LETTER TO THE EDITOR
Data on ancient microorganisms causes skepticism
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12414
The critiques by Weyrich et al. (2014) and any other colleague on our paper analyzing the evolutionary history of genes involved in quorum sensing are certainly welcome, although the former seem to be based on several erroneous assumptions. The skepticism associated with any publication dealing with apparently ancient bacteria is completely understood because of the possible problems associated with contamination. However, and in contradiction to what Weyrich et al. mention, we did not fail to address previous publications; we are very well aware of them. We carry out these experiments as carefully as any microbiologist would: granted, contamination may still occur, but our own procedures indicate directly or indirectly that there is no contamination by extant bacteria or remaining DNA. Arguments for or against demonstration of the resilience of bacteria and biological molecules will go on, as it would be impossible to answer every single one of the critiques by doing yet further experiments. As responsible scientists, we can only present data obtained from carefully designed experiments, present the data to peer review, and let the readers draw their own conclusions. We were very aware that our results would be met with skepticism, given the belief in the apparent impossibility of the survival of microorganisms over a certain period of time. A similar response occurred when other colleagues reported the viable but nonculturable state. However, as we dig deeper into this area, we ourselves are surprised at the resilience of microorganisms. There are several publications focusing on the impossibility of isolating microorganisms from amber. Weyrich et al. (2014) indicate that contamination might have occurred during the original isolation of the microorganisms. However, precisely because of the skepticism, later isolations, including those bacteria used in our article, were not obtained at a university research laboratory, but rather at an industrial facility, earmarked exclusively for ancient microorganisms and where extensive controls were conducted routinely – as needed to satisfy investors. As part of a further control, independent consultants were brought in to validate the isolations. We did not include this detailed information in our manuscript, although we do indicate that proper controls were used throughout the experiments. We do not think a disclaimer type of statement should be included with every manuscript. In fact, this very same point was brought up during the peer-review process. FEMS Microbiol Lett && (2014) 1–2
Weyrich et al. (2014) also reference papers indicating the inability to obtain DNA from unfossilized amber. We have not published data on naked DNA in amber, as it would seem that this macromolecule would be rapidly degraded under these conditions. Although we have also attempted to isolate DNA directly from amber, we are still in the process of developing a method to do so, as this is not an easy task. We may have to include a DNAdesorbing compound to extract DNA from these matrices (Alvarez et al., 1998). Bacteria, on the other hand, actually protect their DNA from degradation, and given the low water activity in amber, bacteria may be rapidly dehydrated, thus preventing DNase activity. Survival experiments would have to be conducted to eliminate this skepticism. We should note that the inability to isolate DNA or bacteria from a specific matrix does not automatically translate into their absence from this matrix. Greenblatt et al. (2004) resuscitated the bacteria for 15 min, thus making it possible to analyze the rDNA and determine terminal restriction patterns (TRFs) that correlated with those of the isolates after more prolonged culture, thus bypassing culture conditions and eliminating the possibility of contamination. Addressing the apparent contradiction to which Weyrich et al. refer in the identification of the isolates, all we can say is that we are not working with the bacteria isolated in 1995. Also, our colleagues seem to misinterpret their own reference where the authors did in fact not conclude that the 1995 Bacillus pumilus was a contaminant. Actually, the authors indicated they could not disprove the results. We have several amber isolates now as part of our current strain collection. At that time, it was thought that mainly spore-forming bacteria could survive and that only these structures could withstand taphonomic conditions for such a long time. However, it has long been demonstrated that even nonsporulating, Gramnegative bacteria have evolved strategies for long-term survival (Amy et al., 1983; Greenblatt et al., 2004). In addition, studies characterizing the process of resuscitation in dormant microorganisms are ever more frequent (Kaprelyants & Kell 1993; Koltunov et al., 2010). Fortunately, we know more about bacterial survival now than we did back in 1995. In regard to the strain identification, we are aware that our sequences could only be compared to those currently available in the database. Identification ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
2
at the species level using universal primers for the 16S ribosomal RNA gene should be carefully considered, as is stated in our publication. As for the inconsistency in gene phylogenies, there seems to be confusion on the argument addressed by Weyrich et al. (2014). We concluded that all ancient luxS sequences clustered closely together with Firmicutes because it is simply correct, whether or not in disagreement with the 16S rRNA phylogeny. The discrepancy on gene phylogenies exists, and it was a surprising finding, which we believe is best explained by horizontal gene transfer (HGT) from Bacillus species. The fact that not all ancient luxS sequences fall basal to modern luxS sequences only fails to support our hypothesis of HGT if diversification had occurred more recently than the age of our amber samples. If diversification happened over 25–40 million years ago, there is no reason for the ancient sequences to be all basal or for the modern sequences to be all nested. Regarding the possible contamination, we are also completing other types of ancient DNA work, which has clearly indicated the low probability of cross-contamination, as Weyrich et al. mention (Santiago-Rodrıguez et al., 2013). We are painfully aware of many preconceptions regarding the resilience of bacteria and in fact any other biological molecules, and as such, we are very careful to include controls in each and every one of our experiments. As experienced microbiologists, we practice a discipline where contamination is always possible, but we have devised means to discriminate between contaminants and relevant organisms. Without this knowledge and the developed skills, clinical microbiology would have been impossible. As we look into ancient DNA, we are surprised by the results. We are also sequencing ancient DNA, and this is an indirect way of finding contamination. We are aware that in our area of research skepticism is not easily eliminated, or in many cases even addressed, and we welcome well-placed skepticism. We do not, however, disregard the published literature, nor do we try to confirm previous findings with new data, as mentioned by our colleagues. In addition, we do not see the need to write disclaimers to please those on one side, or the other of this debate each time, we send a manuscript for review. Doing so would do a disservice to the peer-review system.
Letter to the Editor
Amy PS, Pauling C & Morita RY. (1983) Starvation-survival processes of a marine Vibrio. Appl Environ Microbiol 45: 1041–1048. Greenblatt CL, Baum J, Klein BY, Nachshon S, Koltunov V & Cano RJ. (2004) Micrococcus luteus – survival in amber. Microb Ecol 48: 120–127. Kaprelyants AS & Kell DB. (1993) Dormancy in stationaryphase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59: 3187. Koltunov V, Greenblatt CL, Goncharenko AV, Demina GR, Klein BY, Young M & Kaprelyants AS. (2010) Structural changes and cellular location of resuscitation-promoting factor in environmental isolates of Micrococcus luteus. Microb Ecol 59: 296–310. Santiago-Rodriguez TM, Narganes-Storde YM, Chanlatte L, Crespo-Torres E, Toranzos GA, Jimenez-Flores R, Hamrick A & Cano RJ. (2013) Microbial communities in pre-Columbian coprolites. PLoS One 8: e65191. Weyrich LS, Llamas B & Cooper A (2014) Reply to SantiagoRodriguez et al: Was luxS really isolated from 25- to 40million-year-old bacteria? FEMS Microbiol Lett DOI: 10. 1111/1574-6968.12415.
Raul J. Cano Department of Biology, California Polytechnic State University, San Luis Obispo, CA, USA Gary A. Toranzos Department of Biology, University of Puerto Rico, San Juan, Puerto Rico Tasha M. Santiago-Rodriguez Department of Pathology, University of California, San Diego, CA, USA Ana R. Patricio Centre for Ecology & Conservation, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, UK Jessica I. Rivera Department of Biology, University of Puerto Rico, San Juan, Puerto Rico E-mail: [email protected]
References Alvarez AJ, Khanna M, Toranzos GA & Stotzky G. (1998) Amplification of DNA bound on clay minerals. Mol Ecol, 7: 775–778.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett && (2014) 1–2
Data on ancient microorganisms causes skepticism
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12414
The critiques by Weyrich et al. (2014) and any other colleague on our paper analyzing the evolutionary history of genes involved in quorum sensing are certainly welcome, although the former seem to be based on several erroneous assumptions. The skepticism associated with any publication dealing with apparently ancient bacteria is completely understood because of the possible problems associated with contamination. However, and in contradiction to what Weyrich et al. mention, we did not fail to address previous publications; we are very well aware of them. We carry out these experiments as carefully as any microbiologist would: granted, contamination may still occur, but our own procedures indicate directly or indirectly that there is no contamination by extant bacteria or remaining DNA. Arguments for or against demonstration of the resilience of bacteria and biological molecules will go on, as it would be impossible to answer every single one of the critiques by doing yet further experiments. As responsible scientists, we can only present data obtained from carefully designed experiments, present the data to peer review, and let the readers draw their own conclusions. We were very aware that our results would be met with skepticism, given the belief in the apparent impossibility of the survival of microorganisms over a certain period of time. A similar response occurred when other colleagues reported the viable but nonculturable state. However, as we dig deeper into this area, we ourselves are surprised at the resilience of microorganisms. There are several publications focusing on the impossibility of isolating microorganisms from amber. Weyrich et al. (2014) indicate that contamination might have occurred during the original isolation of the microorganisms. However, precisely because of the skepticism, later isolations, including those bacteria used in our article, were not obtained at a university research laboratory, but rather at an industrial facility, earmarked exclusively for ancient microorganisms and where extensive controls were conducted routinely – as needed to satisfy investors. As part of a further control, independent consultants were brought in to validate the isolations. We did not include this detailed information in our manuscript, although we do indicate that proper controls were used throughout the experiments. We do not think a disclaimer type of statement should be included with every manuscript. In fact, this very same point was brought up during the peer-review process. FEMS Microbiol Lett && (2014) 1–2
Weyrich et al. (2014) also reference papers indicating the inability to obtain DNA from unfossilized amber. We have not published data on naked DNA in amber, as it would seem that this macromolecule would be rapidly degraded under these conditions. Although we have also attempted to isolate DNA directly from amber, we are still in the process of developing a method to do so, as this is not an easy task. We may have to include a DNAdesorbing compound to extract DNA from these matrices (Alvarez et al., 1998). Bacteria, on the other hand, actually protect their DNA from degradation, and given the low water activity in amber, bacteria may be rapidly dehydrated, thus preventing DNase activity. Survival experiments would have to be conducted to eliminate this skepticism. We should note that the inability to isolate DNA or bacteria from a specific matrix does not automatically translate into their absence from this matrix. Greenblatt et al. (2004) resuscitated the bacteria for 15 min, thus making it possible to analyze the rDNA and determine terminal restriction patterns (TRFs) that correlated with those of the isolates after more prolonged culture, thus bypassing culture conditions and eliminating the possibility of contamination. Addressing the apparent contradiction to which Weyrich et al. refer in the identification of the isolates, all we can say is that we are not working with the bacteria isolated in 1995. Also, our colleagues seem to misinterpret their own reference where the authors did in fact not conclude that the 1995 Bacillus pumilus was a contaminant. Actually, the authors indicated they could not disprove the results. We have several amber isolates now as part of our current strain collection. At that time, it was thought that mainly spore-forming bacteria could survive and that only these structures could withstand taphonomic conditions for such a long time. However, it has long been demonstrated that even nonsporulating, Gramnegative bacteria have evolved strategies for long-term survival (Amy et al., 1983; Greenblatt et al., 2004). In addition, studies characterizing the process of resuscitation in dormant microorganisms are ever more frequent (Kaprelyants & Kell 1993; Koltunov et al., 2010). Fortunately, we know more about bacterial survival now than we did back in 1995. In regard to the strain identification, we are aware that our sequences could only be compared to those currently available in the database. Identification ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
2
at the species level using universal primers for the 16S ribosomal RNA gene should be carefully considered, as is stated in our publication. As for the inconsistency in gene phylogenies, there seems to be confusion on the argument addressed by Weyrich et al. (2014). We concluded that all ancient luxS sequences clustered closely together with Firmicutes because it is simply correct, whether or not in disagreement with the 16S rRNA phylogeny. The discrepancy on gene phylogenies exists, and it was a surprising finding, which we believe is best explained by horizontal gene transfer (HGT) from Bacillus species. The fact that not all ancient luxS sequences fall basal to modern luxS sequences only fails to support our hypothesis of HGT if diversification had occurred more recently than the age of our amber samples. If diversification happened over 25–40 million years ago, there is no reason for the ancient sequences to be all basal or for the modern sequences to be all nested. Regarding the possible contamination, we are also completing other types of ancient DNA work, which has clearly indicated the low probability of cross-contamination, as Weyrich et al. mention (Santiago-Rodrıguez et al., 2013). We are painfully aware of many preconceptions regarding the resilience of bacteria and in fact any other biological molecules, and as such, we are very careful to include controls in each and every one of our experiments. As experienced microbiologists, we practice a discipline where contamination is always possible, but we have devised means to discriminate between contaminants and relevant organisms. Without this knowledge and the developed skills, clinical microbiology would have been impossible. As we look into ancient DNA, we are surprised by the results. We are also sequencing ancient DNA, and this is an indirect way of finding contamination. We are aware that in our area of research skepticism is not easily eliminated, or in many cases even addressed, and we welcome well-placed skepticism. We do not, however, disregard the published literature, nor do we try to confirm previous findings with new data, as mentioned by our colleagues. In addition, we do not see the need to write disclaimers to please those on one side, or the other of this debate each time, we send a manuscript for review. Doing so would do a disservice to the peer-review system.
Letter to the Editor
Amy PS, Pauling C & Morita RY. (1983) Starvation-survival processes of a marine Vibrio. Appl Environ Microbiol 45: 1041–1048. Greenblatt CL, Baum J, Klein BY, Nachshon S, Koltunov V & Cano RJ. (2004) Micrococcus luteus – survival in amber. Microb Ecol 48: 120–127. Kaprelyants AS & Kell DB. (1993) Dormancy in stationaryphase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59: 3187. Koltunov V, Greenblatt CL, Goncharenko AV, Demina GR, Klein BY, Young M & Kaprelyants AS. (2010) Structural changes and cellular location of resuscitation-promoting factor in environmental isolates of Micrococcus luteus. Microb Ecol 59: 296–310. Santiago-Rodriguez TM, Narganes-Storde YM, Chanlatte L, Crespo-Torres E, Toranzos GA, Jimenez-Flores R, Hamrick A & Cano RJ. (2013) Microbial communities in pre-Columbian coprolites. PLoS One 8: e65191. Weyrich LS, Llamas B & Cooper A (2014) Reply to SantiagoRodriguez et al: Was luxS really isolated from 25- to 40million-year-old bacteria? FEMS Microbiol Lett DOI: 10. 1111/1574-6968.12415.
Raul J. Cano Department of Biology, California Polytechnic State University, San Luis Obispo, CA, USA Gary A. Toranzos Department of Biology, University of Puerto Rico, San Juan, Puerto Rico Tasha M. Santiago-Rodriguez Department of Pathology, University of California, San Diego, CA, USA Ana R. Patricio Centre for Ecology & Conservation, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, UK Jessica I. Rivera Department of Biology, University of Puerto Rico, San Juan, Puerto Rico E-mail: [email protected]
References Alvarez AJ, Khanna M, Toranzos GA & Stotzky G. (1998) Amplification of DNA bound on clay minerals. Mol Ecol, 7: 775–778.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett && (2014) 1–2
