We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used ...
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Antonie van Leeuwenhoek 71: 143–150, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures David M. Ward , Cecilia M. Santegoeds, Stephen C. Nold, Niels B. Ramsing, Michael J. Ferris & Mary M. Bateson
Department of Microbiology, Montana State University Bozeman, MT 59717, USA ( author for correspondence)
Key words: 16S rRNA, enrichment culture, microbial biodiversity, community ecology
Abstract We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the ‘New Pit’ Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54 C and 56 to 63 C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adapted to the habitat temperature (50 C), while populations adapted to 65 C and 70 C were 102 - and 104 -fold less abundant, respectively. However, enrichment at 70 C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotrophic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit. Introduction In a paper on ureolytic bacteria published in 1901, Martinus W. Beijerinck emphasized the value of a new technique he introduced to microbiology, the elective enrichment culture: The present phase in the development of microbiology can be called the ‘systematic’ phase, because in microbiology, as in every young science, it is first necessary to describe the material to be studied, and to place it into an orderly classification. For this phase of microbiology, the enrichment culture experiment has a special importance. (Beijerinck, 1901 translated by Brock, 1961)
The same could be said today about the special importance of molecular techniques that are being used to systematically describe microorganisms which inhabit natural habitats, and to provide an orderly classification of their existence in nature. Beijerinck (and also Winogradsky), realized that enrichments would fail to recover all microorganisms from nature and could bias our view of the material to be studied (i.e. of native populations). Likewise, we must keep in mind the limitations of molecular methods (Ward et al., 1992a; Kopczynski et al., 1994; Robison-Cox et al., 1995; Reysenbach et al., 1992; Rainey et al., 1994). But even with this caveat, it is clear that molecular approaches can provide interesting new information about microorganisms in natural communities. At the
MENNEN/Preproof: Pipsnr.: 114524; Ordernr.:233899-bb; Sp.code:anto894-BEI; Ass. Nr.:F 2263 BIO2KAP anto894.tex; 28/11/1996; 15:40; v.5; p.1
144 time of this Beijerinck Centennial we have the advantage that molecular approaches are also providing an understanding of the evolutionary relationships among microorganisms (Woese, 1987; Olsen et al., 1994). By using similar molecular approaches microbial ecologists can determine both ecological and evolutionary patterns of the occurrence of microbial populations in nature. Both are important to a complete understanding of microbial biodiversity and community structure, which are the result of natural selection, the link between ecology and evolution. Our work on hot spring microbial mats is an example of a large and growing number of studies in which molecular cell components are being used to study the composition and structure of microbial communities. We have used 16S rRNA sequences as the marker to discover many new populations and their evolutionary relations (Ward et al., 1990; Weller et al., 1991, 1992). We have used probes against 16S rRNA sequences to begin to examine the distribution and behavior of these populations (Ruff-Roberts et al., 1994; Ferris et al., 1996a). Some of the major findings to date are that 1. populations sampled by cultivation and molecular approaches are completely different (Ward et al., 1990, 1994; Ferris et al., 1996b) 2. multiple phylogenetically related populations occur simultaneously, at least in some cases constituting guild structure within the community (Ward et al., 1994; Ferris et al., 1996b) 3. such populations appear to be adapted to specific environmental features (Ruff-Roberts et al., 1994; Nold and Ward, 1995; Ferris et al., 1996a), and 4. ecological and evolutionary patterns suggest that intra-guild diversity may be a result of radiations to fill niche space (Ward & Ferris, unpublished) Recently, we have begun to evaluate the basis of the incongruence between the sets of populations observed by either culture methods or 16S rRNA methods, with the following questions in mind. Why do enrichment cultures fail to provide the microorganisms whose molecular signatures we observe in the same habitat? Is it possible to obtain pure cultures of the organisms whose 16S rRNA signatures we see? What is the relevance of microorganisms which we can cultivate, and of the microorganisms whose 16S rRNA sequences we can detect within communities? The purpose of this paper is to review the highlights of several recent and ongoing studies in our lab which follow on an idea expressed by Dr. Norbert Pfennig at the 1990 Mole-
cular Microbial Ecology Conference (Braunschweig, Germany). As he said, Beijerinck and Winogradsky had originally suggested monitoring enrichments so that we could observe whether an approach favored the desired species. If not, conditions could then be modified to achieve success. In the early 1900s monitoring of enrichments had to be done by microscopy and isolation on solidified media. Now it is possible to apply molecular methodology to monitoring enrichment cultures. The method we have chosen is denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993; Ferris et al., 1996a) because it permits simultaneous analysis of numerous (theoretically all) populations inhabiting an enrichment culture. We have so far emphasized two fundamental approaches to attempt to enhance enrichment of more relevant species: (1) extincting dilution of inoculum, and (2) use of medium resources and incubation conditions more relevant to the natural environment from which the sample came. Our long-range goals are to erode the concept that bacteria may be uncultivatable and to ultimately be able to predict how specific populations, which are as yet uncultivated may be recovered. It should then be possible to better understand some of the phenotypic potential of the organisms whose molecular signatures predominate in microbial communities.
Materials and methods Samples Samples for Chloroflexus enrichments were obtained in July 1994 from the 55.2 C ‘New Pit’ Spring mat (Ward et al., 1987) using a cork borer to sample the top 1 mm. Samples for cyanobacterial enrichments were obtained from the Octopus Spring cyanobacterial mat (Brock, 1978). The top cyanobacterial layers at a 44 to 53 C site in the shoulder region and at a 55 to 62 C site in the effluent channel closest to Great Fountain Geyser were sampled on 30 September 1995. Samples for aerobic chemoorganotrophic enrichments were obtained from Octopus Spring using a cork borer to core the top 1 cm of a 50 to 55 C site in the shoulder region on 30 September and 29 October 1992 (pure culture work) and 5 November 1994 (DGGE experiments). All samples were transported to the lab in water from the site, maintaining temperature near the habitat temperature, and were then processed immediately. Samples were homogenized in a Dounce tissue homogenizer, then homogenates were diluted 10-fold
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145 directly into liquid culture media. Direct microscopic counts were done by phase contrast microscopy using a Petroff-Hausser chamber to determine the density of Chloroflexus-like filament fragments in ‘New Pit’ Spring mat homogenate or unicellular cyanobacteria (Synechococcus) in Octopus Spring mat homogenates. Approximate population densities (per ml Octopus Spring mat) were estimated from the highest dilution in which individual populations were detected (by DGGE or by subsequent plating on solidified media) by dividing by the number of Synechococcus cells present in the inoculum (determined by considering dilutions used to prepare homogenates), then multiplying by the density of Synechococcus cells in the mat (determined by direct counts, 1010 cells/ml mat). Cultivation Photoheterotrophic enrichments for Chloroflexus were performed in anoxically prepared Castenholz medium D (Pierson and Castenholz, 1992) amended with 0.2 g/l NH4 Cl and 0.025% yeast extract (w/v), incubated under low intensity tungsten light at 52 C. Enrichments for cyanobacteria were performed in Castenholz medium D, incubated at 50 C or 58 C and illuminated by low-intensity fluorescent and incandescent lights. Enrichments for aerobic chemoorganotrophic populations were performed in 0.1%, 0.01% or 0.001% tryptone and yeast extract (w/v) in Castenholz medium D, incubated in flask cultures shaken at 150 RPM at 50 C or 70 C in the dark. The specific media for enrichment and isolation of Thermus strains was described in detail previously (Nold and Ward, 1995). DGGE analysis All procedures were detailed previously (Ferris et al., 1996a; Santegoeds et al., submitted). Briefly, DNA was extracted and purified from cell pellets of enrichments and pure cultures following French Press (Chloroflexus), bead beater (cyanobacteria) or enzymatic lysis (aerobic chemoorganotrophs). DGGE involved the PCR-amplification of a fragment of the 16S rRNA gene between Escherichia coli positions 1070 and 1392, using a GC clamp on the 1392 primer. Separation based on differences in sequence was done by electrophoresis on a 6.5% polyacrylamide gel containing a linear gradient of 35% to 80% urea and formamide. Individual DGGE bands were excised, reamplified and sequenced. Band identity was based on co-migration with DGGE bands generated from pure
cultures and by comparison to sequence data available in the Ribosomal Database Project (Maidak et al., 1994) or in our Octopus Spring sequence database. The image of the DGGE gel for cyanobacterial enrichments was enhanced using the shadowing option of the NIH Image program Version 1.59 (by Wayne Rasband); all lanes were treated identically.
Results and discussion Numerically relevant Chloroflexus populations of the ‘New Pit’ Spring mat This mat and its predominant Chloroflexus spp. are of interest because of the antiquity of this lineage and the possibility that the earliest stromatolites might have been formed by photosynthetic bacteria rather than by cyanobacteria (Ward et al., 1989, 1992b). As shown in Figure 1, DGGE analysis reveals that we have succeeded in enriching from a highly diluted sample of the ‘New Pit’ Spring mat a population whose molecular signature is prominent among those recovered directly from the mat. We have confirmed by sequencing that this population has a Chloroflexus-like 16S rRNA signature. The organism enriched has so far resisted growth on solidified media. Numerically relevant cyanobacteria of the Octopus Spring mat Through direct cloning and DGGE analysis of 16S rRNA sequences in the mat we have so far detected 7 cyanobacterial populations (Weller et al., 1992; Ferris et al., 1996b), none of which corresponds to Synechococcus lividus, formerly thought to be the major mat forming cyanobacterium. These include types we designate A, A’, B, B’, I, J and P. As shown in Figure 2, a preliminary DGGE analysis shows that enrichments inoculated with highly diluted mat samples appear to contain bands which can be tentatively identified as some of these populations, based on co-migration with standards. For example, an enrichment from a 44 to 54 C mat sample incubated at 50 C appears to contain population B, as well as a population likely to be B’ on the basis of its position just above population B (see Ferris et al., 1996a). This was confirmed by isolation of Synechococcus cultures from this enrichment which were characterized as B and B’ populations by partial 16S rRNA sequence analysis. There is also an indication of the type-P cyanobacterial population. A band
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Figure 2. DGGE profiles of cyanobacterial enrichment cultures inoculated with 44 to 54 C and 56 to 63 C Octopus Spring mat samples and incubated at 50 or 58 C. Estimated population densities are 1.25 x 108 /ml for the 44 to 54 C sample and 1.25 x 103 /ml for the 56 to 63 C samples. Reference bands (Ref.) are from cyanobacterial 16S rRNA populations recovered by cloning or cultivation (P, C1, B, A and C9) and C. aurantiacus (Y400). Band positions are indicated on both sides of the gel. The image was inverted and bands were intensified electronically.
Figure 1. DGGE profiles of the ‘New Pit’ Spring mat (Mat) and an enrichment culture (En) for photoheterotrophic bacteria inoculated with a 10 5 -fold diluted sample from this mat (the extincting dilution for growth). Direct microscopic counts gave estimates of 2 x 106 filament fragments/ml. Arrow indicates band with Chloroflexus-like 16S rRNA sequence.
which appears to co-migrate with C. aurantiacus might also be the green nonsulfur bacteria-like population C, which is known to have a similar band position (Ferris et al., 1996a). All of these populations occur at densities at least as high as 1.25 x 108/ml mat. An enrichment from a 56 to 63 C sample incubated at 58 C appears to contain populations B’, A and P. The population density is much lower, possibly due to the effect of temperature reduction on population size (see Ward, 1978 for evidence that mat populations may exhib-
it such temperature sensitivity). The A- and B-like populations detected in these two samples correspond nicely with results from DGGE analysis (Ferris et al., 1996a), which also showed populations B and B’ in 48 to 54 C mat samples and B’ and A in 50 to 65 C mat samples. However, when an enrichment from the 56 to 63 C sample was incubated at 50 C populations B and B’ (and P) appeared to develop, indicating that lower incubation temperature favors selection of populations found in the lower temperature sample. The pattern is as predicted, given our understanding of possible temperature specializations of A- and B-like populations, and demonstrates how, as Beijerinck suggested, enrichments might be perfected through a better understanding of the ‘materials’ (i.e., populations) and their specializations (in this case to temperature). In addition to B and B’ populations reported above, in earlier work we isolated three other populations of Synechococcus in pure culture by plating on solidified media from similar high dilution enrichments (Ferris et al., 1996b). Fast-growing strains whose 16S rRNA sequence is identical to all culture collection strains of S. lividus were obtained from the low- dilution inocu-
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Figure 3. Estimated densities of 50 to 55 C Octopus Spring mat Thermus populations detected by DGGE analysis of aerobic chemoorganotrophic enrichments incubated at 50 or 70 C. The shaded portion of each bar represents the dilutions in which populations were actually detected. The phylogenetic affiliation and optimum growth temperature of isolates with identical 16S rRNA sequences are indicated.
la, whereas slow-growing Synechococcus isolates were obtained from highly diluted inocula. The isolate with the highest estimated population density (4 x 108 /ml mat) had a 16S rRNA sequence identical to the typeP sequence previously cloned from the mat (Ferris et al., 1996b) and tentatively identified in DGGE analysis. Another isolate, population C9 whose density was slightly lower (4 x 107 /ml mat), had a 16S rRNA sequence not previously detected in the mat by cloning or DGGE. The samples used in the DGGE and earlier cultivation studies may have had different predominating cyanobacterial populations. Temperature-specialized Thermus populations of the Octopus Spring mat DGGE analysis of 0.1% tryptone and yeast extract enrichments for aerobic chemoorganotrophic populations inhabiting the Octopus Spring mat gave even more clear evidence that the particular conditions during enrichment affect the population selected. As shown in Figure 3, enrichments from a 50 to 55 C mat
sample incubated at 50 C or 70 C contained three populations detectable by DGGE analysis, which could be identified as Thermus populations based on comigration with standards of Thermus isolates and by sequencing of DGGE bands (Santegoeds et. al., submitted). These isolates were previously recovered from similar enrichments followed by plating (Nold & Ward, 1995) and showed a similar pattern of relative abundance (3.8 x 105 , 3.8 x 102 and 1.1 x 102 per ml mat for T. ruber-like, T. aquaticus-like and Thermus sp. populations, respectively). Because the temperature optima for growth of the isolates has been characterized (Nold & Ward, 1995) it is possible to understand the results presented in Figure 3 more fully. Incubation at 70 C with low-dilution inoculum led to selection of only one population, Thermus sp., whose optimum growth temperature was best-suited to the incubation temperature (i.e. 70 C). However, dilution of inoculum resulted in selective enrichment of a T. aquaticus-like population, whose optimum growth temperature is only 65 C. At low inoculum dilution the numerically inferior population was clearly able to displace the ca. 100-fold numerically superior population because it was more fit under the incubation conditions. Van Niel (1949) quoted Beijerinck as having said that elective enrichments favor specific organisms ...under predetermined conditions, either because they alone can develop, or because they are the more fit and win out over their competitors. This appears to be an example of competitive exclusion. In the enrichment incubated at 50 C, neither of these two populations were detected. However, a T. ruber-like population, whose optimum growth temperature is 50 C, was detected in enrichments inoculated with yet higher inoculum dilutions. Interestingly, the most dense population was the one most fit in terms of the temperature at the sampling site. Beijerinck (1901) distinguished ‘imperfect’ (mixed species) from ‘perfect’ (single species) enrichments. Because the enrichment culture method would obviously become a popular approach he was optimistic: Only a small number of such perfect experiments have been performed, but in the future more of these will be performed, as our understanding improves. In this case, by understanding the community structure and specializations of different populations it is possible to use different incubation temperatures and dilutions of inoculum to favor the recovery of individual populations. Temperature specialization appears to
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Figure 4. Estimated densities of 50 to 55 C Octopus Spring mat populations detected by DGGE analysis of aerobic chemoorganotrophic enrichments at 0.1%, 0.01% and 0.001% tryptone and yeast extract concentrations incubated at 50 C. Arrows indicate populations which were also detected at lower density in enrichments with 10-fold (one arrow) or 100-fold (two arrows) lower substrate concentration. Thermus populations reported in Figure 3 were not included. Stippling indicates populations whose 16S rRNA sequence has been previously detected in the mat. ‘ ’ indicates a population which was also recovered by isolation on solidified medium. OS-N, Octopus Spring proteobacterial sequence type-N; OS-L, Octopus Spring sequence type-L; Pr, proteobacteria.
be a general pattern of community organization in hot spring microbial mats and might be used to advantage in obtaining organisms of many different physiological types (Peary & Castenholz, 1964; Castenholz, 1974; Bauld & Brock, 1973; Ferris et al., 1996a; RuffRoberts et al., 1994). Other aerobic chemoorganotrophic populations of the Octopus Spring mat In his study of ureolytic bacteria, Beijerinck noted that differences in urea concentration, carbon source and time of incubation also affected the types of organisms selected. We have observed similar results (Santegoeds et al., submitted). For example, DGGE analysis of enrichments over a two order of magnitude range of substrate concentration and at various inoculum dilutions revealed many different 16S rRNA-sequence defined populations (Figure 4). These might all represent species populations, but could also include different 16S rRNA operons of individual species. The most abundant aerobic chemorganotrophs enriched were C. aurantiacus-like and Gram- positive bacterial populations which developed as a pellicle of filamentous bacteria adhering to the flask at the air-liquid interface in enrichments with lower substrate concentrations. Only two of the aerobic chemoorganotrophic populations
have been previously detected by 16S rRNA analysis of the mat. There are many possible explanations why the other populations have not been detected yet in the mat. They may have been absent or at relatively low numerical abundance (perhaps because they occupy a higher trophic level in the community) in samples used for cloning 16S rRNA sequences. Alternatively, molecular procedures (e.g., lysis, amplification or cloning) might have biased against their recovery. Aside from the above-mentioned Thermus populations only one of the aerobic chemoorganotrophic populations detected by DGGE was isolated from similar enrichments on solidified medium (indicated in Figure 4 by ‘ ’). Their absence among cultures could also reflect differences in populations of samples used in DGGE or plating experiments. However, this result might also reflect plating bias.
Conclusions 1. Numerically inferior populations may overgrow numerically superior populations that are less fit under liquid enrichment conditions. 2. Dilution of inoculum to extinction may favor the recovery of numerically superior populations that are less fit under liquid enrichment conditions.
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149 Often these populations have molecular signatures matching those detected by direct analysis of the types of samples used for inoculation. 3. Populations enriched in liquid culture may be unable to develop on solidified medium, so that plating may bias recovery. 4. By understanding the phenotypes of populations before enrichment, it may be possible to increase the probability of ‘perfect’ enrichments (sensu Beijerinck). 5. Numerically relevant populations grow poorly in the enrichment culture medium and incubation conditions provided, suggesting that medium environments may not accurately reflect natural environmental resources and conditions. This, of course, might also prevent recovery of many important populations.
Acknowledgements We appreciate financial support from the U.S. (BSR– 9209677) and Danish National Science Foundations, the National Aeronautics and Space Administration (NAGW–2764), and a scholarship to C.M. Santegoeds from the Rotary Foundation of Rotary International, as well as the assistance of the U.S. National Park Service.
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