bluuebunny/crossref_metadata_2025_split 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThenodZgene, 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTGenome 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

ABSTRACT

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

ABSTRACT

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.