bluuebunny/crossref_metadata_2025_split huggingface dataset:
Binding Site of Macrolide Antibiotics on the Ribosome: New Resistance Mutation Identifies a Specific Interaction of Ketolides with rRNA
Authors: Garza-Ramos Georgina , Xiong Liqun , Zhong Ping , Mankin Alexander 
Published: July 2002

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

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

ABSTRACT

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

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

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

ABSTRACT

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

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

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

ABSTRACT

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

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

ABSTRACT

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

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

ABSTRACT

Tests of the survival of
Candida
spp.,
Aspergillus
spp., a
Fusarium
sp.
,
a
Mucor
sp.
,
and a
Paecilomyces
sp. on hospital fabrics and plastics indicated that viability was variable, with most fungi surviving at least 1 day but many living for weeks. These findings reinforce the need for appropriate disinfection and conscientious contact control precautions.