Directed evolution of three-finger toxin to produce serine protease inhibitors.

Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules. Previously, we demonstrated that functional regulatory molecules against interleukin-6 receptor (IL-6R) could be obtained by directed evolution of the three-finger toxin (3F) scaffold. In the present study, trypsin was selected as a target for directed evolution to further explore the potential use of the 3F cDNA display library. After seven rounds of selection, the DNA sequences converged. The recombinant proteins produced by the selected candidates had inhibitory activity against trypsin (Ki of 33-450 nM). Three of the six groups had Ki values that were comparable to bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor. Two of the candidates also had inhibitory effects against chymotrypsin and kallikrein. This study suggests that 3F protein is suitable for the preparation of high-diversity libraries that can be utilized to obtain protease inhibitors. In addition to our previous successful targeting of IL-6R, the technique developed in our studies may have wide applications in the generation of regulatory molecules for targets of interest, such as receptors, enzymes for research, diagnostic applications, and therapeutic uses.

A sero-epidemiological analysis of Coxiella burnetii infection and its risk factors in livestock from Addis Ababa, Adama, and Modjo abattoirs and pastoral areas of Oromia, Ethiopia.

BACKGROUND: Coxiella burnetii is causing infections in both humans and animals, resulting in Q fever and Coxiellosis, respectively. Information on the occurrence of C. burnetii infection is scarce in Ethiopia. This study estimated the sero-prevalence of C. burnetii infection and associated risk factors in four common livestock species from Addis Ababa, Adama, and Modjo abattoirs and pastoral areas of Oromia, Ethiopia. RESULTS/PRINCIPAL FINDINGS: Sera samples were analyzed for the presence of anti-C. burnetii antibodies using an indirect Enzyme Linked Immunosorbent Assay kit. Out of the 4140 serum samples tested, 777 (18.77%; 95% CI: 17.59, 19.99) were found positive for C. burnetii. The sero-prevalence estimate was 27.17% at Addis Ababa abattoir, 19.41% at Adama abattoir, 19.13% at Modjo abattoir and 12.1% in animals tested from pastoral areas. Sera analysis at the animal species level showed that cattle exhibited the lowest sero-prevalence estimate (11.83%; 95% CI, 10.27-13.53%), while the highest was observed in camels (28.39%; 95% CI, 25.16-31.80%). The sero-prevalence estimate was 21.34% (95% CI, 18.86-23.99%) in goats and 20.17% (95% CI, 17.49-23.07%) in sheep. The results of multivariable logistic regression analysis showed that species, age, sex of animals and tick infestation were important risk factors for C. burnetii infection. The odds of infection were 3.22 times higher in camels and almost twice as high in goats and sheep compared to cattle. Adult animals were infected more likely (OR = 3.23) than young ones. Interestingly, a significant difference was observed in the sero-prevalence of infection between animals that were infested with ticks (OR = 16.32) and those which were tick-free. CONCLUSION: This study provides valuable insights into the sero-epidemiology of C. burnetii infection in four common livestock species at major abattoirs and pastoral areas of Ethiopia. The findings highlight the need for further studies and implementing surveillance and biosecurity measures to prevent the spread of the disease in both humans and livestock to safeguard the economical and public health aspects.

Role of messenger RNA-ribosome complex in complementary DNA display.

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips.

Optically amended biosynthesized crystalline copper-doped ZnO for enhanced antibacterial activity.

The emergence and re-emergence of antibiotic-resistant bacteria is a potential threat to treating infectious diseases. This study employed a nanometer-scale green synthesis using an extract of Solanum incanum leaves to obtain nanoparticles (NPs) and nanocomposites (NCs) possessing antibacterial properties. The FESEM-EDS elemental mapping analysis proved the novelty of the green synthesis approach in synthesizing a copper-doped ZnO NCs with good dopant distribution. The crystallinity and ZnO bandgap were adjusted by extrinsic copper doping in the ZnO lattice. The optical property adjustments from 3.04 to 2.97 eV for indirect Kubelka-Munk functions were confirmed from DRS-UV-vis analysis. The dopant inclusion in the host lattice was also confirmed by the angle shift on the XRD pattern analysis relative to single ZnO. In addition to doping, the XRD pattern analysis also showed the development of CuO crystals. The lattice fringe values from HRTEM analysis confirmed the existence of both CuO and ZnO crystals with local heterojunctions. Doping and heterojunctions have crucial values in charge transfer and visible light harvesting behaviour, as proved by the PL analysis. The synergistic effects of the doped NCs showed greater antibacterial activity against both Gram-positive and Gram-negative bacteria as a result of more ROS generation through the bacteria-cell-catalyst interaction and release of metal ions. The antioxidant potential of the doped NCs was found to be higher than that of single NPs, using the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay and is expected to impart protective effects to the host cells by scavenging destructive free radicals. Thus, the overall analysis leads to the conclusion that the potentiality of synthesized materials has a future outlook for biological applications, especially in the development of antimicrobials to combat antibiotic-resistant bacteria and microbes.

Phytochemical Composition, Antioxidant, Antimicrobial, Antibiofilm, and Antiquorum Sensing Potential of Methanol Extract and Essential Oil from Acanthus polystachyus Delile (Acanthaceae).

The evolution of microbes in response to conventional antimicrobials leads to antimicrobial resistance (AMR) and multidrug resistance (MDR), and it is a global threat to public health. Natural products are possible solutions to this massive challenge. In this study, the potential of Acanthus polystachyus extracts was investigated for phytochemical composition and biological properties as antimicrobials. Gas chromatography-mass spectra (GC-MS) analysis of methanol extract (ME) and essential oil (EO) detected 79 and 20 compounds, respectively. The major compounds identified in ME and their abundance were ß-sitosterol acetate (16.06%), cholest-5-en-3-yl (9Z)-9-octadecenoate (9.54%), 1-dodecanol (7.57%), (S)-(E)-(-)-4-acetoxy-1-phenyl-2-dodecen-1-one (6.03%), neophytadiene (5.7%), (E)-2-nonadecene (3.9%), hexanol-4-D2 (2.92%), and decane (2.4%). Most compounds have known bioactive functions. In EO, the major compounds were stearyl alcohol (25.38%); cis-9-tetradecenoic acid, isobutyl ester (22.95%); butyl 9-tetradecenoate (10.62%); 11,13-dimethyl-12-tetradecen-1-ol acetate (10.14%); ginsenol (3.48%); and diisooctyl phthalate (2.54%). All compounds are known to be bioactive. The antioxidant activity of ME and EO ranged from 48.3 to 84.2% radical scavenging activity (RSA) and 45.6 to 82% RSA, respectively, with dose dependency. The disc diffusion assay for the antimicrobial activity of ME revealed high inhibition against Acenetobacter baumannii (130.2%), Pseudomonas aeruginosa (100.3%), and Staphylococcus aureus (87.7%). The MIC, MBC/MFC, and MBIC values for ME were 0.5-1.0, 2-4, and 0.5-1.0 mg/mL and for EO were 0.31-0.62, 1.25-2.5, and 0.31-0.62 µL/mL, respectively, indicating inhibition potential as well as inhibition of biofilm formation. The tolerance test values indicated bactericidal activity against most strains and bacteriostatic/fungistatic activity against A. baumannii, E. faecalis, and C. albicans. The antiquorum sensing activity of ME achieved by pyocyanin inhibition assay on P. aeruginosa showed a 51.6% inhibition at 500 µg/mL. These results suggest that ME and EO derived from A. polystachyus leaves are potent, valuable, cost-effective antioxidants and antimicrobials. Both extracts may effectively combat pathogenic and resistant microbes.

In silico analysis of promoter regions to identify regulatory elements in TetR family transcriptional regulatory genes of Mycobacterium colombiense CECT 3035.

BACKGROUND: Mycobacterium colombiense is an acid-fast, non-motile, rod-shaped mycobacterium confirmed to cause respiratory disease and disseminated infection in immune-compromised patients, and lymphadenopathy in immune-competent children. It has virulence mechanisms that allow them to adapt, survive, replicate, and produce diseases in the host. To tackle the diseases caused by M. colombiense, understanding of the regulation mechanisms of its genes is important. This paper, therefore, analyzes transcription start sites, promoter regions, motifs, transcription factors, and CpG islands in TetR family transcriptional regulatory (TFTR) genes of M. colombiense CECT 3035 using neural network promoter prediction, MEME, TOMTOM algorithms, and evolutionary analysis with the help of MEGA-X. RESULTS: The analysis of 22 protein coding TFTR genes of M. colombiense CECT 3035 showed that 86.36% and 13.64% of the gene sequences had one and two TSSs, respectively. Using MEME, we identified five motifs (MTF1, MTF2, MTF3, MTF4, and MTF5) and MTF1 was revealed as the common promoter motif for 100% TFTR genes of M. colombiense CECT 3035 which may serve as binding site for transcription factors that shared a minimum homology of 95.45%. MTF1 was compared to the registered prokaryotic motifs and found to match with 15 of them. MTF1 serves as the binding site mainly for AraC, LexA, and Bacterial histone-like protein families. Other protein families such as MATP, RR, σ-70 factor, TetR, LytTR, LuxR, and NAP also appear to be the binding candidates for MTF1. These families are known to have functions in virulence mechanisms, metabolism, quorum sensing, cell division, and antibiotic resistance. Furthermore, it was found that TFTR genes of M. colombiense CECT 3035 have many CpG islands with several fragments in their CpG islands. Molecular evolutionary genetic analysis showed close relationship among the genes. CONCLUSION: We believe these findings will provide a better understanding of the regulation of TFTR genes in M. colombiense CECT 3035 involved in vital processes such as cell division, pathogenesis, and drug resistance and are likely to provide insights for drug development important to tackle the diseases caused by this mycobacterium. We believe this is the first report of in silico analyses of the transcriptional regulation of M. colombiense TFTR genes.

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery.

cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

cDNA Display of Disulfide-Containing Peptide Library and In Vitro Evolution.

Directed in vitro evolution (IVE) is now a widely applied technology to obtain molecules that have designed new features of one's demands. We describe here experimental procedures for a cDNA display IVE to select peptide aptamers from a scaffold-based random peptide library. A three-finger (3-F) peptide library is exemplified, which has been shown its pluripotency to various target molecules. Peptide scaffolds including 3-F are refined through evolution, and they are mostly stabilized by disulfide bridges. To utilize such disulfide-containing protein library in IVE, we optimized the translation and folding conditions. Co-translational folding assisted by protein disulfide isomerase was found to have better efficiency than posttranslational refolding in the IVE. Linker is also a key element to make a tight genotype-phenotype linkage. Here, we introduced a whole procedure of IVE to use a newly designed puromycin linker, which was synthesized by a novel branching strategy. The improved linker enabled rapid and highly efficient ligation of mRNA and synthesis of protein fusions.

Molecular design guided by a local map of sequence space: DNA aptamers that inhibit cathepsin E.

It has been strongly demanded by a number of people to elevate activities of molecules of a particular function. Currently, there is no general guide available for this purpose. Here we present a novel approach for this; a local sequence space map-directed method for exploring molecules of a higher activity. This approach exploits the knowledge of a local sequence space so far established and obtains the shape of sequence space (map) by intra- and extrapolating the known landscape, which was drawn through the principal coordinates analysis. In this method, we successfully obtained 16 DNA aptamers of cathepsin E (CE) inhibitory activity that have comparable or higher activities than the ancestral ones on which the designed molecules were based. Some of them had a 30% higher activity than the previously reported top one (SFR-6-3). This high efficiency in obtaining functional molecules (16 out of 21 newly designed ones) is by no means usual because most of molecules generated at random are known to have no function, showing the effectiveness of the map-based approach. The selected molecules were confirmed to have the i-motif structure, consistent to the fact that they have a C-rich sequence and their CE-inhibitory activities were measured at an acidic pH, both of which are favorable for the i-motif. This structure of CE-inhibitory aptamers was inferred to contribute to the structural stability but not to the function itself directly.

Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution.

We developed a method to produce, identify and analyze DNA fragments for the purpose of taxonomic classification. Genome profiling (GP) is a strategy that identifies genomic DNA fragments common to closely related species without prior knowledge of the DNA sequence. Random PCR, one of the key technologies of GP, is used to produce fragments and may be used even when there are mutations at the priming site. These fragments can then be distinguished based on the information of mobility and melting pattern when subjected to temperature gradient gel electrophoresis (TGGE). Corresponding fragments among several species, designated as commonly conserved genetic fragments (CCGFs), likely have the same genetic origin or correspond to the same gene. The criteria for identification of CCGFs has been defined and presented here. To assess this prediction, some of the fragments were sequenced and were confirmed to be CCGFs. We show that genome profiles bearing evolutionarily conserved CCGFs can be used to classify organisms and trace evolutionary pathways, among other profound applications.

A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.

BACKGROUND: Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. RESULTS: We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. CONCLUSIONS: This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

Development of systemic in vitro evolution and its application to generation of peptide-aptamer-based inhibitors of cathepsin E.

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.

A solution for universal classification of species based on genomic DNA.

Traditionally, organisms have been classified on the basis of their phenotype. Recently, genotype-based classification has become possible through the development of sequencing technology. However, it is still difficult to apply sequencing approaches to the analysis of a large number of species due to the cost and labor. In most biological fields, the analysis of complex systems comprising various species has become an important theme, demanding an effective method for handling a vast number of species. In this paper, we have demonstrated, using plants, fish, and insects, that genome profiling, a compact technology for genome analysis, can classify organisms universally. Surprisingly, in all three of the domains of organisms tested, the phylogenetic trees generated from the phenotype topologically matched completely those generated from the genotype. Furthermore, a single probe was sufficient for the genome profiling, thereby demonstrating that this methodology is universal and compact.

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.

Genome analysis technologies: towards species identification by genotype.

Traditional identification of species has been based on phenotypic traits, although it is clear that, theoretically, genotype-based classification is more accurate. This is especially the case for microorganisms which possess less identifiable traits and are more easily influenced by environment. Therefore, technology that allows identification of species based on genotype is highly desirable. Whole genome sequencing can provide a sufficient amount of information and can be determinative for this purpose but is very impractical for routine use. Thus, a competent technology is needed that allows a reproducible reduction in the amount of information required about a whole genome, while still providing sufficiently accurate identification. It is almost imperative for such a technology to be of a high cost-performance and of easy handling. Universality and portability are also strongly desired. Based on these criteria, the current state of genome analysis technologies are reviewed. Among various methodologies discussed here, amplified fragment length polymorphism (AFLP), genome profiling (GP) and microarrays are the subject of particular attention. As species identification is a base for most fields of biology including microbiology, ecology, epidemiology and for various biotechnologies, it is of paramount importance to establish a more efficient, easily handled and more objective methodology, in parallel with conventional phenotype-based methodologies. GP is currently considered to have the most optimal nature for identification of species since it can reproducibly reduce a huge amount of genome information to a manageable size by way of random polymerase chain reaction and can extract a sufficient amount of information for species identification from the DNA fragments thus profiled by temperature gradient gel electrophoresis. The potential ability of DNA microarrays for this purpose is also discussed and promises much for the future.

A database for the provisional identification of species using only genotypes: web-based genome profiling.

BACKGROUND: For a long time one could not imagine being able to identify species on the basis of genotype only as there were no technological means to do so. But conventional phenotype-based identification requires much effort and a high level of skill, making it almost impossible to analyze a huge number of organisms, as, for example, in microbe-related biological disciplines. Comparative analysis of 16S rRNA has been changing the situation, however. We report here an approach that will allow rapid and accurate phylogenetic comparison of any unknown strain to all known type strains, enabling tentative assignments of strains to species. The approach is based on two main technologies: genome profiling and Internet-based databases. RESULTS: A complete procedure for provisional identification of species using only their genomes is presented, using random polymerase chain reaction, temperature-gradient gel electrophoresis, image processing to generate 'species-identification dots' (spiddos) and data processing. A database website for this purpose was also constructed and operated successfully. The protocol was standardized to make the system reproducible and reliable. The overall methodology thus established has remarkable aspects in that it enables non-experts to obtain an initial species identification without a lot of effort and is self-developing; that is, species can be determined more definitively as the database is used more and accumulates more genome profiles. CONCLUSIONS: We have devised a methodology that enables provisional identification of species on the basis of their genotypes only. It is most useful for microbe-related disciplines as they face the most serious difficulties in species identification.