Phylogenetic interrelations between serological variants of Bacillus thuringiensis

Aim. B. thur3ng3ens3s (Bt) are gram-positive spore-forming aerobic or facultative anaerobic bacteria able to form during sporulation species specific crystal-like inclusions of protein nature, consisting of particular thermolabile d-endotoxins. Serological Bt variants produce different entomotoxins; their synthesis in many respects depends on the conditions of cultivation. There was accumulated a vast information on the entomotoxins, their origin, synthesis, structure, toxic properties and mechanisms of action on insects. These bacteria are dominating in the microbiomethods of pest control in plants and animals. There are more than 70 serovariants of Bt selectively specific to the definite groups of host insects. However, the description of new variants not always looks justified considering the phylogenetic systematization based on phenotype signs. Methods. A comparative phylogenetic analysis of the Bt intraspecific interrelations was performed on the basis of the cloned 16S rRNA genes of entomopathogenic bacteria BtÍ1, BtÍ10, BtÍ14. Results. The phylogenetically homogeneous lines were investigated – a homology of 16S rRNA of the strains 1 and 10 ranged from 90,0 to 94,0 %; no distinct genetic isolation among the strains of 14 and 10 serovars was revealed. Conclusions. The comparison of nucleotides sequences of 16S rRNA has shown the existence of strains polymorphism within the group of entomopathogens ÂtÍ1, ÂtÍ10, ÂtÍ14, connected with their entomocide activity.

In tro duc tion.The use of ge nome or its frag ments in the sys tem ati za tion of mi cro or gan isms is of spe cial im por tance due to its rel a tive con ser va tism com pared to any other fea tures; be sides, ge netic dif fer ences better re flect evo lu tion ary formed re la tions be tween mi cro or gan isms, which makes them a good foun da tion for mod ern clas si fi ca tion of bac te ria.
The ap pli ca tion of the meth ods of eval u at ing ge netic sim i lar ity (com par a tive DNA anal y sis and anal y sis of amino acid se quences of pro teins) al lows ex tend ing the pos si bil i ties of re search ers in cre at ing mo lec u lar evo lu tion ary sys tem at ics and phy log eny of bac te ria.The phy log eny of a group of or gan isms is tra di tion ally pre sented in the form of hi er ar chic "tree", re flect ing pos si ble evo lu tion ary re la tions [1,2].The ap pli ca tion of mo lec u lar and ge netic ap proaches in mi cro bi ol ogy re sulted in the es tab lish ment of sev eral di rec tions in the sys tem ati za tion of bac te ria, in par tic u lar, the for ma tion of phylo gen etic sys tem ati za tion of bac te ria, iden ti fi ca tion of strains us ing phylo gen etic and phenotypic in for ma tion, and de tec tion of mi cro or gan isms in the en vi ron ment with out their cul ti va tion.It al lows con trol ling the pu rity of biopreparations, based on ac tive, tech no log i cal strains, and mon i tor ing cul tures in cor re spond ing con di tions.There is a need for foun da tion of fur ther study ing on genotype of cultures, promising for biotechnologies, as well as for the search and detection of genes, predetermining high biological activity.
B. thuringiensis (Bt) are gram-pos i tive spore-form ing aer o bic or fac ul ta tive an aer o bic bac te ria able to form spe cies spe cific crys tal-like in clu sions of pro tein na ture, con sist ing of par tic u lar thermolabile d-endo tox ins, dur ing sporulation.

Serological
Bt vari ants pro duce dif fer ent entomotoxins; in many re spects their syn the sis de pends on the con di tions of cul ti va tion.There was ac cu mu lated a vast in for ma tion on the entomotoxins, their or i gin, syn the sis, struc ture, toxic prop er ties, and mech a nisms of their ac tion on in sects.
There fore, Bt are dom i nat ing in the microbiomethods of pest con trol in plants and an i mals.There are more than 70 serovariants of Bt, se lec tively spe cific to the def i nite groups of host in sects.How ever, the de scrip tion of new vari ants does not al ways seem jus ti fied con sid er ing the phylo gen etic sys tem ati za tion based on phenotype features.
The cur rent work is aimed at phylo gen etic anal y sis of var i ous serological vari ants of entomopathogenic Bt bac te ria, iso lated from nat u ral pop u la tions of in sects, which is based on the study on poly mor phism of nu cle o tide se quences of 16S rRNA genes.
Ma te ri als and Meth ods.The dif fer ent strains of entomopathogenic Bt bac te ria, iso lated from nat u ral pop u la tions of in sects, were used in the work, namely, exotoxinogenic Bt var.thuringiensis (BtH 1 ), Bt var.darmstadiensis (BtH 10 ), Bt var.israelensis (BtH 14 ) (Table ).
The DNA of entomopathogenic bac te ria was ex tracted us ing the method, de scribed in [4].Af ter elec tro pho retic sep a ra tion in 1% agarose gel the sam ples of DNA ob tained were vi su ally de tected as de scribed in [5].Poly mer ase chain re ac tion (PCR) for 16S rRNA was per formed ac cord ing to the stan dard pro ce dure in the am pli fier BioRad My Cycler, us ing oligonucleotides SSU-642-F HAATHYGTGCCAGCAGC and SSU-1445-R GTCRTCCYDCCTTCCTC.Af ter ex trac tion from 1% agarose gel the prod ucts of am pli fi ca tion were used for clon ing in vec tor pAL-TA [6,7].Re sults and Dis cus sion.The phylo gen etic anal y sis is based on the com par i son of genes or pro teins, sim i lar in struc ture or func tions, start ing with their pri mary se quences.The anal y sis of 16S rRNA proved to be a more ef fi cient mo lec u lar and tax o nomic means in eval u at ing ge netic va ri ety and re la tions be tween bac te rial kinds (serotypes) of Bt entomopathogens, com pared to ap proaches, based on phenotypic data (e.g.serotyping by H-an ti gen).How ever, it is note wor thy that re con struc tions of phylo gen etic re la tions be tween strains, con ducted on the ba sis of dif fer ent mo lec u lar data are not al ways in good agree ment with "mor pho log i cal" clas si fi ca tion or among them selves.The main rea sons of di ver gence of mor pho log i cal and mo lec u lar re con struc tions are based on the fact that only a small part of ge netic in for ma tion, en closed in the ge nome of the or gan ism, is re vealed on the mor pho log i cal level.The com bi na tion of data of the study on 16S rRNA may be used to ob tain vi su ally ev i dent fin ger prints and to confirm species membership of strains and phylogenetic interrelations inside the specific species.
The dendrogram, built on the ba sis of data ob tained (Fig ure ), dem on strates ex ist ing in ter re la tions be tween Bt strains of the 1 st , 10 th , and 14 th serotypes.Al most all Bt types may be di vided into groups; they dem on strate phylo gen etic sim i lar ity, how ever, there are some ev i dent iso lated vari ants.The to pol ogy of di vid ing ba cilli types on the dendrogram tes ti fies to the ex is tence of three main clus ters, cor re spond ing to six ge no types.The com par i son of Bt strains showed that ge net i cally-wise there are three closely-re lated ge no types of the first clus ter with the sim i lar ity level of 90.0-93.5% (BtH 1 (thuringiensis), dem on strat ing entomocide ac tiv ity re gard ing Lepidoptera, and BtH 10 (darmstadiensis), ac tive against Coleoptera.Thus, ge netic homology of entomopathogenic strains is also confirmed by the spectrum of their entomocide activity.
The BtH 14 (israelensis) strains, af fect ing the larvea of Culicidae, gnat and herbovorous mos qui toes (Cricotopus silvestris, Licoriella fucorum, etc.) are in the sec ond and the third clus ters with the small est sim i lar ity level of 16S rRNA (less than 70% homology).It is note wor thy that the unique ef fect of israelensis bac te ria on the lar vae of dip ter ans, in clud ing larvicidal ef fect, is solely re lated to the specificities of their crys tal line endotoxin.
Bt strains, pro duc ing var i ous sets of entomotoxins, may vary greatly in their bi o log i cal ac tiv ity re gard ing sus cep ti ble in sects or show con sid er able sim i lar i ties, e.g.some mosquitocide strains, etc.The entomopathogens of the same type are known for their fre quently ob served sim i lar set of patho ge nic ity fac tors as well as for sim i lar bio chem i cal and ge nome char ac ter is tics.How ever, the de scen dants of dif fer ent clones of the same type are spe cific for some vari abil ity both on genotype and phenotype levels.
There fore, the mo lec u lar and ge netic anal y sis of BtH 1 , BtH 10 , BtH 14 , based on the com par i son of nu cle o tide se quences of 16S rRNA, re vealed ex is tence of strain poly mor phism within the group of entomopathogens.The dif fer ences, de fined by mor pho log i cal, phys i o log i cal and bio chem i cal, and serological char ac ter is tics of Bt strains, al low mak ing an in terim con clu sion on their be long ing to at least two phylo gen eti cally ho mo ge neous lines.The sim i lar ity level of 16S rRNA of strains of the 1 st and 10 th serotypes is 90.0-94.0%.There was no dis tinct phylo gen etic iso la tion re vealed be tween the strains of the 10 th and 14 th serotypes.The re li abil ity of phylo gen etic re sults will de pend greatly on the com bi na tion of var i ous data which will give a pos si bil ity in fu ture to in flu ence the con gru ence of phylo gen etic trees, sta tis ti cal support, and definition of their branches, eliminating the systematic error.