Similarity and dissimilarity of primary structures of some Streptomyces spp . genomes and the Streptomyces globisporus 1912-2 chromosomal DNA

L. V. Polishchuk © 2017 L. V. Polishchuk; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 579.873.71 : 57.063.8] : 575.113.26

With the emergence of data indicating that most Streptomycetes contain up to 30 gene clusters involved in the synthesis of secondary metabolites, it has become clear that their biosynthetic potential is underestimated [3,8,9,11,12].Nowadays many efforts are made to estimate this potential and to get access to the natural products, the biosynthetic pathways of which are encoded in the genomes of the corresponding Streptomyces strains [13][14][15].The accumulation of high quality genome sequencing data will strongly contribute to implementation of this task [3,4,[6][7][8][9][10][11].
The primary structures are identified for more than 1000 chromosomes of Actino bacteria.The genus Streptomyces is now one of the most highly sequenced: 50 finished genomic sequences (Complete genome) and some hundreds of draft assemblies are availab le additionally in the GenBank database of server NCBI in September 2016.
The wild type strain S. globisporus 1912 and its variants synthesize some carotenoids, antitumor antibiotics landomycin E and A and a new regulator of the antibiotics biosynthesis and morphogenesis [7,12,16].The nucleotide sequence of S. globisporus 1912-2 chromosomal DNA was identified [7].
The aim of this research was in silico definition of similarity and dissimilarity of the nucleotide sequences of the S. globisporus 1912-2 chromosome and primary structures of some Streptomycetes genomes.

Materials and Methods
The sequencing of the genomic DNA of strain S. globisporus 1912-2 was carried out in BaseClear B.V., Leiden, Netherlands using described previously procedures [7]

Results and Discussion
Chromosomes of prokaryotes are one doublestrand DNAs, which are considered as bacterial genomes.Genetic information coded by the genomes of microorganisms is necessary to provide their metabolism, growth, development and differentiation.Thousands of housekeeping genes (required for the cell vital acti-vi ty) -for example tRNA-genes, rRNA-gene clusters and many others -are obligatory presented in the genome.The presence of additional optional genes (such as genes of anti biotic resistances, genes of antibiotic biosynthesis clusters and others) were reported for the Streptomycetes genomes.Besides, the co ding sequences (structural and regulatory genes) in the genome are identified in many non-coding DNA regions (more than 10 %) [1,2,17].
Genetic instability is widespread within the genus Streptomyces [8,[17][18][19].This important phenomenon includes a high frequency loss (>0.1 % of plated spores) of certain species specific traits, several of which are mapped to the chromosome [20][21][22].The insertion and deletions of microbial genomes sequences appeared to be important events in the genome evolution.The balance between these processes is not always kept.As a result, an expansion of some genomes and reduction of others happen [22].According to the established knowledge of microbial genetics three major natural strategies can be distinguished in the spontaneous generation of genetic variations in bacteria.These strategies are: (1) small local changes in the nucleotide sequence of genome, (2) intra genomic reshuffling of segments of genomic sequences and (3) the acquisition of DNA sequences from another organism [21].
This investigation was carried out by comparative analysis of the primary structures of genomes of some Streptomyces strains.We picked by in silico analysis of the GenBank database Genome the genomes of 15 Strep tomy ces strains (Complete genome).The criterion for their selection was a similarity grade of nucleotide sequences of their 16S rRNAgenes and the same genes of S. globisporus 1912-2 (AJ132630) [23].Their range of identity exceeded 97.8 %.The Strains S. xiamenensis 318 (95.0 %), S. bingchenggensis BCW-1 (97.0 %) and S. violaceusniger Tu 4113 (97 %) were used as the out-group in this in silico analysis (Fig. 1).
The dendrogramme in Fig. 1, based on the analysis of 16S rRNA fragment sequences, reflects the relationships between some strains Streptomycetes The similarity of 16S rRNA-genes primary structures of S. fuvissimus DSM 40593, S. globisporus C-1027, S. pratensis ATCC 33331 and S. griseus NBRC 13350 to 16S rRNA gene of S. globisporus 1912-2 was within the range 99.8 % -100 %.The trains are members of the S. griseus clade [24].The members of same clade have to share the 16S rRNA gene similarities within the range 97.8 % -100 % [25].We assumed the strain S. globisporus 1912 to be a member of this S.griseus clade (S. albovinaceus subgroup).
We chose 6 genomes of Streptomyces strains for further research.A high similarity of four primary structures of Streptomycetes genomes and nucleotide sequences of chromosomal DNA of S. globisporus 1912-2 was defined in silico (Tabl.1).Strains S. xiamenensis 318 and S. violaceusniger Tu 4113 were used as the out-group.
An existence of difference in the genomic DNAs maps of different strains of the same species is well known.The presence of different assortments of addition genes (or gene clusters) and different location of homologous genes in chromosomes of such strains were indicated.However, there are certain models in the construction of chromosomes: for example, the same order of localization of homologous genes in similar clusters (synteny), the accumulation of obligatory genes in the central part of the chromosome [26].
The genus Streptomyces members served as a source of new natural products and antibiotics in particular for a long time [1,2].Most Streptomycetes contain up to 50 gene clusters involved in the synthesis of secondary metabolites [3,8,10].For example, the genomes  of S. griseus and S. avermitilis contain 34 and 30 secondary metabolite clusters, respectively [18].Some of them are cryptic [9,11].We assume that the strain S. globisporus 1912 has a high potential to produce secondary metabolites because its genome may also contain several biosynthetic gene clusters.As previously reported, some biosynthetic gene clusters were identified in the chromosome of the S. globisporus 1912-2.The clusters of genes for syntheses of antibiotics landomycin E and A, carotenoides and gas vesicle proteins were identified in silico in the genome of the S. globisporus 1912-2 [7,12,27].The biosynthetic lnd-genes cluster was unique and absent in the genomes of all tested Streptomycetes.Only three Streptomycetes cultures are able to produce the landomycins [7].The biosynthetic crt-genes clusters were identified in genomes of S. coelicolor A3(2), S. griseus NBRC13350, S. setonii ISP5395, S. chrysomallus var.carotenoides and some others [28,29].Carotenoge ne sis of S. globisporus 1912-2 does not require induction by illumination or other inducers [12].The biosynthetic gvp-genes clusters were identified in genomes of S. pratensis ATCC 33331, S. xiamenensis 318 and S. violaceusniger Tu 4113 but not in the genome of S. griseus NBRC13350 [27].
The strain S. globisporus C-1027 is a producer of the antitumor antibiotic C-1027 (lidamycin) [5].Alignment of the nucleotide sequences of the S. globisporus 1912-2 and the lidamycin biosynthetic gene cluster (AY048670.1,85163 bp) was done by the program bl2seq: megablast.There was not found any genomic DNA fragment of S. globisporus 1912-2 homologous by the primary structure to the nucleotide sequence of the li-damycin biosynthetic gene cluster of the strain S. globisporus C-1027.
The Strains of the species S. griseus are well known as the producers of antibiotic streptomycin [3].Alignment of the nucleotide sequences of the S. globisporus 1912-2 and the streptomycin biosynthetic cluster of S. griseus N2-3-11 (Y00459.1, 12203 bp) was done.No genomic DNA fragment of S. globisporus 1912-2 homologous by the primary structure to the nucleotide sequence of the str-cluster was identified.
A great deal of homologous fragments spread along the whole length of the genomic sequences of two S. globisporus strains (1912-2 and C-1027) (Fig. 2).A few of non-homologous fragments (59 contigs) in the sequences of genomes of two strains were found.It might be due to the absence of special S. globisporus 1912-2 genes in the S. globisporus C-1027 genome (and vice versa) or incomplete definition of the primary structure of the S. globisporus 1912-2 chromosomal DNA (which can be done by the next sequencing).
82 contigs of S. globisporus 1912-2 had entire non-homologous nucleotide sequences in the genomes of S. griseus NBRC13350 but only 37 contigs were not found in any of them.Total molecular size of these 37 S. globisporus 1912-2 contigs with non-homologous primary structures to the S. globisporus C-1027 and S. griseus NBRC13350 amounted to 7.59 % of total nucleotide sequences of tested strain contigs.The identity of nucleotide sequences of the genomes of S. globisporus C-1027 and S. griseus NBRC13350 was 94 %.In total 70 % of the nucleotide sequence (in fragments) of S. griseus NBRC 13350 genome was homologous in their primary structures to the structure of the S. globisporus C-1027 genome (overlap).Many other S. globisporus 1912-2 contigs had the fragments with non-homologous nucleotide sequences.For example the homologous fragments in an individual contig (N 2 -75588 bp) amounted to only 26 % (S. globisporus C-1027) and 24 % (S. griseus NBRC13350).
Interestingly, there were no homologous nucleotide sequences in the genomes of Streptomyces spp.(database Nucletide collection, taxide: 1883 (genus Streptomyces)) to 7 contigs of S. globisporus 1912-2 (0.001 %).Further in silico studies of the nucleotide sequences of all these non-homologous contigs of the strain S. globisporus 1912-2 are necessary to identify new clusters that determine synthesis of unique metabolites.Localization of the unique lnd-genes in contigs N 3 and N 220 of S. globisporus 1912-2 can be given as an example.

Conclusions
The strain S. globisporus 1912-2 was identified as a member of the S. griseus clade.Many fragments with unique nucleotide sequences were found in chromosomal DNA of this strain (9.35 % of all primary structure of its chromosome).We assumed a big biosynthetic potential of this strain.

Fig. 2 .
Fig. 2. The localization scheme of chromosomal fragments of the S. globisporus 1912-2 (the bottom line) and the S. globisporus C-1027 (the upper line) with homologous primary structures.