Unculturable Bacterium Research Articles

English

2Embrapa Agrobiology, BR 465, km07, 23890-000, Seropedica, Rio de Janeiro, Brazil. Accepted 26 March, 2013 The objective of the study was to characterize culturable diazotrophic endophytic bacteria from ‘Champaka’ pineapple plants during the fruiting period in an organic system. Micropropagated plants were inoculated with the diazotrophic endophytic bacterium, strain AB 219a, during acclimatization phase. In the field, the plants were fertilized with different dosages and sources of organic compost. After 17 months of field cultivation, roots and stems were collected for the isolation of endophytes, DNA extraction, 16S rDNA amplification and molecular analysis. A total of nine bacterial groups were identified, and species Burkholderia silvatlantica, Azorhizobium caulinodans, Pantoea eucrina, Erwinia sp. and unculturable bacterium occurred in inoculated plants. In contrast, non-inoculated plants were associated endophytes related to Burkholderia cenocepacia, Azospirillum brasilense, Enterobacter oryzae, Erwinia sp. and Sphingobium yanoikuyae. Segments of 360 base pair from nifH gene were amplified from representative endophytes within identified species, except from the Pantoea eucrina strain AB 295. 16S rDNA phylogenetic analysis of endophytes isolated from inoculated plants revealed distinct families: Xanthobacteraceae, Burkholderiaceae and Enterobacteriaceae; and from noninoculated plants, Rodospirillaceae and Sphingomonadaceae families were also identified. The ‘Champaka’ pineapple plants at fruiting stage associate with endophytes related to α, β and γ Proteobacteria, after plantlets inoculation or not with the diazotrophic bacterium Burkholderia silvatlantica (AB 219a). Most of those endophytes present nifH gene, a characteristic for nitrogen-fixing bacteria.

Identification of the unculturable bacteria Candidatus arthromitus in the intestinal content of trouts using Dot blot and Southern blot techniques

The rainbow trout gastroenteritis (RTGE) syndrome is affecting progressively more sites in various European countries causing significant economical losses for the rainbow trout industries. As Candidatus arthromitus, a not yet culturable bacteria, is the microorganism responsible for the RTGE, molecular methods have been identified as an important tool for the identification of this microorganism. An early, fast and specific detection of C. arthromitus using specific primers and probes, are important goals for the aquaculture sector. In the present study were used: a polymerase chain reaction (PCR), using universal primers annealing the 16S rRNA gene, to obtain amplification products representatives of the microbiota present in the intestinal content of trouts, including C. arthromitus when present; a denaturing gradient gel electrophoresis (DGGE) to produce DNA finger printings corresponding to the microbiota present in the intestinal contents of each trout analysed; a specific DNA probe designed within the 16S rDNA that was used both in Dot blot and Southern blot techniques to detect the unculturable bacterium C. arthromitus. Dot blot applied directly onto DNA extracted from the intestinal contents allowed a fast identification of the samples containing C. arthromitus. Southern blotting allowed the identification of the specific DNA band corresponding to C. arthromitus among the various DNA bands obtained by the utilisation of the universal primers and run in DGGE. Molecular methods such as Dot blot and Southern blot are useful methods for a fast identification of the unculturable C. arthromitus.

Interaction of Epulopiscium Methyl‐Accepting Chemotaxis Proteins with E. Coli CheW and CheA

Epulopiscium sp. Type B is a large (up to 250 μm length) unculturable gram positive bacterium that grows symbiotically in the gut of the surgeonfish Naso tonganus. To further understand the unique biology of this organism, we recombinantly expressed GST fusions of Epulo methyl accepting chemotaxis sensory transducers (MCP) in E.coli. MCPs are responsible for sensing and binding extracellular ligands as well as recognizing pH and temperature changes. In E. Coli the MCP interacts indirectly with CheA (sensory kinase) through CheW (coupling protein). The degree of sensory kinase activity controls the motor complex resulting in taxis towards or away from the stimulus. Aligned with the Tar MCP in E. Coli, Epulo MCP 3 displays a 16.2% identity and 28.8% similarity. Epulopiscium MCP 6 has an 18.6% identity and 33.7% similarity. Epulo CheW was aligned with E. Coli CheW and shows 44.9% conservation and 26.9% identity. Epulo CheA has 30.8% identity and 48% similarity when compared with CheA from E. Coli. As a result Epulo MCPs should theoretically interact with CheA through CheW in an in vitro pull‐down assay. These protein interaction studies lay the foundation for future study of Epulo chemotaxis in E. Coli using motility “swarm” assays. This may help to elucidate the environment in which Epulo lives, and its role in symbiosis with the surgeonfish. This project was funded by the Hartwick College Department of Chemistry.

Evidence of two lineages of the symbiont ‘Candidatus Erwinia dacicola’ in Italian populations of Bactrocera oleae (Rossi) based on 16S rRNA gene sequences

The close association between the olive fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) and bacteria has been known for more than a century. Recently, the presence of a host-specific, hereditary, unculturable symbiotic bacterium, designated 'Candidatus Erwinia dacicola', has been described inside the cephalic organ of the fly, called the oesophageal bulb. In the present study, the 16S rRNA gene sequence variability of 'Ca. E. dacicola' was examined within and between 26 Italian olive fly populations sampled across areas where olive trees occur in the wild and areas where cultivated olive trees have been introduced through history. The bacterial contents of the oesophageal bulbs of 314 olive flies were analysed and a minimum of 781 bp of the 16S rRNA gene was sequenced. The corresponding host fly genotype was assessed by sequencing a 776 bp portion of the mitochondrial genome. Two 'Ca. E. dacicola' haplotypes were found (htA and htB), one being slightly more prevalent than the other (57%). The two haplotypes did not co-exist in the same individuals, as confirmed by cloning. Interestingly, the olive fly populations of the two main Italian islands, Sicily and Sardinia, appeared to be represented exclusively by the htB and htA haplotypes, respectively, while peninsular populations showed both bacterial haplotypes in different proportions. No significant correlation emerged between the two symbiont haplotypes and the 16 host fly haplotypes observed, suggesting evidence for a mixed model of vertical and horizontal transmission of the symbiont during the fly life cycle.

Cloning of the unculturable parasite Pasteuria ramosa and its Daphnia host reveals extreme genotype–genotype interactions

The degree of specificity in host-parasite interactions has important implications for ecology and evolution. Unfortunately, specificity can be difficult to determine when parasites cannot be cultured. In such cases, studies often use isolates of unknown genetic composition, which may lead to an underestimation of specificity. We obtained the first clones of the unculturable bacterium Pasteuria ramosa, a parasite of Daphnia magna. Clonal genotypes of the parasite exhibited much more specific interactions with host genotypes than previous studies using isolates. Clones of P. ramosa infected fewer D. magna genotypes than isolates and host clones were either fully susceptible or fully resistant to the parasite. Our finding enhances our understanding of the evolution of virulence and coevolutionary dynamics in this system. We recommend caution when using P. ramosa isolates as the presence of multiple genotypes may influence the outcome and interpretation of some experiments.

Retinal production from β-carotene by β-carotene 15,15′-dioxygenase from an unculturable marine bacterium

The conversion of beta-carotene to retinal by a recombinant beta-carotene 15,15'-dioxygenase (Blh protein) from an unculturable marine bacterium was optimized in aqueous solution. Toluene was optimal solvent for the dissolution of beta-carotene and the optimal solution for the conversion reaction contained 2.4% (w/v) Tween 20, 0.15 U enzyme/ml, and 350 mg beta-carotene/l. Under these conditions, the enzyme produced 181 mg retinal/l after 20 h. This is the highest reported value for the retinal concentration from beta-carotene.

Selection of indicator bacteria based on screening of 16S rDNA metagenomic library from a two-stage anoxic–oxic bioreactor system degrading azo dyes

Dye degradation has gained attention of late due to indiscriminate disposal from user industries. Enhancing efficiency of biological treatment provides a cheaper alternative vis-à-vis other advanced technologies. Dye molecules are metabolized biologically via anoxic and oxic treatments. In this study, bacterial community surviving on dye effluent working in anoxic–oxic bioreactor was analyzed using 16S rDNA approach. Azo-dye decolorizing and degrading bacterial community was enriched in lab-scale two-stage anoxic–oxic bioreactor. 16S rDNA metagenomic libraries of enriched population were constructed, screened and phylogenetically analyzed separately. Removal of ∼35% COD with complete decolorization was observed in anoxic bioreactor. Process was carried out by uncultured gamma proteobacterium constituting 48% of the total population and 12% clones having homology to Klebsiella. Aromatic amines generated during partial treatment under anoxic bioreactor were treated by aerobic population having 72% unculturable unidentified bacterium and rest of the population consisting of Thauera sp., Pseudoxanthomonas sp., Desulfomicrobium sp., Ottowia sp., Acidovorax sp., and Bacteriodetes bacterium sp.

Prevalence of Mycoplasma suis in slaughter pigs, with correlation of PCR results to hematological findings

Porcine infectious anemia is a well-known disease that occurs worldwide and is caused by the unculturable hemotrophic bacterium Mycoplasma suis. The actual prevalence and impact of M. suis infections, however, remain fairly unknown. This study examined the prevalence of M. suis in post-weaning pigs by employing a quantitative real-time LightCycler PCR. M. suis infections were detected in 164 out of 1176 feeder pigs (20–30 kg; 13.9%) as well as on 79 out of 196 pig farms (40.3%). The comparison of PCR results with microscopic investigation of acridine-orange-stained blood smears revealed a considerably lower sensitivity of the microscopic method: only 35 out of 1176 blood smears were microscopically positive. The microscopic detection of M. suis was shown to be closely linked to the bacterial load in the blood. M. suis infection is associated with significantly decreased hematocrit, erythrocyte counts and hemoglobin concentrations as well as significantly higher bilirubin concentrations. Furthermore, M. suis blood loads were significantly associated with erythrocyte count, hematocrit, hemoglobin, glucose and iron concentrations indicating that high M. suis loads are connected with clinical anemia. In conclusion, this study has shown, that M. suis infections are often under-diagnosed in pig husbandry and can therefore lead to considerable economic profit losses in pig husbandry. Furthermore, our study has shown that the LightCycler PCR could be an appropriate tool for a sufficiently coherent identification of M. suis in latent carrier animals in view of introducing effective treatment and disease control measures.

Acquisition of uncharacterized sequences from Candidatus Liberibacter, an unculturable bacterium, using an improved genomic walking method

An effective PCR-based genomic walking approach is described to discover previously unknown flanking genomic DNA sequences from Candidatus Liberibacter asiaticus, an unculturable, phloem-limited bacterium. Using this technique, 8564 bp of new DNA sequences were obtained from three genomic loci; tufB-secE-nusG-rplKAJL-rpoBC gene cluster, omp gene (outer membrane protein, Omp) and 16/23S rRNA gene in Ca. L. asiaticus. These, together with publicly available Ca. Liberibacter sequences, are clustered into five contigs and two singlets representing 24,477 non-redundant base pairs. BLAST annotation predicts 12 full-length genes, two partial genes and one pseudogene among these sequences. The sequences obtained in this study provide new genome information about Ca. Liberibacter that will facilitate development of new genome-based detection tools. The technique described here can also be employed to acquire new genomic information for other unculturable or fastidious organisms for which available sequences are limited or for filling sequence gaps between known flanking genomic DNA sequences.

Citrus Huanglongbing: Understanding the Vector-Pathogen Interaction for Disease Management.

Introduction Citrus Huanglongbing (HLB), better known as citrus greening disease, is a recently introduced disease to the Americas (Brazil and Florida). Numerous reviews on this important disease are available (2,5,6) and include a recent article by Gottwald et al. published jointly in Plant Health Progress and as an APSnet feature article (8). A PDF file has been developed that provides detailed information about the disease and its vectors. The article by Gottwald et al. addressed the pathogen and its impact, with an emphasis on the history and distribution of the disease, symptoms, the causal organism, its insect vectors, impacts for Brazil and Florida, and potential threats to other citrus producing regions in the Americas. The article extensively dealt with the epidemiology of the disease and the challenges that the temporal increase and spatial spread of the disease have on disease control and management. In this article we will discuss the currently known facts of vector (psyllid) transmission of the citrus greening pathogen and how an understanding of the vector-pathogen interaction affects the current management strategies for HLB. As previously reviewed by Gottwald et al. (8) HLB is putatively caused by an unculturable bacterium belonging to the α proteobacter. Three types of the disease—Asian, African and American (Brazil)— have been described, with causal bacteria tentatively named Candidatus Liberibacter asiaticus (2), Ca. L. africanus (2) and Ca. L. americanus (17,18,19), respectively. As described, the two known insect vectors of HLB are Diaphorina citri and Trioza erytreae, which inhabit different environmental niches, yet can apparently vector both the Asian or African HLB bacteria. In Brazil D. citri has been found to be the vector of the Ca. L. americanus bacterium (28), limited to Brazil thus far, as well as the Ca. L. asiaticus bacterium. In Florida, HLB is caused by Ca. L. asiaticus, which is vectored by D. citri.

소 반추위 메타게놈에서 비배양 세균의 α-amylase 유전자 클로닝

미생물 메타게놈은 특이한 생체촉매의 다양한 원료로 제공된다. 한우의 반추위에서 게놈 DNA를 분리한 후 메타게놈 은행을 구축하고 <TEX>$\alpha$</TEX>-amlylase를 암호화하는 유전자를 클로닝하여 DNA 및 아미노산 서열을 밝히고 생화적 특징을 조사하였다. amyA유전자는 1,893 bp로 631개의 아미노산 잔기를 가진 단백질을 암호화였으며 효소의 분자량은 단백질 전기영동 결과 약 71,000 Da으로 확인되었다. 이 효소를 다른 아밀라제와 비교한 결과 <TEX>$21-59\%$</TEX>의 상동성을 보였다. AnyA는 pH <TEX>$6.40\%$</TEX>에서 최적 활성을 나타내었고, pl값은 5.87이었다. E. coliDH5<TEX>$\alpha$</TEX>에서 발현된 AmyA의 활성은 <TEX>$\Mg^{2+}$</TEX>(20mM), <TEX>$Ca^{2+}$</TEX> (30 mM) 존재 시 그 활성이 증가하였고, <TEX>$Fe^{2+}$</TEX>, <TEX>$Cu^{2+}$</TEX> 존재 시 저해되었다. amyA 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR분석한 결과 해당하는 벤드를 확인할 수 없었다. AmyA는 현재로 배양할 수 없는 반추 미생물에서 온 것으로 추정된다. The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular <TEX>$\alpha$</TEX>-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5<TEX>$\alpha$</TEX> and sequenced. The <TEX>$\alpha$</TEX>-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to <TEX>$59\%$</TEX> sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and <TEX>$40\%$</TEX>. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5<TEX>$\alpha$</TEX> was enhanced in the presence of <TEX>$Mg^{2+}$</TEX> (20 mM) and <TEX>$Ca^{2+}$</TEX> (30 mM) and inhibited in the presence of <TEX>$Fe^{2+}$</TEX> and <TEX>$Cu^{2+}$</TEX>. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

Cloning and expression analysis of Phytoplasma protein translocation genes.

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.

Reassessment of the phylogenetic position of the bacterium associated with Whipple's disease and determination of the 16S-23S ribosomal intergenic spacer sequence.

Whipple's disease is a rare chronic illness associated with an unculturable bacterium that is constantly present in affected tissues. This bacterium was previously characterized at the molecular level by PCR and sequencing of the 16S rRNA gene. On the basis of 1,321 nucleotides of the sequence of its gene coding for 16S rRNA (16S rDNA), a phylogenetic relationship to the actinomycetes was established. In this study, we determined an almost complete 16S rDNA sequence (1,495 nucleotides), the 16S-23S ribosomal intergenic spacer sequence, and 200 nucleotides of the 23S rRNA gene. The 16S rDNA sequence was compared with the large number of actinomycete sequences that have been added to the database since the original study. Phylogenetic analysis revealed a branching position as the deepest branch of the cluster comprising the actinomycetes with group B peptidoglycan between this group and the family Cellulomonadaceae. This provides additional information on the phylogenetic position of this bacterium and some clues as to its characteristics. The spacer region between the 16S and 23S rRNA genes is 294 nucleotides long and does not contain tRNA genes. As has been shown in other instances, the increased variability of the ribosomal intergenic spacer compared with the 16S rRNA gene makes it a potential target for use in the differentiation of strains of the bacterium associated with Whipple's disease.

Development of a technique for detecting antimicrobial susceptibility of unculturable gastric spiral bacterium Gastrospirillum hominis.

Development of a technique for detecting antimicrobial susceptibility of unculturable gastric spiral bacterium Gastrospirillum hominis.