bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
Bacillus subtilis
HBsu Protein Modifies the Effects of α/β-Type, Small Acid-Soluble Spore Proteins on DNA
Authors: Ross Margery A. , Setlow Peter 
Published: July 2002

ABSTRACT

HBsu, the
Bacillus subtilis
homolog of the
Escherichia coli
HU proteins and the major chromosomal protein in vegetative cells of
B. subtilis
, is present at similar levels in vegetative cells and spores (∼5 × 10
4
monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the α/β-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with α/β-type SASP on the nucleoid, suggesting that HBsu could modulate α/β-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered α/β-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing α/β-type SASP-mediated positive supercoiling, and greatly ameliorated the α/β-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the α/β-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of α/β-type SASP on the properties of DNA in the developing and dormant spore.
Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin β-Ketoacyl-Acyl Carrier Protein Synthase
Authors: He Min , Varoglu Mustafa , Sherman David H. 
Published: July 2002

ABSTRACT

A three-dimensional model of the
Streptomyces coelicolor
actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related
Escherichia coli
fatty acid synthase condensing enzyme β-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production.
3
H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.
Role of the
Eikenella corrodens pilA
Locus in Pilus Function and Phase Variation
Authors: Villar Maria T. , Hirschberg Rona L. , Schaefer Michael R. 
Published: July 2002

ABSTRACT

The human pathogen
Eikenella corrodens
expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate
E. corrodens
VA1. Earlier work defined the
pilA
locus which includes
pilA1
,
pilA2
,
pilB
, and
hagA
. Both
pilA1
and
pilA2
predict a type IV pilin, whereas
pilB
predicts a putative pilus assembly protein. The role of
hagA
has not been clearly established. That work also confirmed that
pilA1
encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the
pilA
locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different
pilA
mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of
pilA1
yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of
pilA1
and proper export and assembly of PilA1. Inactivation of
pilA2
yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that
pilA2
is not essential for biosynthesis of functionally normal pili. A mutant inactivated for
pilB
was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of
hagA
, which may encode a tellurite resistance protein, had no effect on pilus structure or function.
Cell Wall Core Galactofuran Synthesis Is Essential for Growth of Mycobacteria
Authors: Pan Fei , Jackson Mary , Ma Yufang , McNeil Michael 
Published: July 2002

ABSTRACT

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In
Mycobacterium tuberculosis
the
glf
gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that
glf
can be knocked out in
Mycobacterium smegmatis
by allelic replacement only in the presence of two rescue plasmids carrying functional copies of
glf
and Rv3808c. The
glf
rescue plasmid was designed with a temperature-sensitive origin of replication and the
M. smegmatis glf
knockout mutant is unable to grow at the higher temperature at which the
glf
-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the
glf
-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both
glf
and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.
Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates
csrB
Transcription in
Escherichia coli
Authors: Gudapaty Seshagirirao , Suzuki Kazushi , Wang Xin , Babitzke Paul , Romeo Tony 
Published: July 2002

ABSTRACT

The global regulator CsrA (carbon storage regulator) of
Escherichia coli
is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5′ end of the CsrB transcript was mapped, and a
csrB
::
cam
null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic
csrA
,
csrB
,
rpoS
, or
csrA rpoS
mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (∼10-fold) in the
csrA
mutants. CsrB transcript stability was unaffected by
csrA
. The expression of a
csrB-lacZ
transcriptional fusion containing the region from −242 to +4 bp of the
csrB
gene was decreased ∼20-fold by a
csrA
::
kanR
mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating
csrB
transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB.
Campylobacter fetusUses Multiple Loci for DNA Inversion within the 5′ Conserved Regions ofsapHomologs
Authors: Tu Zheng-Chao , Ray Kevin C. , Thompson Stuart A. , Blaser Martin J. 
Published: July 2002

ABSTRACTCampylobacter fetuscells possess multiple promoterlesssaphomologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique promoter present on a 6.2-kb invertible element. Eachsaphomolog includes a 626-bp 5′ conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because the FCRs are identical. In mutant strain 23D:ACA2K101, in whichsapAandsapA2flanking the invertible element in opposite orientations were disrupted by promoterless chloramphenicol resistance (Cmr) and kanamycin resistance (Kmr) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inverted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream ofsapA2in 23D:ACA2K101 by introducingNotI andKpnI sites to create strains 23D:ACA2K101Nand 23D:ACA2K101K, respectively. Alternatively selecting colonies for Cmror Kmrshowed that mutagenizing the IR or CLS had no apparent effect on the frequency of the DNA inversion. However, mapping the uniqueNotI orKpnI site in relation to the Cmror Kmrcassette in the cells that changed phenotype showed that splices occurred both upstream and downstream of the mutated sites. PCR and sequence analyses also showed that the splice could occur in the 425-bp portion of the FCR downstream of the cassettes. In total, these data indicate thatC.fetuscan use multiple sites within the FCR for itssap-related DNA inversion.
Regulatory Architecture of the Iron-Regulated
fepD-ybdA
Bidirectional Promoter Region in
Escherichia coli
Authors: Christoffersen Catherine A. , Brickman Timothy J. , Hook-Barnard India , McIntosh Mark A. 
Published: July 2002

ABSTRACT

The overlapping and opposing promoter elements for the
Escherichia coli fepDGC
operon and the
ybdA
gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their −10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the
E. coli
consensus sequence, both promoters were poorly conserved at the −35 position and mutations which strengthened the −35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the
fepD
and
ybdA
primary promoters were shown to require a 5′-TGn-3′ upstream extension of their −10 elements for optimal activities. Secondary promoters were identified for both
fepD
and
ybdA
, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping
fepD
and
ybdA
promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.
Thermoadaptation of α-Galactosidase AgaB1 in
Thermus thermophilus
Authors: Fridjonsson Olafur , Watzlawick Hildegard , Mattes Ralf 
Published: July 2002

ABSTRACT

The evolutionary potential of a thermostable α-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid α-galactosidase genes of
agaA
and
agaB
from
Bacillus stearothermophilus
KVE39, designated
agaA1
and
agaB1
, were cloned into an autonomously replicating
Thermus
vector and introduced into
Thermus thermophilus
OF1053GD (Δ
agaT
) by transformation. This selector strain is unable to metabolize melibiose (α-galactoside) without recombinant α-galactosidases, because the native α-galactosidase gene,
agaT
, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded
agaA1
gene but unable when harboring a plasmid-encoded
agaB1
gene. With incubation of the
agaB1
plasmid-harboring strain under selective pressure at a restrictive temperature (67°C) in a minimal melibiose medium, spontaneous mutants as well as
N
-methyl-
N′
-nitro-
N
-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant α-galactosidase genes were amplified by PCR, cloned in
Escherichia coli
, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant
agaB1
genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.
Comparison of DNA Sequencing of the Protein A Gene Polymorphic Region with Other Molecular Typing Techniques for Typing Two Epidemiologically Diverse Collections of Methicillin-Resistant
Staphylococcus aureus
Authors: Oliveira Duarte C. , Crisóstomo Inês , Santos-Sanches Ilda , Major Peter , Alves C. Rute , Aires-de-Sousa Marta , Thege Marianne K. , de Lencastre Hermı́nia 
Published: July 2002

ABSTRACT

The aim of this study was to compare the recently developed typing approach for methicillin-resistant
Staphylococcus aureus
(MRSA) based on the DNA sequencing of the protein A gene polymorphic region (
spaA
typing) with a combination of three well-established molecular typing techniques:
Cla
I-
mecA
vicinity polymorphisms,
Cla
I-Tn
554
insertion patterns, and
Sma
I pulsed-field gel electrophoresis (PFGE) profiles. In order to evaluate the applicability of this typing technique in different types of studies, two groups of MRSA clinical isolates were analyzed: a collection of 185 MRSA isolates circulating in Hungary recovered from 17 hospitals in seven cities during a 3-year period (1994 through 1996), and a selection of 53 MRSA strains isolated in a single hospital in Hungary between 1997 and 1998. The 238 MRSA clinical strains from Hungary were first classified in clonal types (defined as
Cla
I-
mecA
::
Cla
I-Tn
554
::
Sma
I-PFGE patterns), and 65 of the 238 strains, representing major MRSA clones and some sporadic clones, were further analyzed by
spaA
typing. Our results showed that the lineages most recently introduced in the hospital setting showed little variability in
spaA
types, whereas the MRSA clones circulating for a longer period of time and spread among several hospitals showed a higher degree of variability. The implementation of the
spaA
typing method was straightforward, and the results obtained were reproducible, unambiguous, and easily interpreted. This method seems to be adequate for outbreak investigations but should be complemented with other techniques in long-term surveillance or in studies comparing distant clonal lineages.
Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing
Authors: Angeletti Silvia , Lorino Giulia , Gherardi Giovanni , Battistoni Fabrizio , De Cesaris Marina , Dicuonzo Giordano 
Published: July 2002

ABSTRACT

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with

ddl
E. faecalis

and

ddl
E. faecium

primers and another with
vanC-1
and
vanC-2/3
primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.
Resistance-Associated Mutations in the Human Immunodeficiency Virus Type 1 Subtype C Protease Gene from Treated and Untreated Patients in the United Kingdom
Authors: Cane Patricia A. , de Ruiter Annemiek , Rice Philip , Wiselka Martin , Fox Ray , Pillay Deenan 
Published: July 2002

ABSTRACT
This work reports the variability of human immunodeficiency virus type 1 (HIV-1) protease from treated and untreated patients infected with HIV-1 subtype C in the United Kingdom. The most common primary mutation observed in treated patients was L90M. D30N, M46I, V82A/F, and I84V were seen rarely. M36I and I93L mutations were observed in nearly all samples from both treated and untreated patients and so cannot be considered as resistance-associated mutations in this subtype.
Stx2 Subtyping of Shiga Toxin-Producing
Escherichia coli
Isolated from Cattle in France: Detection of a New Stx2 Subtype and Correlation with Additional Virulence Factors
Authors: Bertin Yolande , Boukhors Karima , Pradel Nathalie , Livrelli Valerie , Martin Christine 
Published: July 2002

ABSTRACT

At least 11 Stx2 variants produced by Shiga toxin-producing
Escherichia coli
(STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for
stx
2
(
n
= 104) or
stx
2
and
stx
1
(
n
= 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring
stx
1
and one or two
stx
2
genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring
stx
2
-EDL933 alone (78%) or a combination of
stx
2
-EDL933 and
stx
2
vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.
Immunoglobulin M (IgM)-Glycoinositolphospholipid Enzyme-Linked Immunosorbent Assay: an Immunoenzymatic Assay for Discrimination between Patients with Acute Toxoplasmosis and Those with Persistent Parasite-Specific IgM Antibodies
Authors: Giraldo Mónica , Portela Ricardo W. D. , Snege Mirian , Leser Paulo G. , Camargo Mário E. , Mineo José Roberto , Gazzinelli Ricardo T. 
Published: July 2002

ABSTRACTIn the present study we developed an enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for glycoinositolphospholipids (GIPL) derived from tachyzoite membrane (IgM-GIPL ELISA). The sensitivity and specificity of the assay were compared with those of commercially availableToxoplasma-specific IgM serological tests, namely, immunofluorescence assay (IFA) with fixed tachyzoites and capture ELISA employing tachyzoite extracts. Our results show that all patients with acute toxoplasmosis, as determined by clinical data and conventional serological tests, were also positive by the IgM-GIPL ELISA. Interestingly, many patients that were classified as indeterminate, who had IgG with high avidity but positive results in the IgM-specific IFA and capture ELISA, were negative by the IgM-GIPL ELISA. Finally, we tested the sera from patients with rheumatoid arthritis and various parasitic infections and found no evidence of false positives in the IgM-GIPL ELISA.
Identification of a Novel Methicillin-Resistant
Staphylococcus aureus
Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections
Authors: Sola Claudia , Gribaudo Germán , Vindel Ana , Patrito Luis , Bocco José Luis 
Published: July 2002

ABSTRACT

Infections caused by methicillin-resistant
Staphylococcus aureus
(MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.