Biomedical subjects

J M Bove

Publications and source records attributed to J M Bove.

At least 19 recordsLinked to original sources

PubMed2000

Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon.

Transposon Tn4001 mutagenesis of Spiroplasma citri wild-type (wt) strain GII-3 led to the isolation and characterization of non-phytopathogenic mutant GMT 553. In this mutant, transposon Tn4001 is inserted within the first gene of the fructose operon. This operon comprises three genes. The first gene (fruR) codes for a putative transcriptional regulator protein belonging to the deoxyribonucleoside repressor (DeoR) family. Sequence similarities and functional complementation of mutant GMT 553 with different combinations of the wt genes of the fructose operon showed that the second gene (fruA) codes for the permease of the phosphoenolpyruvate:fructose phosphotransferase system (fructose PTS), and the third, fruK, for the 1-phosphofructokinase (1-PFK). Transcription of the fructose operon in wt strain GII-3 resulted in two messenger RNAs, one of 2.8kb and one of 3.8kb. Insertion of Tn4001 in the genome of mutant GMT 553 abolished transcription of the fructose operon, and resulted in the inability of this mutant to use fructose. Functional complementation experiments demonstrated that fructose utilization was restored with fruR-fruA-fruK, fruA-fruK or fruA only, but not with fruR or fruR-fruA. This is the first time that an operon for sugar utilization has been functionally characterized in the mollicutes.

Amino Acid Sequence↗

PubMed1997

Comparison of the 16S/23S ribosomal intergenic regions of "Candidatus Liberobacter asiaticum" and "Candidatus Liberobacter africanum," the two species associated with citrus huanglongbing (greening) disease.

16S/23S intergenic spacer regions from the rRNA operons of two strains of "Candidatus Liberobacter asiaticum" and one strain of "Candidatus Liberobacter africanum" were cloned and sequenced. The intergenic spacers of the two "Candidatus L. asiaticum" strains studied are identical and contain the genes for isoleucine tRNA (tRNA(Ile)) and alanine tRNA (tRNA(Ala)) separated by 11 nucleotides. The intergenic spacer of the "Candidatus L. africanum" strain contains only one tRNA gene (tRNA(Ala)). The level of homology between the intergenic spacers of the two liberobacter species is 79.46%. Ribosomal operons with 16S/23S spacer regions other than those studied might be present in the two "Candidatus Liberobacter" species.

Base Sequence↗

PubMed1997

Spiroplasma diabroticae sp. nov., from the southern corn rootworm beetle, Diabrotica undecimpunctata (Coleoptera:Chrysomelidae).

Spiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32 degrees C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41 degrees C but failed to grow at 5 or 43 degrees C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 +/- 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.

Animals↗

PubMed1996

Insusceptibility of members of the class Mollicutes to rifampin: studies of the Spiroplasma citri RNA polymerase beta-subunit gene.

In order to study the mechanism of insusceptibility of Spiroplasma citri to rifampin, we have cloned and sequenced its rpoB gene, which encodes the beta subunit of RNA polymerase. By comparison of the deduced amino acid sequence with sequences of beta subunits from susceptible and resistant bacteria, it was possible to identify several differences in the so-called Rif region (encompassing rpoB codons 500 to 575 in the Escherichia coli sequence). We constructed a chimeric rpoB gene made of the E. coli rpoB gene in which the Rif region was replaced by the equivalent region from S. citri. E. coli cells harboring this chimeric gene were resistant to rifampin. Subsequent experiments involving site-directed mutagenesis demonstrated that a single amino acid substitution (asparagine at position 526) was able to provide high-level rifampin resistance in E. coli.

Amino Acid Sequence↗

PubMed1996

Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum.

In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.

Amino Acid Sequence↗

PubMed1994

The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria.

Using the PCR, we amplified the 16S ribosomal DNAs (rDNAs) of an Asian strain and an African strain of the uncultured, gram-negative, walled, phloem-limited bacterium-like organism (BLO) associated with citrus greening disease. We evaded coamplification of chloroplast 16S rDNA by using restriction enzymes; the chloroplast 16S rDNA was sensitive to BclI digestion and resistant to EcoRI digestion, while the 16S rDNA of the BLO was resistant to BclI digestion and sensitive to EcoRI digestion. The 16S rDNA of the African BLO strain was amplified from BclI-digested DNA extracted from infected periwinkle leaf midribs. The Asian strain was isolated from plant extract by using a specific monoclonal antibody coated onto the surface of a PCR tube. The 16S rDNAs of the two BLO strains were cloned and sequenced. Comparisons with sequences of 16S rDNAs obtained from the GenBank data base revealed that the two citrus greening disease BLOs belong to the alpha subdivision of the class Proteobacteria. Even though their closest relatives are members of the alpha-2 subgroup, these BLOs are distinct from this subgroup as we observed only 87.5% homology between the 16S rDNAs examined. Therefore, the two BLOs which we studied probably are members of a new lineage in the alpha subdivision of the Proteobacteria. We propose the trivial name "liberobacter" for this new group of bacteria and will wait until additional characteristics have been determined before we propose a formal name.

Base Sequence↗

PubMed1993

Evidence for a phosphoenolpyruvate dependent sugar-phosphotransferase system in the mollicute Acholeplasma florum.

In order to confirm the presence of a phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS) in the mollicute Acholeplasma florum we studied the ability of cell free extracts of this organism to phosphorylate glucose and/or fructose in the presence of PEP. We also cloned and sequenced a DNA fragment coding for a putative polypeptide showing significant similarity with the enzyme II of the beta-glucoside PTS of Escherichia coli. Taken together, these results show that A florum possesses a fructose-PTS, but not a glucose-PTS, and that the amino acid sequence deduced from the DNA fragment is related to beta-glucoside and sucrose enzymes II of PTS from various bacteria.

Acholeplasma↗

PubMed1993

Expression in Spiroplasma citri of an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for cloning and expressing foreign genes in S. citri, an organism which reads UGA as a tryptophan codon (C. Stamburski, J. Renaudin, and J.M. Bové, J. Bacteriol. 173:2225-2230, 1991). We now report cloning and expression in S. citri of the G fragment of cytadhesin P1 gene from Mycoplasma pneumoniae. The G fragment was inserted in the SpV1 RF downstream of a synthetic ribosome binding site and introduced into S. citri by electroporation. Northern (RNA) blot analyses showed that in S. citri, the G fragment was transcribed from an SpV1 RF promoter as a 1.2-kb mRNA. The translation product was detected by Western blotting (immunoblotting) with a rabbit antiserum raised against total proteins from M. pneumoniae (strain FH) and was proved to be P1 specific by using monoclonal antibodies specific for the G region of the P1 protein. The apparent molecular mass of the polypeptide (24.5 kDa) indicates that in S. citri, the G fragment was fully translated in spite of the seven UGA codons present in the reading frame.

Adhesins, Bacterial↗

PubMed1992

A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets.

Mycoplasma hyopneumoniae is the primary agent of swine enzootic pneumonia. Because of fastidious growth requirements and its serological cross-reactions with other porcine mycoplasmas, we developed a specific DNA probe for its detection. A partial genomic library of M. hyopneumoniae was constructed in plasmid pBR 322 using Hind III chromosomal fragments. The recombinant plasmids were screened by differential hybridization with M. flocculare and M. hyorhinis genomic DNA probes. One non-hybridizing recombinant plasmid was selected and its 1.65 kbp insert (designated I141) tested for specificity against genomic DNA from numerous mycoplasmas, other bacteria species and DNA from lung tissue of specific pathogen free (SPF) piglets. The 32P labelled I141 could detect specifically down to 400 pg of M. hyopneumoniae genomic DNA. To test the suitability of the I141 probe for the laboratory diagnosis of M. hyopneumoniae infections, we used clinical tracheobronchial specimens from piglets which were experimentally infected with M. hyopneumoniae. The results with hybridization on each specimen were compared to findings with an immunofluorescence test. Of the clinical specimen tested, there was agreement in the two tests of 63%.

Animals↗

PubMed1991

First step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read UGA as a tryptophan codon: synthesis of chloramphenicol acetyltransferase in Spiroplasma citri.

Spiroplasmas are wall-less procaryotes in which the UGA codon serves not as a stop signal but as a code for the amino acid tryptophan. Spiroplasma genes that contain UGA codons thus cannot be studied in the usual Escherichia coli cloning and expression systems. Although this problem can be circumvented by using UGA-suppressor strains of E. coli, spiroplasmas themselves would provide a more efficient cloning and expression host. We have now successfully employed the replicative form (RF) of a filamentous spiroplasma virus (SpV1) to clone and express the E. coli-derived chloramphenicol acetyltransferase (CAT) gene in Spiroplasma citri. The CAT gene was inserted in one of the four intergenic regions of the SpV1 RF and introduced into cells by electroporation. Both the RF and the virion DNA produced by the transfected cells contained the CAT gene sequences. Northern blot analysis, primer extension, and S1 mapping showed that transcription of the CAT gene started from a promoter located on the SpV1 RF and was terminated downstream of the CAT gene, still within the viral RF. Expression of the CAT gene was demonstrated by acetylation of chloramphenicol by cell-free extracts from the transfected spiroplasmas.

Base Sequence↗

PubMed1990

Characterization of a promoter and a transcription terminator of Spiroplasma melliferum virus SpV4.

Spiroplasma virus 4 (SpV4) is an isometric virus with single-stranded, circular DNA infecting the helical mollicute Spiroplasma melliferum, a honeybee pathogen. Previous studies in our laboratory led to the determination of the base sequence of the SpV4 DNA. Nine open reading frames and three promoterlike sequences (P1, P2, and P3) were identified. An inverted repeat leading to the formation of a hairpin structure on the transcription product was also found and predicted to be a transcription terminator (T). We have now studied the in vivo transcription of the SpV4 genome by Northern (RNA) blot analysis of the total RNAs extracted from SpV4-infected spiroplasma cells. Transcripts of 7.8, 4.4, 3.4, and 2.7 kilobases (kb) were detected. The 3.4-kb RNA was the major transcript. The 5' and 3' ends of this transcript were determined by S1 mapping and primer extension. Characterization of the 3' end by S1 mapping showed that the 3.4-kb transcript terminates within the stretch of uridine residues following the hairpin structure of terminator T. Characterization of the 5' end by S1 mapping indicated that transcription proceeds from a newly recognized promoter, P0, located 36 nucleotides upstream of P1. Primer extension resulted in two cDNA signals. The short cDNA was probably a primer extension artifact due to the presence of a hairpin structure on the transcript. When reverse transcriptase stopped at this hairpin or read through, the short or the long cDNA, respectively, was obtained. The size of the long cDNA identified P0 as the transcription promoter. Promoter P0 was also shown to be functional in Escherichia coli. Indeed, when inserted upstream of the chloramphenicol acetyltransferase gene of a promoter selection vector, it promoted transcription of this gene. As in the case of S. melliferum, two cDNAs were obtained by primer extension, the longer cDNA identifying P0 as the promoter.

Bacteriophages↗

PubMed1990

Spiralins of Spiroplasma citri and Spiroplasma melliferum: amino acid sequences and putative organization in the cell membrane.

Spiralin is the major membrane protein of the helical mollicute Spiroplasma citri. A similar protein occurs in the membrane of Spiroplasma melliferum, an organism related to S. citri. The gene encoding spiralin has been sequenced. A restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding S. melliferum spiralin. A 4.6-kilobase-pair ClaI DNA fragment from S. melliferum strongly hybridized with the probe. This fragment was inserted in pBR322 and cloned in Escherichia coli. It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991). An open reading frame showing 88.6% base sequence homology with the S. citri spiralin gene could be identified and was assumed to be the gene encoding S. melliferum spiralin. The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence. In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix. On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas.

Amino Acid Sequence↗

PubMed1988

Isolation of Mycoplasma salivarium from swine.

Mycoplasma salivarium, a common human oropharyngeal mycoplasma, was isolated from nasal and pharyngeal secretions of 14 of 284 swine in a barrier-maintained, disease-free herd. M. salivarium was recovered from one boar 6 times over a 26-month period and one time only from 13 other swine. Human isolates of M. salivarium were compared with the swine isolates by DNA-DNA hybridization and SDS-PAGE of the cell proteins and the strains were shown to be closely related. One of eight of the swine from which M. salivarium was isolated had complement-fixing antibodies and another culture-positive animal had metabolic-inhibiting antibodies to M. salivarium. Overt disease was not associated with the organism. These results support previous findings that mycoplasmas closely related to M. salivarium may be isolated from the nasopharynges of swine and they further indicate that the organism can establish persistently in swine without evidence of overt disease.

Animals↗

PubMed1987

New spiroplasmas from insects and flowers: isolation, ecology, and host association.

Eight spiroplasma strains from insects and one from spring flowers failed to react with antisera specific for any of the 11 described spiroplasma groups, with sera directed against spiroplasma Group I subgroups, or with sera directed against two unnumbered groups previously reported to occur in tabanid flies. Strains, all from Maryland, were isolated from the hemolymph of the spotted cucumber beetle Diabrotica undecimpunctata and the lampyrid beetle Ellychnia corrusca, and the guts of the cantharid beetles Cantharis bilineatus and C. carolinus. Other strains were obtained from a tabanid fly, Tabanus gladiator and from the firefly Photuris pennsylvanica in Maryland and from the mosquito Culex tritaeniorhynchus in Taiwan. An isolate from pooled Cicadulina bipunctella leafhoppers in Syria apparently represented a unique group. A single isolate from spring flowers in Oklahoma also appeared to be unrelated to existing groups or subgroups. One-way deformation tests using sera prepared against known beetle and tabanid spiroplasmas showed each of the above strains to be unique. Although these results strongly indicate that the nine strains studied are representatives of unique new spiroplasma groups, the formal designation of new groups awaits fulfillment of recently proposed criteria.

Animals↗

PubMed1987

Epidemiology of Spiroplasma citri in the Old World.

Epidemics of citrus stubborn disease (CSD) have been known in the Mediterranean citrus-growing areas as early as 1928. One such outbreak of stubborn disease occurred around 1980 in newly established citrus nurseries on the Syrian coast. Natural transmission of Spiroplasma citri could be shown to be involved. Enzyme-linked immunosorbent assay (ELISA) and culture of S. citri were used to detect the presence of S. citri in leafhoppers. Over 50 leafhopper species were submitted to these analyses, not only in Syria but also in Morocco and France (Corsica) from 1978 to 1985. Neoaliturus (Circulifer) haematoceps was the only species found to harbor the spiroplasma in Morocco, Syria and France (Corsica). Leafhoppers of this species were collected in Corsica, raised in Bordeaux and shown to be vectors of S. citri. The search for N. haematoceps in nature has revealed that Salsola kali (Chenopodiacae) is a favored host plant of this leafhopper. This plant has a wide geographical distribution. In Iran, for instance, it is well known, and grows close to sugar beet and citrus-growing areas. In such areas, sugar beet is known to be infected by curly top virus, and citrus by CSD. N. haematoceps is a vector of both of these diseases. N. tenellus, the vector of S. citri in the USA, is present in the Mediterranean area, but in view of its paucity it does not seem to be a major vector there. In Iran, even though less abundant than N. haematoceps, it could play a role in S. citri transmission.

Animals↗

PubMed1987

Further characterization of an unusual plant Mollicutes species of uncertain taxonomic status.

PPAV is a type of Mollicutes that was isolated in SP4 medium from seeds of apples affected by proliferation, a disease in which mycoplasma-like organisms (MLOs) are involved. However, PPAV is probably not the etiological MLO agent for at least two reasons: 1) optimal temperature growth is 43 C, and 2) in spite of numerous isolation attempts over several years, no second PPAV culture could be obtained. PPAV is surrounded by a single cytoplasmic membrane and forms typical fried egg-shaped colonies on solid medium. The organism grows in simplified mycoplasma media, such as BSR. In growth inhibition, it shows no serological relationships with any other mycoplasmas or acholeplasmas, including those cultured from the surfaces of plants. The absence of relatedness of PPAV to other Mollicutes was confirmed by DNA hybridization studies. The genome of PPAV is close to 10(9) daltons and contains 25.2 mol % G + C. Its genome size is similar to that of Acholeplasma spp. and Spiroplasma spp. It has, however, a clearcut sterol requirement and therefore cannot be an acholeplasma. Neither is it a spiroplasma since, though filamentous in BSR medium, it has never shown signs of helicity. Hence, PPAV is a taxonomical paradox.

Acholeplasma↗

PubMed1987

Cloning and sequencing of the genome of spiroplasma virus 4.

Spiroplasma virus 4 (Sp V4) has a circular single-stranded DNA. The replicative form (RF) of Sp V4 has been purified from infected cells of Spiroplasma melliferum, strain G1, and cloned in Escherichia coli (HB101) using plasmid pBR328 as the vector. The cloned RF was shown to be infectious by transfection. The Sp V4 RF was randomly subcloned in E. coli (TG1) using the M1 3 mp8 RF as the vector and sequenced by the dideoxy chain termination technique. We found that UGA is probably not a termination codon, but codes for tryptophan. Eight open reading frames, including that for the 65,000-dalton capsid protein, have been detected; they involve all three reading frames.

Bacteriophages↗

PubMed1987

Spiroplasmas: gene structure and expression.

Upon sequencing of the SpV4 genome, eight putative open reading frames (ORFs) including that for the 65-kilodalton (kDa) capsid protein were detected. They involve all three reading frames. Three promoter sequences were found, as well as a transcription terminator and the initiation site for complementary strand synthesis. Ribosome binding sites and regulatory sequences are closely related to those of Eubacteria. Codon usage analysis showed that A and T terminated codons are preferably used. UAA is the major termination codon. Upon cloning of the full-size SpV4 replicative form, the capsid protein gene could not be expressed in Escherichia coli, whereas the spiralin gene cloned in the same bacterium is expressed. These results suggest that in spiroplasmas, as in Mycoplasma capricolum, UGA is not a termination codon, but very probably codes for tryptophan. Spiralin contains no tryptophan. Hence, its gene contains no UGA codons and can thus be expressed in E. coli. On the other hand, the gene for capsid protein has nine UGA codons and cannot be fully expressed in the bacterium. Our results fully support the bacterial origin of spiroplasmas.

Bacteria↗