine the presence of
Bartonella henselae
bacteremia in six cats, we compared isolation using blood culture with direct immunofluorescence on blood smears. Three cats that were positive by blood culture were also positive by direct immunofluorescence, and laser confocal microscopy confirmed the intraerythrocytic location of
B. henselae
.
Quantitative Detection of
Streptococcus pneumoniae
in Nasopharyngeal Secretions by Real-Time PCR
Authors: Greiner Oliver , Day Philip J. R. , Bosshard Philipp P. , Imeri Fatime , Altwegg Martin , Nadal David
Published: July 2002

Streptococcus pneumoniae
is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of
S. pneumoniae
may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of
S. pneumoniae
cells in which the quantities of DNA ranged from the amounts extracted from 1 to 10
6
cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of
S. pneumoniae
following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from
Haemophilus influenzae
,
Moraxella catarrhalis
, or alpha-hemolytic streptococci other than
S. pneumoniae
were not amplified or were only weakly amplified when there were ≥10
6
cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of
S. pneumoniae
organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of
S. pneumoniae
present in NPSs. This assay may serve as a tool to study changes in the amounts of
S. pneumoniae
during lower respiratory tract infections.
Type IV Longus Pilus of Enterotoxigenic
Escherichia coli
: Occurrence and Association with Toxin Types and Colonization Factors among Strains Isolated in Argentina
Authors: Pichel Mariana G. , Binsztein Norma , Qadri Firdausi , Girón Jorge A.
Published: July 2002

The longus type IV pilus structural gene (
lngA
) was sought among 217 clinical enterotoxigenic
Escherichia coli
(ETEC) strains isolated in Argentina.
lngA
was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors. The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.
GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes
Authors: van den Brule Adriaan J. C. , Pol René , Fransen-Daalmeijer Nathalie , Schouls Leo M. , Meijer Chris J. L. M. , Snijders Peter J. F.
Published: July 2002

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (
n
= 196) and biopsies of cervical premalignant lesions (
n
= 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.
Detection of Mammalian Reovirus RNA by Using Reverse Transcription-PCR: Sequence Diversity within the λ3-Encoding L1 Gene
Authors: Leary Thomas P. , Erker James C. , Chalmers Michelle L. , Cruz Andrea T. , Wetzel J. Denise , Desai Suresh M. , Mushahwar Isa K. , Dermody Terence S.
Published: July 2002

Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 ± 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.
Profile of
Salmonella enterica
subsp.
enterica
(Subspecies I) Serotype 4,5,12:i:− Strains Causing Food-Borne Infections in New York City
Authors: Agasan Alice , Kornblum John , Williams George , Pratt Chi-Chi , Fleckenstein Phylis , Wong Marie , Ramon Alex
Published: July 2002

Strains of newly emerging
Salmonella enterica
subsp.
enterica
(subspecies I) serotype 4,5,12:i:− causing food-borne infections, including a large food poisoning outbreak (
n
= 86) characterized by persistent diarrhea (14% bloody), abdominal pain, fever, and headache, were examined. The organisms were found in the stool samples from the patients. The biochemical profile of the organisms is consistent with that of
S. enterica
subsp. I serotypes, except for decreased dulcitol (13%) and increased inositol (96%) utilization. Twenty-eight percent of the strains showed resistance to streptomycin, sulfonamides, or tetracycline only; all three antimicrobial agents; or these agents either alone or in combination with ampicillin, trimethoprim, and trimethoprim-sulfamethoxazole. None of the serotype 4,5,12:i:− strains showed resistance or decreased susceptibility to chloramphenicol or ciprofloxacin. On pulsed-field gel electrophoresis (PFGE), the strains showed 11 or 12 resolvable genomic fragments with 18 banding patterns and three PFGE profile (PFP) clusters (i.e., PFP/A, PFP/B, and PFP/C). Seventy-five percent of the isolates fingerprinted were closely related (zero to three band differences; similarity [Dice] coefficient, 86 to 100%); 63% of these were indistinguishable from each other (PFP/A
1
). PFP/A
1
was common to all strains from the outbreak and 11 hospital sources. Strains from six other hospitals shared clusters PFP/B and PFP/C. PFP/C
4
, of the environmental isolate, was unrelated to PFP/A and PFP/B. Nine band differences (similarity coefficient, 61%) were noted between PFP/A
1
and PFP/E of the multidrug-resistant
S. enterica
subsp.
enterica
serotype Typhimurium definitive type 104 strains. Whether these emerging
Salmonella
strains represent a monophasic, Dul
−
variant of serotype Typhimurium or
S. enterica
subsp.
enterica
serotype Lagos or a distinct serotype of
S. enterica
subsp. I is not yet known. Some of the phenotypic and genotypic properties of the serotype 4,5,12:i:− strains are described here.
In Vitro Activity of Caspofungin (MK-0991) against
Candida albicans
Clinical Isolates Displaying Different Mechanisms of Azole Resistance
Authors: Bachmann Stefano P. , Patterson Thomas F. , López-Ribot José L.
Published: July 2002

Caspofungin inhibits the synthesis of 1,3-β-
d
-glucan, a key step in fungal cell wall biosynthesis. Here we report on its potent in vitro activity (MIC at which 90% of the isolates tested are inhibited = 1 μg per ml of RPMI medium) against 32
Candida albicans
fluconazole-susceptible and -resistant clinical isolates irrespective of the underlying resistance mechanism (alterations in
ERG11
and/or upregulation of
MDR
and
CDR
genes encoding efflux pumps) and provide further evidence that caspofungin is not a substrate for multidrug transporters.
A Novel Polyomavirus (Goose Hemorrhagic Polyomavirus) Is the Agent of Hemorrhagic Nephritis Enteritis of Geese
Authors: Guerin Jean-Luc , Gelfi Jacqueline , Dubois Luc , Vuillaume Aimé , Boucraut-Baralon Corine , Pingret Jean-Luc
Published: July 2002

We have identified the etiological agent of hemorrhagic nephritis enteritis of geese (HNEG), a fatal disease of European geese. HNEG has been recognized in almost all goose breeding areas, with an epizootic pattern, and up to now, the infectious agent has remained unknown. In order to identify the causative agent, infected tissues from HNEG-affected geese were inoculated to 1-day-old goslings, which then developed clinical signs typical of HNEG. Tissue homogenates from these birds were subjected to Freon extraction followed by sucrose density gradient ultracentrifugation. The resulting main band was examined by electron microscopy and consisted of spherical, naked, papovavirus-like particles approximately 45 nm in diameter. The virus was isolated and propagated in goose kidney cell primary culture. Tissue- or culture-purified virus allowed the experimental reproduction of the disease in goslings. Random PCR amplification of viral nucleic acid produced a 1,175-bp fragment which was shown to be associated with field samples collected from geese affected by HNEG on commercial farms in France. Sequence analysis of the PCR product revealed a unique open reading frame, showing 63 to 72% amino acid similarity with the major capsid protein (VP1) of several polyomaviruses. Finally, based on phylogenetic analysis, we conclude that the causative agent of HNEG is closely related to but clearly distinct from other polyomaviruses; we thus have named this newly identified virus
Goose hemorrhagic polyomavirus
.