bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic and Biochemical Characterization of a Novel
umuD
Mutation: Insights into a Mechanism for UmuD Self-Cleavage
Authors: Sutton Mark D. , Kim Melanie , Walker Graham C. 
Published: July 2002

ABSTRACT

Most translesion DNA synthesis (TLS) in
Escherichia coli
is dependent upon the products of the
umuDC
genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA–single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD′. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD′ with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD′ homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD′ proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD
2
and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD
2
for self-cleavage.
Identification and Functional Characterization of Arylamine
N
-Acetyltransferases in Eubacteria: Evidence for Highly Selective Acetylation of 5-Aminosalicylic Acid
Authors: Deloménie Claudine , Fouix Sylvaine , Longuemaux Sandrine , Brahimi Naı̈ma , Bizet Chantal , Picard Bertrand , Denamur Erick , Dupret Jean-Marie 
Published: July 2002

ABSTRACT

Arylamine
N
-acetyltransferase activity has been described in various bacterial species. Bacterial
N
-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time
N
-acetyltransferase activity in 11 species of
Proteobacteriaceae
from seven genera:
Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides
, and
Vibrio cholerae
. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast,
para
-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied,
Pseudomonas aeruginosa
was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a
Salmonella enterica
serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.
Complete Nucleotide Sequence and Organization of the Atrazine Catabolic Plasmid pADP-1 from
Pseudomonas
sp
.
Strain ADP
Authors: Martinez Betsy , Tomkins Jeffrey , Wackett Lawrence P. , Wing Rod , Sadowsky Michael J. 
Published: July 2002

ABSTRACT

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from
Pseudomonas
sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of
s
-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPβ plasmid previously isolated from
Enterobacter aerogenes
. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn
5053
. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS
1071
and TnpA from
Pseudomonas pseudoalcaligenes
were identified and flank each of the
atzA, atzB,
and
atzC
genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes,
atzD
,
atzE
, and
atzF,
which participate in atrazine catabolism. Crude extracts from
Escherichia coli
expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from
Pseudomonas
sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid,
atzE
and
atzF
, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator.
E. coli
strains bearing
atzE
and
atzF
were shown to encode a biuret hydrolase and allophanate hydrolase, respectively.
atzDEF
are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.
RNA Polymerase Sigma Factor That Blocks Morphological Differentiation by
Streptomyces coelicolor
Authors: Gehring Amy M. , Yoo Narie J. , Losick Richard 
Published: July 2002

ABSTRACT

The filamentous bacterium
Streptomyces coelicolor
undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed
bld
(bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a
bld
mutant harboring an insertion in a previously uncharacterized gene,
SCE59.12c
, renamed here
rsuA
. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The
rsuA
gene encodes an apparent anti-sigma factor and is located immediately downstream of
SCE59.13c
, renamed here
sigU
, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of
rsuA
in a strain that contained
sigU
caused a block in development, and the overexpression of
sigU
in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both
rsuA
and
sigU
had been deleted was able to undergo morphological differentiation normally. We conclude that the
rsuA
-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by
sigU
. We also conclude that the
sigU
-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.
The
rlmB
Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in
Escherichia coli
Authors: Lövgren J. Mattias , Wikström P. Mikael 
Published: July 2002

ABSTRACT

In
Saccharomyces cerevisiae
, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposed
Escherichia coli
ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a Δ
rlmB
mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in
E. coli
.
Natural Genetic Transformation of
Streptococcus mutans
Growing in Biofilms
Authors: Li Yung-Hua , Lau Peter C. Y. , Lee Janet H. , Ellen Richard P. , Cvitkovitch Dennis G. 
Published: July 2002

ABSTRACT

Streptococcus mutans
is a bacterium that has evolved to be dependent upon a biofilm “lifestyle” for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in
S. mutans
and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes,
comC
,
comD
, and
comE
, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of
comC
and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown
S. mutans
cells were transformed at a rate 10- to 600-fold higher than planktonic
S. mutans
cells. Donor DNA included a suicide plasmid,
S. mutans
chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in
S. mutans
and that the system functions optimally when the cells are living in actively growing biofilms.
Identification of an
Actinobacillus pleuropneumoniae
Consensus Promoter Structure
Authors: Doree Scott M. , Mulks Martha H. 
Published: July 2002

ABSTRACT

Actinobacillus pleuropneumoniae
promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless
luxAB
genes of
Vibrio harveyi
. The degree of expression was quantifiable, and only high-expression or “hot” promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for
A. pleuropneumoniae
was identified. The consensus promoter sequence for
A. pleuropneumoniae
was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the
A. pleuropneumoniae
consensus with other prokaryotic consensus promoters showed that the
A. pleuropneumoniae
consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical −10 element and a similar but truncated −35 element. However, the
A. pleuropneumoniae
consensus promoter is unique in the spacing between the −10 and −35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an
A. pleuropneumoniae
promoter is shorter than the spacing identified for
Escherichia coli
and
Bacillus subtilis
promoters.
Construction and Characterization of Mutations at Codon 751 of the
Escherichia coli gyrB
Gene That Confer Resistance to the Antimicrobial Peptide Microcin B17 and Alter the Activity of DNA Gyrase
Authors: del Castillo Francisco J. , del Castillo Ignacio , Moreno Felipe 
Published: July 2002

ABSTRACT

Microcin B17 is a peptide antibiotic that inhibits DNA replication in
Escherichia coli
by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same
gyrB
point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.
Characterization of a
Mycoplasma pneumoniae hmw3
Mutant: Implications for Attachment Organelle Assembly
Authors: Willby Melisa J. , Krause Duncan C. 
Published: July 2002

ABSTRACT

The proteins required for adherence of the pathogen
Mycoplasma pneumoniae
to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.
Global Transcriptional Analysis ofclpPMutations of Type 2Streptococcus pneumoniaeand Their Effects on Physiology and Virulence
Authors: Robertson Gregory T. , Ng Wai-Leung , Foley Joseph , Gilmour Raymond , Winkler Malcolm E. 
Published: July 2002

ABSTRACTStreptococcus pneumoniaeis an important human pathogen that contains single copies of genes encoding the ClpP and FtsH ATP-dependent proteases but lacks the Lon and HslV proteases. We constructed and characterized the phenotypes ofclpP,clpC, andclpXdeletion replacement mutants, which lack the ClpP protease subunit or the putative ClpC or ClpX ATPase specificity factor. A ΔclpPmutant, but not a ΔclpCor ΔclpXmutant, of the virulent D39 type 2 strain ofS. pneumoniaegrew poorly at 30°C and failed to grow at 40°C. Despite this temperature sensitivity, transcription of the heat shock regulon determined by microarray analysis was induced in a ΔclpPmutant, which was also more sensitive to oxidative stress by H2O2and to puromycin than itsclpP+parent strain. A ΔclpPmutant, but not a ΔclpCmutant, was strongly attenuated for virulence in the murine lung and sepsis infection models. All of these phenotypes were complemented in a ΔclpP/clpP+merodiploid strain. Consistent with these complementation patterns,clpPwas found to be in a monocistronic operon, whose transcription was induced about fivefold by heat shock inS. pneumoniaeas determined by Northern and real-time reverse transcription-PCR analyses. BesidesclpP, transcription ofclpC,clpE, andclpL, but notclpXorftsH, was induced by heat shock or entry into late exponential growth phase. Microarray analysis of ΔclpPmutants showed a limited change in transcription pattern (≈80 genes) consistent with these phenotypes, including repression of genes involved in oxidative stress, metal ion transport, and virulence. In addition, transcription of the early and late competence regulon was induced in the ΔclpPmutant, and competence gene expression and DNA uptake seemed to be constitutively induced throughout growth. Together, these results indicate that ClpP-mediated proteolysis plays a complex and central role in numerous pneumococcal stress responses, development of competence, and virulence.
ZipA Is Required for Recruitment of FtsK, FtsQ, FtsL, and FtsN to the Septal Ring in
Escherichia coli
Authors: Hale Cynthia A. , de Boer Piet A. J. 
Published: July 2002

ABSTRACT

The septal ring in
Escherichia coli
consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.
Characterization of
Mycobacterium bohemicum
Isolated from Human, Veterinary, and Environmental Sources
Authors: Torkko Pirjo , Suomalainen Sini , Iivanainen Eila , Suutari Merja , Paulin Lars , Rudbäck Eeva , Tortoli Enrico , Vincent Véronique , Mattila Rauni , Katila Marja-Leena 
Published: July 2002

ABSTRACT

Chemotaxonomic and genetic properties were determined for 14 mycobacterial isolates identified as members of a newly described species
Mycobacterium bohemicum
. The isolates recovered from clinical, veterinary, and environmental sources were compared for lipid composition, biochemical test results, and sequencing of the 16S ribosomal DNA (rDNA) and the 16S-23S rDNA internal transcribed spacer (ITS) regions. The isolates had a lipid composition that was different from those of other known species. Though the isolates formed a distinct entity, some variations were detected in the features analyzed. Combined results of the phenotypic and genotypic analyses were used to group the isolates into three clusters. The major cluster (cluster A), very homogenous in all respects, comprised the
M. bohemicum
type strain, nine clinical and veterinary isolates, and two of the five environmental isolates. Three other environmental isolates displayed an insertion of 14 nucleotides in the ITS region; they also differed from cluster A in fatty alcohol composition and produced a positive result in the Tween 80 hydrolysis test. Among these three, two isolates were identical (cluster B), but one isolate (cluster C) had a unique high-performance liquid chromatography profile, and its gas liquid chromatography profile lacked 2-octadecanol, which was present in all other isolates analyzed. Thus, sequence variation in the 16S-23S ITS region was associated with interesting variations in lipid composition. Two of the isolates analyzed were regarded as potential inducers of human or veterinary infections. Each of the environmental isolates, all of which were unrelated to the cases presented, was cultured from the water of a different stream. Hence, natural waters are potential reservoirs of
M. bohemicum
.
Differentiation of
Candida dubliniensis
from
Candida albicans
on Staib Agar and Caffeic Acid-Ferric Citrate Agar
Authors: Al Mosaid Asmaa , Sullivan Derek , Salkin Ira F. , Shanley Diarmuid , Coleman David C. 
Published: July 2002

ABSTRACT

The methods currently available for the identification of the pathogenic yeast
Candida dubliniensis
all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschhäuser, Mycoses 42:521–524; 1999) of 14
C. dubliniensis
and 11
C. albicans
isolates, it was suggested that the ability of
C. dubliniensis
to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative
C. albicans
. In the present investigation, we examined the colony morphology and chlamydospore production of 130
C. dubliniensis
and 166
C. albicans
isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the
C. dubliniensis
and
C. albicans
isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the
C. albicans
isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the
C. dubliniensis
isolates produced chlamydospores on SA and CAF, respectively. All of the
C. albicans
isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30°C, while 97.7% of the
C. dubliniensis
isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the
C. albicans
and 93.8% of the
C. dubliniensis
isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between
C. dubliniensis
and
C. albicans
, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.
Genetic Diversity of
Histoplasma capsulatum
Strains Isolated from Soil, Animals, and Clinical Specimens in Rio de Janeiro State, Brazil, by a PCR-Based Random Amplified Polymorphic DNA Assay
Authors: Muniz Mauro de Medeiros , Pizzini Claudia Vera , Peralta José Mauro , Reiss Errol , Zancopé-Oliveira Rosely M. 
Published: July 2002

ABSTRACT

Little is known about the genetic strain diversity and geographical range of
Histoplasma capsulatum
isolated in Rio de Janeiro State, Brazil. We characterized 13 environmental, 7 animal, and 28 clinical
H. capsulatum
isolates by using a PCR-based random amplified polymorphic DNA (RAPD) assay. DNA fingerprinting of these soil, animal, and clinical specimens was performed with four primers (1253, 1281, D-9355, and D-10513) and generated amplicons with considerable polymorphism. Although all of the isolates exhibited more than 80% genetic relatedness, they could be clustered into four to six genotypes for each primer. The RAPD profiles of
H. capsulatum
isolated from Rio de Janeiro State could be distinguished from those of the U.S. strains included in this study (Downs, G222B, G-186B, and FLS1) by showing less than 70% similarity to each primer. The genetic polymorphisms between
H. capsulatum
strains isolated from animals and soil obtained in the same geographic areas were 100% similar, suggesting that an environmental microniche could be acting as a source of infection for animals and the local human population.