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Reply to “Isolating Viable Ancient Bacteria: What You Put In Is What You Get Out” A. Goncharov,a,b,c S. Grigorjev,e A. Karaseva,a V. Kolodzhieva,a,b D. Azarov,b Y. Akhremenko,e L. Tarasova,e A. Tikhonov,d A. Masharskiy,c L. Zueva,b A. Suvorova,c FSBSI Institute of Experimental Medicine, Saint Petersburg, Russiaa; North-West State Medical University named after I. I. Mechnikov, Saint Petersburg, Russiab; Saint Petersburg State University, Saint Petersburg, Russiac; Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russiad; M. K. Ammosov North-Eastern Federal University, Yakutsk, Russiae
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nvestigation and description of viable bacteria isolated from ancient permafrost are an essential part of modern paleomicrobiology (1, 2), despite the difficulties with evidence of autochthony of isolates. In our work, we have described the draft genome of an unusual strain of Enterococcus faecium isolated from a paleontological specimen (3). The possibility of contamination with modern microbiota, of course, was considered by us. In this regard, we have used the term “putative ancient” for determination of the isolates. However, we cannot agree with the opinion that multilocus sequence typing (MLST) allelic profile investigation is sufficient for the estimation of absolute age of bacterial strains, as suggested by Eisenhofer et al. (4). It is clear that housekeeping genes coding for proteins with important functions for bacterial survival are considered for use in MLST schemes because they are stable with respect to rapid genetic modifications. Regarding Enterococcus faecium, it has been assumed by Galloway-Peña et al. (5) that divergence of this species into the hospital and commensal phylogenetic clusters occurred no earlier than 300,000 years ago. However, nucleotide differences between sequence types representing the hospital and commensal clusters are relatively small. For example, we have found by MLST profile comparison between ST17 (belonging to the hospital clade) and ST32 (commensal clade) that four of the seven MLST genes are 100% intact and the other three have only 11 single nucleotide polymorphisms (SNPs) in total (95, C ¡ T; 128, C ¡ T; 314, T ¡ C; and 542, T ¡ G in the internal fragment of atpA; 171, T ¡ G; 204, G ¡ A; 327, T ¡ C; 348, C ¡ T; 387, C ¡ A; and 435, G ¡ A in ddl; and 115, C ¡ T in purK). This fact demonstrates that the molecular clock for different housekeeping genes artificially chosen for MLST analysis might go with different speeds depending on the evolutional pressure on the bacteria. As we believe that the rate of molecular evolution is low, we do not expect significant accumulation of mutations in these genes over the last 28,500 years. Besides, the isolation of ST32 among taxonomically distant species of animals and the wide distribution of ST32 in geographically distant regions may indicate that this sequence type is relatively ancient.
July/August 2016 Volume 4 Issue 4 e00734-16
Irrespective of the age of the E. faecium 58m strain, the presence of genetic regions in its genome that do not have any significant similarity with the genomes of modern prokaryotes (for example, genomic regions from positions 13577 to 23686 in contig 46 and positions 22200 to 23200 in contig 43) is of considerable interest for further study. REFERENCES 1. Steven B, Léveillé R, Pollard WH, Whyte LG. 2006. Microbial ecology and biodiversity in permafrost. Extremophiles 10:259 –267. http://dx.doi.org/ 10.1007/s00792-006-0506-3. 2. Gilichinsky DA, Wilson GS, Friedmann EI, McKay CP, Sletten RS, Rivkina EM, Vishnivetskaya TA, Erokhina LG, Ivanushkina NE, Kochkina GA, Shcherbakova VA, Soina VS, Spirina EV, Vorobyova EA, Fyodorov-Davydov DG, Hallet B, Ozerskaya SM, Sorokovikov VA, Laurinavichyus KS, Shatilovich AV, Chanton JP, Ostroumov VE, Tiedje JM. 2007. Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology. Astrobiology 7:275–311. http:// dx.doi.org/10.1089/ast.2006.0012. 3. Goncharov A, Grigorjev S, Karaseva A, Kolodzhieva V, Azarov D, Akhremenko Y, Tarasova L, Tikhonov A, Masharskiy A, Zueva L, Suvorov A. 2016. Draft genome sequence of Enterococcus faecium strain 58m, isolated from intestinal tract content of a woolly mammoth, Mammuthus primigenius. Genome Announc 4(1):e01706-15. http://dx.doi.org/10.1128/ genomeA.01706-15. 4. Eisenhofer R, Cooper A, Weyrich LS. 2016. Isolating viable ancient bacteria: what you put in is what you get out. Genome Announc 4(4): e00712-16. http://dx.doi.org/10.1128/genomeA.00712-16. 5. Galloway-Peña J, Roh JH, Latorre M, Qin X, Murray BE. 2012. Genomic and SNP analyses demonstrate a distant separation of the hospital and community-associated clades of Enterococcus faecium. PLoS One 7:e30187. http://dx.doi.org/10.1371/journal.pone.0030187. Published 25 August 2016 Citation Goncharov A, Grigorjev S, Karaseva A, Kolodzhieva V, Azarov D, Akhremenko Y, Tarasova L, Tikhonov A, Masharskiy A, Zueva L, Suvorov A. 2016. Reply to “Isolating viable ancient bacteria: what you put in is what you get out.” Genome Announc 4(4):e00734-16. doi:10.1128/genomeA.00734-16. Copyright © 2016 Goncharov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to A. Karaseva, [email protected]. This is a response to a letter by Eisenhofer et al. (doi:10.1128/genomeA.00712-16).
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Reply to “Isolating Viable Ancient Bacteria: What You Put In Is What You Get Out” A. Goncharov,a,b,c S. Grigorjev,e A. Karaseva,a V. Kolodzhieva,a,b D. Azarov,b Y. Akhremenko,e L. Tarasova,e A. Tikhonov,d A. Masharskiy,c L. Zueva,b A. Suvorova,c FSBSI Institute of Experimental Medicine, Saint Petersburg, Russiaa; North-West State Medical University named after I. I. Mechnikov, Saint Petersburg, Russiab; Saint Petersburg State University, Saint Petersburg, Russiac; Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russiad; M. K. Ammosov North-Eastern Federal University, Yakutsk, Russiae
I
nvestigation and description of viable bacteria isolated from ancient permafrost are an essential part of modern paleomicrobiology (1, 2), despite the difficulties with evidence of autochthony of isolates. In our work, we have described the draft genome of an unusual strain of Enterococcus faecium isolated from a paleontological specimen (3). The possibility of contamination with modern microbiota, of course, was considered by us. In this regard, we have used the term “putative ancient” for determination of the isolates. However, we cannot agree with the opinion that multilocus sequence typing (MLST) allelic profile investigation is sufficient for the estimation of absolute age of bacterial strains, as suggested by Eisenhofer et al. (4). It is clear that housekeeping genes coding for proteins with important functions for bacterial survival are considered for use in MLST schemes because they are stable with respect to rapid genetic modifications. Regarding Enterococcus faecium, it has been assumed by Galloway-Peña et al. (5) that divergence of this species into the hospital and commensal phylogenetic clusters occurred no earlier than 300,000 years ago. However, nucleotide differences between sequence types representing the hospital and commensal clusters are relatively small. For example, we have found by MLST profile comparison between ST17 (belonging to the hospital clade) and ST32 (commensal clade) that four of the seven MLST genes are 100% intact and the other three have only 11 single nucleotide polymorphisms (SNPs) in total (95, C ¡ T; 128, C ¡ T; 314, T ¡ C; and 542, T ¡ G in the internal fragment of atpA; 171, T ¡ G; 204, G ¡ A; 327, T ¡ C; 348, C ¡ T; 387, C ¡ A; and 435, G ¡ A in ddl; and 115, C ¡ T in purK). This fact demonstrates that the molecular clock for different housekeeping genes artificially chosen for MLST analysis might go with different speeds depending on the evolutional pressure on the bacteria. As we believe that the rate of molecular evolution is low, we do not expect significant accumulation of mutations in these genes over the last 28,500 years. Besides, the isolation of ST32 among taxonomically distant species of animals and the wide distribution of ST32 in geographically distant regions may indicate that this sequence type is relatively ancient.
July/August 2016 Volume 4 Issue 4 e00734-16
Irrespective of the age of the E. faecium 58m strain, the presence of genetic regions in its genome that do not have any significant similarity with the genomes of modern prokaryotes (for example, genomic regions from positions 13577 to 23686 in contig 46 and positions 22200 to 23200 in contig 43) is of considerable interest for further study. REFERENCES 1. Steven B, Léveillé R, Pollard WH, Whyte LG. 2006. Microbial ecology and biodiversity in permafrost. Extremophiles 10:259 –267. http://dx.doi.org/ 10.1007/s00792-006-0506-3. 2. Gilichinsky DA, Wilson GS, Friedmann EI, McKay CP, Sletten RS, Rivkina EM, Vishnivetskaya TA, Erokhina LG, Ivanushkina NE, Kochkina GA, Shcherbakova VA, Soina VS, Spirina EV, Vorobyova EA, Fyodorov-Davydov DG, Hallet B, Ozerskaya SM, Sorokovikov VA, Laurinavichyus KS, Shatilovich AV, Chanton JP, Ostroumov VE, Tiedje JM. 2007. Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology. Astrobiology 7:275–311. http:// dx.doi.org/10.1089/ast.2006.0012. 3. Goncharov A, Grigorjev S, Karaseva A, Kolodzhieva V, Azarov D, Akhremenko Y, Tarasova L, Tikhonov A, Masharskiy A, Zueva L, Suvorov A. 2016. Draft genome sequence of Enterococcus faecium strain 58m, isolated from intestinal tract content of a woolly mammoth, Mammuthus primigenius. Genome Announc 4(1):e01706-15. http://dx.doi.org/10.1128/ genomeA.01706-15. 4. Eisenhofer R, Cooper A, Weyrich LS. 2016. Isolating viable ancient bacteria: what you put in is what you get out. Genome Announc 4(4): e00712-16. http://dx.doi.org/10.1128/genomeA.00712-16. 5. Galloway-Peña J, Roh JH, Latorre M, Qin X, Murray BE. 2012. Genomic and SNP analyses demonstrate a distant separation of the hospital and community-associated clades of Enterococcus faecium. PLoS One 7:e30187. http://dx.doi.org/10.1371/journal.pone.0030187. Published 25 August 2016 Citation Goncharov A, Grigorjev S, Karaseva A, Kolodzhieva V, Azarov D, Akhremenko Y, Tarasova L, Tikhonov A, Masharskiy A, Zueva L, Suvorov A. 2016. Reply to “Isolating viable ancient bacteria: what you put in is what you get out.” Genome Announc 4(4):e00734-16. doi:10.1128/genomeA.00734-16. Copyright © 2016 Goncharov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to A. Karaseva, [email protected]. This is a response to a letter by Eisenhofer et al. (doi:10.1128/genomeA.00712-16).
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