Dereplication for biotechnology screening: PyMS analysis and PCR–RFLP–SSCP (PRS) profiling of 16S rRNA genes of marine and terrestrial actinomycetes

The search for exploitable biology is a major task for biotechnology-based industries. In this context, discrimination between previously tested or recovered micro-organisms (dereplication) is imperative, in order to reduce screening costs by sorting large collections of isolates, which are then subjected to further detailed evaluation. Pyrolysis mass spectrometry (PyMS) is a whole-cell fingerprinting technique that enables the rapid and reproducible sorting of micro-organisms, uses small samples and has the advantage of being fully automated. In this study, we compare chemometric fingerprinting with a ribotyping fingerprinting method, in order to investigate the extent to which pyrogroups formed by PyMS analysis relate to genetic diversity, using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS). A mixture of environmental strains of mycolic acid containing actinomycetes was used to mimic the selection of colonies from primary isolation plates. The congruence found between the clusters defined by the chemometric and molecular fingerprinting techniques was very high and demonstrated the effectiveness of PyMS as a rapid sorting and dereplicating procedure for putatively novel strains, criteria that are critical for biotechnological screens. Moreover, PyMS analysis revealed significant variation within pyrogroups that contained strains with the same genotypic (PRS) characteristics, thus emphasising its discriminatory capacity at the infraspecies level.     

Effect of soil bradyrhizobia on the success of soybean inoculant strain CB 1809

Four decades of soybean [Glycine max (L.) Merr.] cultivation in South Africa has resulted in the establishment of populations of bradyrhizobia against which the recently introduced inoculant strain CB 1809 must compete. Serological and DNA fingerprinting methods were used to study the diversity of nodule isolates from soils at Bergville, Koedoeskop and Morgenzon. Dominant serogroups included Bradyrhizobium elkanii serotype 76 at Bergville (67%), Bradyrhizobium japonicum serotype 123 at Morgenzon (81%) and B. japonicum serotype 135 at Koedoeskop (100%). Their origin is unknown as they do not correspond in serotype to strains used in previous inoculants. A small percentage of isolates from Bergville (13%) and Morgenzon (16%) were serologically homologous to strain WB 1 (serotype 31/76), applied for two decades before CB 1809 (serotype 122). Nitrogen-fixing effectiveness of CB 1809 was superior to 60% of the isolates tested from Bergville and Morgenzon, but similar to 73% of the Koedoeskop isolates. Seed and liquid-in-furrow application methods increased CB 1809 nodule occupancy at least three-fold above background levels at Bergville (pH 5.16) and Morgenzon (pH 6.33). Inoculation did not, however, increase CB 1809 nodule occupancy at Koedoeskop (pH 7.76), possibly because alkaline soil conditions favoured the serotype 135 population predominant at this site.     

Rapid characterisation of deep-sea actinomycetes for biotechnology screening programmes

A continual need in natural product discovery is dereplication, that is the ability to exclude previously tested microorganisms from screening programmes. Whole-cell fingerprinting techniques offer an ideal solution to this problem because of their rapidity and reproducibility, dependence on small samples, and automation. One such technique, Curie-point pyrolysis mass spectrometry (PyMS), has been deployed for the characterisation of a unique collection of actinomycetes recovered from Pacific Ocean sediments approximately 2000 to 6500 m below sea level. This paper addresses the question: to what extent are pyrogroups, defined on the basis of PyMS fingerprinting, related to classifications derived from more conventional microbial systematics? A collection of 44 randomly chosen deep-sea rhodococci were coded and subjected to a double-blind PyMS and numerical taxonomic (NT) analysis; the latter sorted the strains into clusters (taxospecies) using large sets of equally weighted phenotypic data. At the end of the experiment the codes were disclosed and the NT classification shown to generate 6 homogeneous clusters corresponding to different deep-sea sites. The matching of these clusters with the resulting pyrogroups was very high with an overall congruence of nearly 98%. Thus, PyMS characterisation is directly ascribable to the phenotypic variation being sought for biotechnology screens. Moreover, the exquisite discriminatory power of PyMS readily revealed infraspecific diversity in these industrially important bacteria.     

Discrimination of higher plant calluses based on embryogenic capacity and taxonomic classification by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum, and has been widely applied to the discrimination of closely related microbial strains. Minimally prepared samples of embryogenic and non-embryogenic calluses derived from various higher plants (sweet potato, morning glory, Korean ginseng, Siberian ginseng, and balloon flower) were subjected to PyMS for spectral fingerprinting. A dendrogram based on the unweighted pair group method, with arithmetic mean of pyrolysis mass spectra, divided the calluses into Siberian ginseng embryogenic callus and the others, which were subsequently divided into embryogenic and non-embryogenic callus groups, regardless of plant species from which the calluses were derived. In the non-embryogenic callus group, the dendrogram was in agreement with the known taxonomy of the plants. These results indicate that PyMS analysis could be applied for discriminating plant calluses based on embryogenic capacity and taxonomic classification.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

The value of pyrolysis mass spectrometry to investigate nosocomial outbreaks caused by Serratia marcescens

Simultaneous outbreaks of S. marcescens infection going on in the Neonatal Intensive Care Unit and the Surgical Department of the same hospital were investigated by pyrolysis mass spectrometry (PyMS). The PyMS analysis of the strains clearly demonstrated that the two outbreaks were caused by different strains. The 14 S. marcescens isolates from the first outbreak were closely related, with the exception of one environmental isolate, which did not harbour the ESBL plasmid, which was present in all other isolates. However, the phage type of all 14 isolates was the same. Among the 9 S. marcescens isolates from the second outbreak, PyMS clearly distinguished 3 that exhibited gentamicin resistance from the remaining 6 gentamicin-susceptible isolates. Phage typing was unhelpful in this case, as none of the isolates were typable. The PyMS typing of nosocomial outbreak strains can reach the level of discrimination approaching that achieved by molecular genetic analysis.     

Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Performance of phaseolus bean rhizobia in soils from the major production sites in the Nile Delta

The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P. vulgaris were studied in silty loam and clayey soils. The experiments were carried out to address the performance of two rhizobia strains (CE3 and Ph. 163] and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt. Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars. The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight. This response was also positive in dry matter and biomass accumulation by the plants. The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph. 163. However, the mixed inoculant strains were not always as good as single strain inoculants. The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting. The nodule occupancy by inoculant strain Ph. 163 in both soils occupied 30-40% and 38-50 of nodules of cultivar Bronco. The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph. 163 in clayey soil. The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar. For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains. Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean.     

Nodulation Efficiency of Legume Inoculation as Determined by Intrinsic Antibiotic Resistance

Patterns of intrinsic resistance and susceptibility to different levels of antibiotics were determined for strains of both fast- and slow-growing rhizobia. These patterns were stable to plant passage when they were used to identify Rhizobium strains in nodule suspensions or nodule isolates. The method of identification by intrinsic resistance and susceptibility patterns was reliable for identifying strains in field nodules when strains were first isolated from the nodules to provide a standard inoculum size and then typed on antibiotic-containing media. Other patterns of resistance were encountered during identification of field isolates; these patterns may have resulted from acquired resistance to certain antibiotics or from mixed infections of the nodules. The occurrence of resistance patterns identical to those of inoculant strains among native strains was directly related to the size of the soil population. High strain recovery was associated directly with high rates of inoculation.     

Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.     

Changes in electrophoretic profiles of lipopolysaccharides from competitive strains of Bradyrhizobium spp. induced by soybean roots

The soybean Bradyrhizobium strain Semia 566 was introduced into soils of the Cerrados (Brazilian edaphic savannas) in the late 1960s. Then, nodule occupancy by this strain was not greater than 2%. Recently, this serogroup has been found in approximately 60% of nodules formed on soybeans cultivated in the Cerrados, replacing the strains 29W and Semia 587, the Brazilian commercial inoculant for soybean. Although some re-isolates of Semia 566, adapted to Cerrado soils, were more competitive than 29W under both field and aseptic conditions, they did not differ from the parental strain, based on their lipopolysaccharide (LPS) electrophoretic profile. The only exceptions were the isolates 4A-5 and CPAC-15 which presented an additional polysaccharidic band of low molecular weight or higher mobility. On the other hand, this same band may be induced and intensified in LPS extracted from competitive strains (29W, 220, 204, 370, 372, 516, 122 and CPAC-15) after bacterial contact with soybean roots for 6 or 12 h. In addition, a 29W Tn5 mutant with a phenotype of delayed nodulation showed a delayed induction of this polysaccharidic band. Conversely, the LPS of less competitive strains was not modified or showed a weak intensification of this band. As this band alteration was correlated with the concurrent elevation of dominance in nodules, it may be suggested that LPS plays a role in the competitive ability of rhizobia strains for nodulation.     

Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity,Adaptations, and Host Specificity

The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizobia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX repetitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sampling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqingense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates'' performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations.     

Novel rhodococci and other mycolate actinomycetes from the deep sea

A large number of mycolate actinomycetes have been recovered from deep-sea sediments in the NW Pacific Ocean using selective isolation methods. The isolates were putatively assigned to the genus Rhodococcus on the basis of colony characteristics and mycolic acid profiles. The diversity among these isolates and their relationship to type strains of Rhodococcus and other mycolate taxa were assessed by Curie point pyrolysis mass spectrometry (PyMS). Three major (A, C, D) and two minor (B, E) groups were defined by PyMS. Cluster A was a large group of isolates recovered from sediment in the Izu Bonin Trench (2679 m); Cluster C comprised isolates from both the Izu Bonin Trench (6390 and 6499 m) and from the Japan Trench (4418, 6048 and 6455 m). These Cluster C isolates showed close similarity to Dietzia maris and this was subsequently confirmed using molecular methods. Cluster D contained isolates recovered from a sediment taken from a depth of 1168m in Sagami Bay and were identified as members of the terrestrial species Rhodococcus luteus. Clusters B and E had close affinities with members of the genera Gordonia and Mycobacterium. The presence of Thermoactinomyces in certain of the deep-sea sediments studied was indicative of the movement of terrestrial material into the ocean depths.16S ribosomal RNA gene sequence analyses produced excellent definition of most genera of the mycolata, and indicated that the among the deep sea isolates (1) were novel species of Corynebacterium, Gordonia and Mycobacterium, and (2) a Sea of Japan isolate the phylogenetic depth of which suggests the possibility of a new genus. Polyphasic taxonomic analysis revealed considerable diversity among the deep sea rhodococci and evidence for recently diverged species or DNA groups.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Methods To Alter the Recovery and Nodule Location of Bradyrhizobium japonicum Inoculant Strains on Field-Grown Soybeans

Three strains of Bradyrhizobium japonicum, I17, 110, and 61A76, were evaluated for their ability to form nodules on field-grown soybeans in soil with a highly competitive indigenous B. japonicum population. The predominant indigenous strain, 0336, in the field site used was unlike the more common isolates from Midwestern soils which belong to the 123 or 138 serogroups. This strain persisted in the soil for at least 30 years without any soybean crops. The three inoculant strains differed in their ability to compete with indigenous strains for nodule formation. Four different inoculation treatments were tested in three adjacent fields. When the amount of inoculum was increased, a higher proportion of nodules contained the inoculant strain. The most competitive inoculant strain was I17, a recent field isolate. Strain 61A76 was better than 110. There was no difference in recovery of the inoculant strains on the Hodgson or Corsoy soybean cultivars, nor was there a difference in recovery of the inoculant strains during the growing season. The vertical distribution of nodules containing the inoculant strains was affected by the method of adding the inoculant to the soil. Inoculant added to the seed furrow produced nodules mainly in the top region of the soybean root. Inoculant tilled into the soil produced nodules primarily in the bottom part of the root. The nodules that were produced in the bottom part of the root are younger and may contribute significant amounts of fixed nitrogen to the soybean during seed formation.     

Identification and Discrimination of Oral Asaccharolytic Eubacterium spp. by Pyrolysis Mass Spectrometry and Artificial Neural Networks

Curie-point pyrolysis mass spectra were obtained from 29 oral asaccharolytic Eubacterium strains and 6 abscess isolates previously identified as Peptostreptococcus heliotrinreducens. Pyrolysis mass spectrometry (PyMS) with cluster analysis was able to clarify the taxonomic position of this group of organisms. Artificial neural networks (ANNs) were then trained by supervised learning (with the back-propagation algorithm) to recognize the strains from their pyrolysis mass spectra; all Eubacterium strains were correctly identified, and the abscess isolates were identified as un-named Eubacterium taxon C₂ and were distinct from the type strain of P. heliotrinreducens. These results demonstrate that the combination of PyMS and ANNs provides a rapid and accurate identification technique.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.