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B Pettersson

Publications and source records attributed to B Pettersson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp. and Haemobartonella spp.

The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria. Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.

Anaplasmataceae↗
PubMed1999

Truncated fragments in polymerase chain reaction-based DNA sequencing.

Reconstitution experiments were performed by using an ordinary dye-primer protocol of a template spiked with known amounts of truncated fragments. We observed that as little as 0.2 mole-percentage of the truncated fragment caused sequence interpretation problems. Two protocols were developed for sequencing with dye-labeled terminators; this eliminates the problems with truncated fragments, which are adapted to a one-dye chemistry. One was designed for single extension sequencing using T7 DNA polymerase and one for cycle sequencing. To avoid precipitation and centrifugation and to facilitate automation, the dye-terminator protocols included the use of a biotinylated sequencing primer. Thus, the Sanger fragments were recovered and, by magnetic separation, washed and released by formamide, EDTA, and heat treatment before loading on the electrophoresis gel. Integrated procedures for sequencing PCR products using one-dye-labeled terminators suitable for automation are described. High quality data in terms of long reads and detection of polymorphisms is obtained. The protocols serve as attractive alternatives to internal labeling and dye-primer approaches.

Base Sequence↗
PubMed1999

Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli.

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method.

Chromatography, Gel↗
PubMed1999

Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae.

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly beta-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.

Animals↗
PubMed1999

Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.

A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.

Amino Acid Sequence↗
PubMed1999

Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

Almost complete 16S rRNA gene sequences were generated for the type strains of the obligate insect pathogens Bacillus lentimorbus and Bacillus popilliae and a second strain of Bacillus popilliae (NRRL B-4081) received as 'Bacillus popilliae var. melolonthae'. A phylogenetic tree was constructed which grouped these strains into a well defined subcluster within the genus Paenibacillus. Bacillus popilliae NRRL B-4081 occupied an intermediate position between the type strains of Bacillus lentimorbus and Bacillus popilliae but with a marked clustering to the latter. The phylogenetic assignment of these strains to Paenibacillus is in contrast to earlier studies which placed these bacteria in the genus Bacillus, close to Bacillus subtilis. Indeed, the rRNA sequences generated in this study share less than 88% similarity to the deposited sequences for Bacillus popilliae ATCC 14706T and Bacillus lentimorbus ATCC 14707T. The results obtained by using different tree algorithms, bootstrap analysis, branch lengths and verification by signature nucleotide analysis supported the reclassification of these species in the genus Paenibacillus as Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

Animals↗
PubMed1999

Diagnosis of contagious bovine pleuropneumonia by PCR-laser- induced fluorescence and PCR-restriction endonuclease analysis based on the 16S rRNA genes of Mycoplasma mycoides subsp. mycoides SC.

As contagious bovine pleuropneumonia (CBPP) is spreading fast in many African countries, there is an increasing demand for rapid and sensitive diagnostic methods that can be used to confirm the initial diagnosis based on clinical symptoms or pathological findings. Two PCR-based diagnostic systems for identification of the infectious agent, Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC), in various samples are presented. Both systems involve group-specific amplification of the two 16S rRNA genes from mycoplasmas of the M. mycoides cluster. The laser-induced fluorescence assay is based on a unique sequence length difference between the two 16S rRNA genes in M. mycoides SC. This region was amplified by PCR, and the products were separated by polyacrylamide gel electrophoresis in a DNA sequencer. The resulting electropherogram showed two peaks for strains of M. mycoides SC and one peak for all other members of the M. mycoides cluster. The second system was based on restriction endonuclease analysis and agarose gel electrophoresis. Restriction of amplicons from a region containing a polymorphism, which is found in M. mycoides SC only, resulted in an extra band on the agarose gel because an AluI site is lacking in the rrnA operon. Specimens from cows with postmortem signs of CBPP were analyzed with the two PCR systems. M. mycoides SC was clearly identified in pleural fluid and lung tissue, and the methods were found to be robust and rapid. The results were in agreement with those obtained by conventional diagnostic techniques.

Animals↗
PubMed1999

Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, a novel analogue of cladribine in human leukemic cells.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

Adenine Nucleotides↗
PubMed1998

Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii.

The nucleotide sequences of the 16S rRNA genes from the type strains of four goat mycoplasmas, Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii, were determined by direct solid-phase DNA sequencing. Polymorphisms were found in two of the 16S rRNA gene sequences, showing the existence of two different rRNA operons. Three polymorphisms were found in M. adleri, and one was found in M. yeatsii. The sequence information was used for the construction of phylogenetic trees. M. adleri was included in the Mycoplasma lipophilum cluster within the hominis group. M. auris was comprised in the Mycoplasma hominis cluster of the hominis group. M. cottewii and M. yeatsii were found to be very closely related with only four nucleotide differences, and they grouped with Mycoplasma putrefaciens in the Mycoplasma mycoides cluster within the spiroplasma group. Sequencing of two field isolates of M. cottewii and M. yeatsii, geographically distant from the type strains, showed that the 16S rRNA gene from the field isolate of M. cottewii was identical to the one from the type strain. The field isolate of M. yeatsii had only two nucleotide differences to the type strain and these were present in only one of the two rRNA operons. Sequencing of the 16S rRNA genes from two unidentified mycoplasma isolates from Nepal indicated that they should both be regarded as M. auris strains.

Animals↗
PubMed1998

Phylogeny of the family Moraxellaceae by 16S rDNA sequence analysis, with special emphasis on differentiation of Moraxella species.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity of > or = 95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (< or = 93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.

DNA Primers↗
PubMed1998

Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes.

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.

Africa↗
PubMed1998

PCR-introduced loop structure as primer in DNA sequencing.

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.

Base Sequence↗
PubMed1998

Pharmacokinetics of cladribine in plasma and its 5'-monophosphate and 5'-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia.

The pharmacokinetic parameters of cladribine (CdA) in patient plasma and its intracellular nucleotides CdA 5'-monophosphate (CdAMP) and CdA 5'-triphosphate (CdATP) were delineated in circulating leukemia cells in 17 patients with chronic lymphocytic leukemia, after the last dose intake and up to 72 h thereafter. Patients were treated with 10 mg/m2 CdA p.o. on 3 consecutive days. A novel and specific ion-pair liquid chromatographic method, which separates the intracellular CdA nucleotides, was used. The area under the concentration versus time curve (AUC) of CdAMP in leukemia cells was generally higher (median, 47 micromol/liter x h) than the AUC of CdATP (median, 22 micromol/liter x h); however, in some patients (3 of 17), the reverse relationship was seen. The median ratio between the AUC values for CdATP and CdAMP was 0.60 (95% confidence interval, 0.4-1.0). The median half-life (t(1/2)) of CdAMP was 15 h, and that of CdATP was 10 h. The median terminal t(1/2) of CdA in plasma was 21 h. A significant correlation was found between the maximum plasma CdA and cellular CdAMP concentrations (r = 0.56, P = 0.02). There was no correlation between the AUC values of cellular CdAMP and CdATP (r = 0.224, P = 0.55). No correlation was found between deoxycytidine kinase activity and intracellular pharmacokinetic parameters of CdAMP or CdATP. The response to treatment was not significantly related to intracellular concentration of CdAMP or active metabolite CdATP. There is great heterogeneity among patients in terms of AUC and t(1/2) of CdAMP and CdATP. Furthermore, the results emphasize the differences between the pharmacokinetics of plasma CdA and those of the metabolites in circulating leukemic cells.

Adenosine Monophosphate↗
PubMed1997

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.

Animals↗
PubMed1997

Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.

Animals↗
PubMed1997

Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR.

A PCR system for the detection and identification of group IV spirochetes (Serpulina pilosicoli) was designed to complement biochemical tests, e.g., the hippurate hydrolysis and beta-glucosidase tests, and to verify the accuracy of a previously proposed biochemical classification system. The PCR assay was based on amplification of a segment of the 16S rRNA gene. Both primers were constructed to selectively amplify the 16S rRNA gene of Serpulina pilosicoli. All analyzed Serpulina strains exhibiting the capacity to hydrolyze hippurate and lacking beta-glucosidase activity, including the type strain for spirochetal diarrhea, P43, were amplified with the PCR system. All other tested strains, including type and field strains of different phenotypes of Serpulina species, as well as Salmonella species, Campylobacter species, and Escherichia coli strains, were negative in the assay. Among the tested strains were 18 Scottish field isolates originating from the mucosae of pigs with colitis. A simple classification scheme, suitable for routine classification of porcine intestinal spirochetes, is also presented. The scheme is based on hemolysis, indole production, and the hippurate hydrolysis test.

Animals↗