Comments by D. A. Klein concerning NEON planning document "Integrating microbial biology into the National Ecological Observatory Network," White paper prepared by G. M. King et al.,
© Donald A. Klein 2008 25 July and 16 Sept., 2008
Ladies and Gentlemen: Please find my comments concerning this proposed program, planned to integrate microbial biology into the National Ecological Observatory Network (NEON). With the wide range of topics and statements that have been included in this proposed program, it has been possible for me only to provide responses to a limited number of the statements; hopefully this will be satisfactory. Hopefully these comments will be of value in the further development of this program. I have provided the major headings (Roman Numerals) from the planning document in bold face, to assure that my comments can be located in the document. I also have provided a set of recommendations for your consideration.
DAK RESPONSE #1: In relation to the term “microbial communities,” used in this initial statement, it might be of value to start with definitions of the word community. Webster’s Third New Unabridged Dictionary” G. and C. Merriam Co., page 460, provides the following definition: “an interacting population of different kinds of individuals (or species) constituting a society or association or simply an aggregation of mutually related individuals in a given location.” In terms of general ecology, a solid definition is given in the classic ecology text by E. P. Odum, ( "Fundamentals of Ecology,” 3rd edition, W. B. Saunders, 1971), on page 140: "A biotic community is any assemblage of populations living in a prescribed area or physical habitat." Ron Atlas and Richard Bartha, in the 4th edition of their text, “Microbial Ecology, Fundamentals and Applications, l998, Benjamin Cummins, Menlo Park, CA., provide the following definition in their Glossary, page 662: “ Highest biological unit in an ecological hierarchy composed of interacting populations.” Words such as living, interacting and active are critical parts of defining a microbial community.
A similar thought has been provided at the beginning of the Society for General Microbiology l987 symposium 41 on microbial communities, edited by M. Fletcher, T. R. G. Gray and J. G. Jones. In the introduction it is noted that “The ultimate aim of the study of microbial ecology is to understand the interactions within microbial communities and between them and their environments” - this quotation, emphasizes interactions as being a characteristic of microbes that are part of a community. A recent ASM “Microbe” paper that contains my views on this important subject appeared in the Dec., 2007 issue of “Microbe,” the ASM monthly magazine. This paper is attached as a web link if it might be of interest. http://www.asm.org/microbe/index.asp?bid=54999 . In this article, the viewpoint is provided that that to be part of a microbial community, an organism must be active. This is not an original thought. This definition of organisms needing to be active and interacting to be a part of a community is supported by the etymology of this word and by a wide range of more ecologically relevant sources.
Based on these definitions of the word community and more specifically of a microbial community, it is critical to be able to establish that the microbes being studied actually are active in situ. The principles for the conduct of this type of study have been available in the literature for almost 90 years. Fungi were of concern to many of these early investigators. Selman Waksman (1916, Science N.S., 44: 320) noted that: “the question is not how many numbers and types of fungi can be found in a soil, but what organisms lead an active life in the soil.” In a similar vein, Harley and Waid (1955, Trans Brit Myc. Soc. 38: 104 ) noted that “one of the first requirements for an ecological study of soil organisms is that the methods must distinguish between organisms which are vegetatively active and playing a part in the soil in the soil processes and those which exist in dormant or inactive form as spores and other propagules.” M. W. Dick, in a study of aquatic microorganisms, noted (Dick, M. W., l976, pp. 513-542 in Jones, E. B. G., Rec. Adv. Aq. Microbiol., John Wiley) that “ the mere isolation of a species from a particular site provides no evidence of its origin or potential activity in relation to that site” (in the present context, this quote by M. W. Dick could be paraphrased to include sequences – “the mere isolation of a sequence from a particular site provides no evidence of its origin or potential activity in relation to that site.”) In addition, the classic study by Warcup (Trans. Brit. Myc Soc., l957, 490: 237) should be reviewed. In this beautiful study, fungi recovered from a soil by direct observation and micromanipulation were compared with those recovered by the use of culture media and plating. The active genera, recovered directly from the soil as active hyphae, largely were sterile species, while those fungi that developed on laboratory growth media were derived from inactive spores of sexual fungi that were playing no role in the microbial community. These studies provide an important principle: it is critical to be certain that organisms being studied are actually active in the natural environment.
A possibly appropriate analogy is that the people buried in the cemetery are not part of the community. They are there, but they make no contributions. In like manner, we have lots of microbes in natural microbial assemblages that may be inactive/uncultured for a variety of reasons – they are there but not doing anything. They also simply may be dead. They are literally present, but in the "microbial cemetery" – they should not be included in studies that are called metagenomic analyses of “microbial communities.”
DAK RESPONSE #2: Bulk extraction-based molecular sequence recovery from soils, waters, etc., does not provide specific information concerning microbial communities or microbial diversity. The sequences may or may not have been derived from a microbe, and the microbe may not have been active in situ. Based on these considerations, as discussed by Klein (2007, “Microbe,” 2 (12): 591), cited earlier, the presence of a recovered sequence cannot be directly assumed to represent a microbe that is part of a community. This is an outmoded approach that cannot be justified as specifically relating a sequence to a phenotype. From my review of the available literature, this has not been shown to be the case when using bulk extractions of nucleic acids. This is merely a notion-based approach that was suggested to be of value in the mid-1980 era. Case et al., (Appl. Environ. Microbiol. 73:278-288, 2007) used this word “notion” to describe historical aspects of the development of the 16S rRNA approach, and why it was adopted so rapidly. These authors noted that "the notion that rRNA genes could identify an organism by reconstructing its phylogeny, along with the possibility of storing sequences in data bases, resulted in the rapid adoption of the 16S rRNA gene by microbiologists. This gene has established itself as the 'gold standard,' not only in bacterial phylogeny but also in microbial ecology studies." It is of interest that the word "notion," as used by these authors, means (1) a belief or opinion, (2) a mental image or representation; an idea or conception, (3) a fanciful impulse: a whim (thefreedictionary.com). Howard Gest ( l999, “ Bacterial classification and taxonomy: a ‘primer” for the new millennium,” “Microbiology Today” 26: 70-72 ) also has used the word “notion” to describe the development of this entire field of research.
This field of molecular microbial ecology, based on the bulk extraction-based recovery of nucleic acids, has been lauded by Carl Woese (C. Woese, 1994, PNAS 91: 1601-1603) and and Jan Sapp (J. Sapp, 2003, “Genesis: the evolution of biology,” Oxford Univ. Press, New York, page 228) without a critical consideration of the source of the nucleic acids that are being used, based on these bulk extraction-based procedures, based on my reading of these articles.
DAK RESPONSE #3: It would be more valuable to provide a broader background related to microbial ecology, the study of microbes and their interactions with their biotic and abiotic environments. Merely being trained to extract nucleic acids and then to spend most effort (I assume) on the processing of sequences is of limited intellectual value. In the era of epigenetics and gene-environmental interactions, the suggested molecular sequence-based program of NEON no longer can be considered to be cutting-edge science.
I. What is the NEON?
II. What is the value of microbial biology for NEON?
STATEMENT: “Recent and rapid progress made in studying microbial diversity, particularly at the community scale.”
DAK RESPONSE #4:
Diversity is a difficult and demanding word. One can be involved with functional diversity, that of organisms that are active in situ, for which a phenotype can be observed, interacting with the environment, or possibly potential diversity, organisms that are present but that show no detectable in situ activity. If an organism is not active in situ, it is not part of functional “microbial diversity.” The problem is that people have mixed these different definitions of the term “microbial diversity;” by the use of in silico growth techniques, microbial phenotypes and activity often can be detected (the recent book by K. Zengler, 2008, “Accessing uncultivated microorganisms: from the environment to organisms and genomes and back,” ASM Press, Washington DC), is of interest, but there is no assurance that the expression of this in silico- expressed growth is actually being expressed in situ. Microbial diversity, in the context of microbial ecology as it occurs in soils, waters, intestinal contents, etc., ultimately, involves the phenotype of organisms that are active in situ. Without a consideration of epigenetics, particularly the role of DNA methylation in the course of gene-environment interactions (the “ecotype” might be able to be invoked at this point), merely having a sequence is not sufficient.
The lack of a consideration of epigenetics in this proposal, in my view, is a major conceptual deficiency. To make the assumption that a genome ───→ organism ───→ active member of a microbial community, particularly in the context of 16S rRNA-based approaches, is unjustifiable in this post-genomic age. This faulty reductionist assumption reflects a classic, 20th century one gene-one product genetics Weltanschauung that still permeates too much of contemporary biology. This problem of a lack of consideration of gene-environment interactions (including epigenetics) has been described by Kenna Shaw et al., in a recent article that appeared in Genetic and Engineering News ( 15 May, 2008, 28 (10) – the weblink for this article is http://www.genengnews.com/articles/chitem.aspx?aid=2471 . They discuss student essays concerned with genetics in the following way: “a significant portion of essays describe genetic determinism, with no role described for gene-environmental interactions.” To disregard gene-environment interactions, as also appears to have been done in this molecular sequence-based proposed NEON study, simply is not acceptable in terms of modern biology (at least this is my view).
III. What is the value of NEON for microbial biology
C. Key variables to observe and essential data products:
STATEMENT: “Measures of diversity and measures of activity or function.”
SEE DAK RESPONSE #4 for main points
F. STATEMENT: provide unprecedented opportunities to understand responses of organisms and communities to environmental changes over time and space at the gene and genomic levels via analysis of massive sequence databases.”
DAK RESPONSE #5: Massive sequence databases, if one does not know the source of the sequences, simply is pseudoscience. DAK has developed a “white paper” of basic statements concerning molecular microbial ecology that are available on the CSU website: “Basic statements concerning molecular microbial ecology,” dated 15 Jan., 2007; rev. 18 June 2007 that might be of interest: http://www.cvmbs.colostate.edu/mip/people/faculty/klein/klein-white-one.htm A major problem is that one has no idea of the source of nucleic acids that are being studied in these “metagenomic” approaches, when bulk extraction-based approaches are used to recover nucleic acids from environmental samples such as soils, waters, intestinal contents, etc., as suggested to be carried out in terms of integrating microbial biology into the NEON program.
As noted in the table 1 of the Dec., 2007 feature article from ASM “Microbe,” cited above, nucleic acids can be recovered from a wide range of pools that are present in natural microbial assemblages. The only nucleic acid source that represents the microbial community is the microbes that are active in situ.
Also present in natural microbial assemblages are microbes that are not active and that will never be active, and from which nucleic acids can be extracted for inclusion in these “metagenomic” analyses. They just may have been transported to the environment being studied through aeolian movement of soil from the Gobi Desert or the Southern Sahara region (Griffin, D. W. et al., 2001, Global Change and Human Health 2: 20), or they may be derived from an animal that has just dropped to the bottom of the ocean, as examples, resulting in the movement of microbes to environments where their functioning is precluded, due to non-permissive abiotic conditions in the environment they are moved to. As an additional example, thermophiles and hyperthermophiles, and their free nucleic acids, can be transported to cooler lower-temperature environments ( as when water flows from thermally heated areas to lower reaches of a stream or river) where these thermophiles and hyperthermophiles find them in cooler waters where the water temperature is below their low temperature growth minimum. In addition, the recovered nucleic acids can be derived from free nucleic acids (Paul, J. H., W. H. Jeffrey. J. P. Cannon, 1990, Appl. Environ. Microbiol. 56: 2957-2962), and different extraction procedures can lead to variations in the sequences that are recovered (Frostegård, A., et al., l999, Appl. Environ. Microbiol. 65: 5409-5420 ; Martin-Laurent, F., et al., 2001, Appl. Environ. Microbiol. 67: 2354- 2359; Steffan, R. J., J. Gøksyr, A. K. Bej, R. M. Atlas, 1988, Appl. Environ. Microbiol. 54: 2908-2915; Steinberger and Holden, 2005, Appl. Environ. Microbiol., 71: 5404) that can be amplified by PCR ( Alvarez, et al., l998, Molec. Ecol., 7:775; Steinberger and Holden, cited above).
As a last important point, there are many “procedural pitfalls in PCR-based rRNA analyses;” this phrase is used in the title of the article by F. von Wintzingerode et al., (FEMS Microbiology Reviews, 1997, 21: 213-229). As noted in the abstract for this paper, concerns include “sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.” Additional aspects of PCR-related problems have been noted in the literature (Baker and Cowan, 2004, Biochem. Soc. Trans. 32(2): 218; Farrelly et al., 1995, Appl. Environ. Microbiol., 61: 2798; Liesack et al., 1991, Microbial Ecol. 21:191; Ravenschlag et al., 2001, Appl. Environ. Microbiol., 67:387; Suzuki and Giovannoni, 1996, Appl. Environ. Microbiol., 62:625; Sharma et al., 2007, J. Microbiol. Meth., 68: 445; Zehr et al., 2003, BioTechniques 35:996). W. G. Wade ( 2004, Adv. Applied Microbiol., 54:93) has provided and exhaustive summary of “biases in molecular analyses,” including a discussion of intragenomic heterogeneity. It also important to recognize that these problems of intragenomic heterogeneity also impact fungal and protozoal studies of ITS and IGS regions ( Brosch et al., 2008, FEMS Microbiology Reviews, 32: 409; Saksouk et al., 2005, Molecular and Cellular Biol. 25:10301). How are these potential problems dealt with in the “typical” molecular microbial ecology paper, where bulk extraction of nucleic acids from environmental samples is used? Simply by noting that nucleic acids “were extracted,” using a single procedure, with no further discussion being provided.
IV. Challenges for implementing microbial observations into NEON
Section A. STATEMENT: Developing educational programs for current and future microbial biolotists to provide relevant training for use of NEON databases and products, and developing educational programs for non-microbial biologists to utilize microbial data.
SEE DAK RESPONSE #2 Microbiological data are much more than sequences and genomes. To emphasize molecular sequences and not to discuss gene-environmental interactions, is an oversimplified reductionism-driven view of biology.
Section C. STATEMENT: “Understanding patterns of microbial biodiversity in space and time is essential for understanding changes through space and time.” See previous comment.
SEE DAK RESPONSE #4
STATEMENT: “In the context of NEON, biodiversity is defined in terms of various sequence databases that provide information on which microbes are present, the abundance of specific microbes, their functional properties, dynamics through space and time of populations and communities, and patterns of diversity through space and time.” See previous comment – this is totally unjustifiable. The phenomenon of epigenetics, of gene-environment interactions, simply has been neglected.
DAK RESPONSE #5: Simply having an organism (or a sequence) present or to measure sequence “abundance” is not enough – one must be able to document that they are active in situ. Many organisms that are present in natural environments will not be able to function where they occur, having been transported from a permissive to the non-permissive environment. This concept has been emphasized by many workers. Please review the points made in the second part of RESPONSE #1. The microbes that are present may be in a resting form or that do not find themselves in a permissive environment. This point has been made based on almost 100 years of microbial ecology.
STATEMENT: Kinds of sequence data:
*16S rRNA gene and ITS for bacteria-archaea and fungi, respectively, along with other genes, etc.
DAK RESPONSE #6:Please see comment #5. Also, what ever happened to protozoa and algae? These microbial groups play major roles as components of “microbial diversity.” Also, what ever happened to viruses? Viruses are now recognized as playing major roles in influencing the phenotypes of microbes and thus are critical to be included in considerations of microbial diversity. Viruses of themselves are considered to be a critical aspect of microbial diversity. As an initial example, check the initial statements given at the UMR 6197 website. http://wwz.ifremer.fr/umr6197_en/recherche/exploration_de_la_diversite/virus_et_plasmides Epigenetic aspects of 16S rRNA gene and ITS for bacteria-archaea and fungi, respectively, must be considered. In that context, the simple use of sequences is not sufficient to provide information on microbial diversity.
* metagenomic sequences
* targeted functional gene sequences
STATEMENT: 16S rRNA gene and ITS for bacteria-archaea and fungi, respectively, sequences, when obtained quantitatively -----
See DAK RESPONSE #5. There is no evidence that sequences can be obtained quantitatively from natural matrices, based on the studies I have reviewed.
STATEMENT: “Relative abundances of bacteria, archaea and fungi can also be obtained as a valuable secondary product of the data”
See DAK RESPONSE #5
STATEMENT: Metagenomic sequences provide critical information on functions and dynamics within microbial communities
See DAK RESPONSE #5
STATEMENT: metagenomic datasets – annotated sequences for partial and complete genome assemblies; community structure as revealed by 16S rRNA, ITS and other phylogenetically and taxononically informative genes; occurrence and diversity of annotated genes.
See DAK RESPONSE #5
STATEMENT: Additional rate measurements should be targeted towards key processes in elemental cycles, including denitrification, ammonia oxidation and nitrogen fixation.
DAK RESPONSE #7. It would appear that processes of denitrification, ammonia oxidation and nitrogen fixation are being suggested to be monitored based on their frequent measurement, and not perhaps on their centrality to global biogeochemical processes. I would suggest that lignin dynamics and turnover processes might also be informative. Assessment of glomalin dynamics might also be of ecological value.
F. Challenges for Sampling
STATEMENT: The effects of global change on biodiversity at local to continental scales represent a fundamental challenge for NEON to address.
DAK RESPONSE: See DAK RESPONSE #5.
STATEMENT: “the temporal and spatial diversity of bacteria, archaea, and fungi is not well known for any environment.”
See DAK RESPONSE #5.
G. Challenges for sample archiving
STATEMENT: “At present, there is no practical method for reliably harvesting microbes from soils for analysis or storage.”
DAK RESPONSE #8. This is incorrect. Please check in the DAK recommendations section #4, concerned with single-cell analyses. The long history of using micromanipulation and other selection approaches makes it possible to recover bacteria and archaea (hopefully also being scored as being active), as well as fungi, protozoa and algae. Check the paper by Ishoey et al., (2008, Current Opinion in Microbiology, 11:198) for a penetrating discussion of the potential of using single microbial cell analyses from environmental samples. It should be possible to isolate, recover and dry single cells; if this is completed rapidly, the individual cells could be held possibly at room temperature under dessicated conditions until analysis. Careful initial studies would be required to assure that this procedure would be satisfactory. What is the major problem? It may be necessary to use a microscope ---------------
To be able to sense the value of this approach, the classic study by Warcup (Trans. Brit. Myc. Soc., l957, 490: 237) should be reviewed. In this beautiful study, fungi recovered from a soil by direct observation and micromanipulation were compared with those recovered by the use of culture media and plating. The active genera, recovered directly from the soil as active hyphae, largely were sterile species, while those fungi that developed on laboratory growth media were derived from inactive spores of sexual fungi that were playing no role in the microbial community. These studies provide an important principle: it is critical to be certain that organisms being studied are actually active in the natural environment. This level of micromanipulation-based research was being carried out over 50 years ago. There is no reason why this approach, in a modern manifestation, cannot be used in these proposed NEON studies.
V. A NEON-microbial biology pilot study to address sampling and archiving.
STATEMENT: “DNA would be isolated from suites of samples from the sites. Analysis of these sequences would suggest the extent of temporal and spatial variability at these two sites and provide a framework to make proposals on sampling.”
DAK RESPONSE: See DAK RESPONSE #5.
STATEMENT: How does microbial diversity change over time in an air-dried archived sample?
B. Initial sampling for microbial diversity at all core sites.
STATEMENT: DNA extracts from each sample will be sequenced by bar-coded 454 pyrosequencing, etc. These data will provide as comlete a picture as possible at low cost of the soil microbial diversity at each site.”
DAK RESPONSE #9: This is a critical problem; one doesn’t know where the nucleic acids are derived from in most metagenomic studies, where the nucleic acids are recovered by using bulk extraction-based approaches to provide “DNA extracts.” Nucleic acid recovery from a natural microbial assemblage also gives many problems; different methods give different recoveries from different components of the assemblage. How is this dealt with in most metagenomic studies? By not discussing the problem and using a single method in a mechanical manner, the most critical part of such a study is being totally neglected. The goal should be to recover the nucleic acids from the active microbial component, to be able to link the results of the molecular analyses to microbial communities and their functioning. Also, the assumption that ALL the nucleic acids can be extracted has nothing to do with the requirements for the study of a microbial community, which would involve only a subset (active) of the microbes present in a natural microbial assemblage. See also DAK response #5.
C. Design of a pilot study.
STATEMENT: “Expertise in designing complex, large-scale experiments.”
DAK RESPONSE #10: Many long-term experiments have been set up over the decades where the microbial role in processes have been evaluated. In my view, it is critical to select an integrative surrogate for microbial processes that reflects longer-term effects of microbial presence and activities. as they interact with various imposed short-term stresses and environmental changes. We have used this approach in studies of nitrogen impacts on plant soil systems, and in studies of longer-term effects of cloud seeding agents (silver) on plant-microbe-soil systems. References can be supplied if desired.
G. Challenges of sample archiving
STATEMENT: At present, there is no practical method for reliably harvesting microbes from soils for analysis or storage.
DAK RESPONSE #11: In a later section, DAK recommendation #4, it will be suggested that the time is now appropriate to move beyond bulk-extracted nucleic acids, to the use single-cell isolations followed by molecular (and other analyses). In this approach, individual bacteria or fungal hyphal units should be able to be archived in multi-well plates, held under dessicated conditions, and then be ready for analysis.
VI. Summary of specific outcomes from the NEON-microbial biology workshop.
STATEMENT: Sequence-based data will provide the most useful information on diversity, which is itself central to understanding microbial distributions and dynamics in the contexts relevant for NEON.
DAK RESPONSE #12: This is a faulty assumption: only active microbes are part of microbial communities. To extract nucleic acids from both active and inactive microbes, as well as possibly from free nucleic acids, and then to assume that these sequences represent the “microbial community, ” is not defendable. Case et al., (Appl. Environ. Microbiol., 2007, 73:278. ) provide a summary of the mechanical problems that can influence nucleic acid extraction processes, and they also note that multiple heterogenous copies of the 16S rRNA gene can occur within a genome. In discussing the results of their study, (bottom of page 284) it is noted that the "16S rRNA gene copy number ranged from 1 to 15, with an average of 4.2 copies per genome." They also note (page 286 lower left) that "intragenomic heterogeneity can be as significant as intergenomic heterogeneity." These workers also note: “However, none of the 16S rRNA-based molecular methods allows for an accurate representation of microbial communities." The use of “community DNA” to construct metagenomic libraries, as described in this planning document, suffers from the same unacceptable limitation. We are now in the post-genomic age as discussed in my Dec., 2007 article that appeared in ASM “Microbe.” The assumption that a sequence describes an organism is a fundamental problem in much of the field of “molecular microbial ecology,” as it has developed based on the use of bulk-extracted nucleic acids.
Step 4 Analysis - The use of sequence-based metagenomics
DAK RESPONSE #13: Simply having information on the nucleic acid sequence in a “gene” is not sufficient to provide information on the phenotype of members of a microbial community or microbial diversity in this “post-genomic age.” There are several concerns: (1) With the recognition of the role of epigenetics in influencing the phenotype of a microbe, independent of a particular nucleic acid sequence, this deterministic, reductionist view of a nucleic acid sequence describing a microbe, the foundation for most studies of “molecular microbial ecology” carried out to date, no longer can be considered to be valid or sufficient in terms of understanding microbial ecology or the phenotypic/functional characteristics of microbial communities. Casadesús and Low (2006 MMBR 70:830-856 ) have provided an excellent summary of this area. The critical role of epigenetics in influencing bacterial phenotypes has been desicribed in several recent papers. These include the study of a “DNA methylation ratchet” influencing cell cycle timing ( Collier et al., PNAS, 2007, 104: 17111) and also the role of DNA methylation and epigenetic inheritance in plants and filamentous fungi (Martienssen and Colot, 2001, Science, 293:1070) as particularly interesting examples. It also will be critical to consider epigenetics and the areas of protozoa and algae, as well as viruses. SNPs also are of concern - variations in base sequence may occur in regions other than the DNA coding for the 16S rRNA that can affect the phenotype. An interesting recent reference related to this phenomenon is “Population dynamics through the lens of extreme environments,” by Rachel J. Whitaker and Jillian F. Banfield ( 2005, Reviews in Mineralogy and Geochemistry; 59 (1 ); 259-277). http://rimg.geoscienceworld.org/cgi/content/full/59/1/259 . Weissmann et al., (2003, Trends in Microbiology 11(3);115) have discussed the role of SNPs in enterobacterial fimbrial adhesion structure and functioning, as an additional perspective on this important topic.
I. STATEMENT: Synoptic analyses of biodiversity -----
DAK RESPONSE #14: To understand biodiversity, particularly in the microbial world, it is necessary to go beyond nucleic acid sequences. See DAK RESPONSE #5. “Biodiversity analyses should be sequence based and include 16SrRNA-ITS, metagenomic and targeted functional gene analyses.”
DAK RESPONSE #15: See comment # 5 above. Merely having a sequence is not sufficient to provide information on biodiversity, with the important role of epigenetic processes in influencing the phenotype of microbes. As noted earlier, Intragenomic heterogeneity has mentioned previously in relation to bacteria (see DAK response # ) is of concern. In relation to the assessment of ITS/IGS information for fungi, there are problems: (1) possible extraction of DNA from sexual and asexual spores that are not functioning in the environment being studied, and thus not a part of functional microbial diversity or microbial ecology, (2) the possibility of primer sets being used that are not specific for fungi and that also will react with DNA from other eucaryotes ( Zhou et al., 2000, Molecular and Cellular Probes, 14: 339), and (3), intragenomic heterogeneity in both ITS and/or IGS regions, as has been observed with Pythium ( Chang et al., 2008, Eukaryotic Cell, 7:721.), as major concerns. In addition, with epigenetic considerations, as noted earlier, simply having a nucleic acid sequence is not sufficient to provide information on microbial diversity, that is influences by gene-environment interactions.
III. STATEMENT: The 30-year lifespan of NEON
DAK RESPONSE #16: I would question whether after 30 years researchers will still be extracting nucleic acids from soils, waters, intestinal contents, etc. As noted earlier, with this bulk extraction-based approach, one has no idea of the source of the sequences that are being analyzed. I would suggest immediately moving beyond this outdated and flawed bulk extraction-based approach for the recovery of nucleic acids from environmental samples. The field of single cell recovery is developing rapidly. Again, check the paper by Ishoey et al., (2008, Current Opinion in Microbiology, 11:198) for a penetrating discussion of the potential of using single microbial cell analyses from environmental samples, as well as citations given in DAK RESPONSE #1.
V. STATEMENT: "NEON's integrated Science and Education Plan."
DAK RESPONSE #17: In my view, all this program will do is to create additional generations of “molecular microbial ecologists” who have only a limited (or essentially no) ability to deal with experimental microbiology or the truly rigorous demands of microbial ecology, the study of microbes and their functioning and interactions with their specific living and non-living microenvironments. With a lack of emphasis of direct observation of individual microbes in situ, it will be difficult to develop a program with a strong emphasis on microbial ecology and functional microbial diversity – single cell analyses, to be discussed under DAK recommendation #4, can provide an entrée into this field.
STATEMENT: “The MBL microbial diversity course should be considered as a model.”
DAK RESPONSE #17: The MBL microbial diversity course, in my understanding, particularly after having worked in Holger Jannasch’s lab in l995, was developed based on isolation of unique microbes from environments such as the Sippewissett Marsh, coupled with careful direct observations of natural materials, including the use of microscopes. Although a molecular component apparently has been added in recent years (based on having looked at the lab schedule while visiting at Woods Hole) it appears that the foundation of the course still is enrichment/observation of natural materials. The proposed NEON program has only a limited emphasis on what can be considered to be “classical microbiological laboratory skills,” including the use of a microscope. This is a major conceptual deficiency of the entire microbial biology component that is being suggested to be a part of the NEON.
D. A. Klein Recommendations - “Integrating microbial biology into the National Ecological Observatory Network:” Based on my reading of this white paper, I would like to make the following recommendations:
1.0 Invite more diverse opinion(s) concerning the broad problem of “Integrating microbial biology into the National Ecological Observatory Network.” In reading this white paper, it appears that only approved viewpoints and suggested analytical approaches, based on the primacy of sequence-based analyses for the characterization of “microbial communities” and “microbial diversity,” centered on the bulk extraction of nucleic acids from environments such as soils, waters and intestinal contents, have been allowed to be expressed at this workshop.
2.0 Emphasize modern genetics, including epigenetics. The program, as developed, is based on outmoded 20th century genetic determinism, based on a one gene- one product reductionist Weltanschauung. Gene-environment interactions (the essence of epigenetics) simply are not discussed. If epigenetics, the essence of gene-environmental interactions is considered, most of the verbiage in this white paper suddenly will be discovered to be intellectually and scientifically wanting, particularly that related to understanding and documenting “microbial diversity.”
3.0 In terms of considering the effects of stress and global change on microbiological processes, it is critical to be certain that prior studies are integrated into the thinking concerning these investigations. There are many longer-term biological experiments that have been carried out. The Rothamsted plots are a first immediate example - that have been described. Work that has been carried out on nitrogen stress effects on plant-microbe-soil systems at many sites should be reviewed, as well as studies of weather modification seeding agent affects on biological systems. In our studies of nitrogen amendment and seeding agent effects on plant-microbe-soil systems , we worked to identify integrative components of ecosystems that would provide a longer-term indicator of changes in microbial functioning.
4.0 How will it be possible to move beyond these discredited, vapid, bulk extraction-based nucleic acid recovery-based studies that have formed the basis of a large portion of what has become “molecular microbial ecology? What is needed at this time is to work at a level resolution where one is certain that the nucleic acids that are being studied are derived from microbes that are active in situ – analyses at the level of single cells.
The conceptual and technical foundation for carrying out studies of microbes and their ecology at the level of single individual cells are becoming available. As noted in my feature article that appeared in the Dec., 2007 issue of “Microbe,” it is now possible to work at the level of the individual in situ active microbe. This approach, described as the field of cytomics (Palková et al., 2004, Cytometry Part A., 59A:246), has been discussed by many individuals, including Waksman and other early workers noted in this communication, and in addition, Gill Geesey made this point in comments on the future of environmental microbiology (Aquatic Microbiology Newsletter, l999, 38(2) 15) where he discussed moving from population-based to individual cell-based scales of observation and study. This concept also is available in a widely used general microbiology text. Prescott, Harley and Klein “Microbiology, 6th edition (2005), chapter 28 (microbial ecology) discuss the use of single cell-based molecular analyses in microbial ecology. In a recent paper by Kowalchuk et al., (2007, Microbial Ecology 53:475) the use of single cell based analyses in studies of microbial ecology is discussed. They note that “amplification technologies provide access to microbial genomes at the level of a single microbial cell,” and that this “presents the possibility of examining microbial community genomes and activities one cell at a time.”
This exciting vision also has been discussed by many other workers, of which only a few can be cited in this letter: (Amann and Fuchs, 2008, Nature Reviews Microbiology 6:339; Amann et al., 1995, Microbiol Rev., 59:143; dePoorter, et al., 2005, Microbiology 151:1697; Le et al., PNAS 102:9160; Davey and Kell, 1996, Microbiological Reviews, 60:641; Fröhlich and König, 2000, FEMS Microbiology Rev. 24:567; Huang et al., 2007, Microbial Ecol., 53:414; Lebaron et al., 2001, Appl. Environ. Microbiol., 67:1775; Ottesen et al., 2006, Science, 314:1464; Posch et al., l997, Appl. Environ. Microbiol., 63:867; Radajewski et al., 2003, Curr, Opin. Biotechnology, 14: 296; Shendure, et al., 2005, Science, 309:1728; Strovan and Lidstrom, 2008, Microbe, 3(5): 239; Walker and Parkhill, 2008, Nature Rev. Microbiol., 6:176; Westphal et al., 2002, Meth. Enzymol., 356:80). In addition, the technology for single cell microbiology now is available commercially; the company Accelr8 provides reagents and technology, indicating how far this field has advanced. Check http://www.accelr8.com/ There may also be other companies out there -----
A paper by Ishoey et al., (2008, Current Opinion in Microbiology, 11:198) provides a valuable discussion of the potential of using single microbial cell analyses from environmental samples. In this paper, the authors note that (my lettering):
Page 199 ( right-hand column)
( a.) “Soil was pretreated by density gradient centrifugation before single-cell isolations.”
(b.) “ Depending on desired throughput and the environment and organisms targeted, single cells have been isolated for use in MDA reactions by dilution, fluorescence activated cell sorting (FACS) micromanipulation and microfluidics”
(c.) “Although micromanipulation is a relatively low-throughput method compared to flow sorting, it is a powerful research tool that allows observation of cell morphology, documentation by imaging, and a high degree of certainty that a single cell was captured and delivered to the reaction vessel for amplification.”
(d.) “Rinsing single cells in buffer allows the removal of free DNA or other contaminants.”
Page 201 (lower right paragraph)
(e.) “metagenomic shotgun sequencing provides gene frequencies at the community level but rarely reveals the genetic linkage within individuals,”
How can one combine these approaches for the study of single cells with the need to study active organisms? At some point in the cell sorting, carry out a RNA/DNA ratio analysis on the single cells to be able to select those that are deemed to be active before completion of the 16S – rRNA or genomic analyses (Anderson and Parkin, 2007, J. Microbiol. Methods, 68:248; Czechowska, et al., 2008, Curr. Opin. Microbiol. 11:205; LeBaron, et al., 2001, Appl. Environ. Microbiol., 67:1775). In this way, it will be possible to assure that one is dealing with microbial communities (active under in situ conditions), based on experimentally determined criteria, and thus contributing to microbial diversity and microbial ecology.
As noted at the end of the paper by Ishoey et al., (2008, Current Opinion in Microbiology, 11:198), “Finally, the analysis of the microbial cell provides an indispensable view of the living organism. As in the case of the human genome, there is much to learn at the level of the individual.”
FINAL COMMENTS: In my view, it is now time to move beyond the use of outmoded and scientifically flawed bulk extraction-based nucleic acid sequence analyses, based on a scientifically unsupportable 25-year old notion, to the next level of biological resolution, the study of in situ active individual microbial cells. I recently presented a seminar entitled “Microbial ecology in the post-genomic age: individual active microbes as the new paradigm” in which concepts that appear to be important for the development of microbial ecology and the analysis of microbial communities in the future are discussed. If the NEON microbial biology planning group might wish additional information concerning this seminar ( or a paper that is being written concerned with this important topic), please let me know.
Thanks very much for your consideration of these comments concerning the planning document “Integrating microbial biology into the National Ecological Observatory Network,” prepared by G. M. King et al., I look forward to your responses to my observations and conclusions.
Sincerely, Donald A. Klein