Biodiversity and Conservation 1,250-262 (1992)

Unculturable microbes detected by molecular sequences and probes W. L I E S A C K and E. STACKEBRANDT* Department of Microbiology, Centre for Bacterial Diversity and Identification, University of Queensland, St Lucia, Queensland 4072, Australia

Received 5 May 1992; accepted 20 May 1992 Nucleic acid probes have revolutionized rapid diagnostics of plant-, animal- and human-pathogenic viroids, viruses, bacteria and protozoa, and have complemented the spectrum of immunological and serological tests. Sequences of genes and rRNA, of proven potential for identification of taxa, are a valuable tool in that both the identity and the phylogenetic position of an isolate can be elucidated. On the other hand, this approach is still laborious and requires sophisticated equipment. Designation of probes does not require information about sequences, although such knowledge is extremely helpful in the formulation of highly specific oligonucleotide probes, e.g. in the case of rDNA probes, where target stretches containing variable sequences are rather short (about 10-30 nucleotides). The application of probe technologies in molecular environmental studies by in situ hybridization, screening of gene libraries and flow cytometry is in its infancy but a logical step towards reconfirmation of the identity of a species in its natural habitat.

Keywords: molecular microbial ecology; unculturable organisms; gene libraries; DNA; rRNA; DNA probes; polymerase chain reaction primers; phylogeny

Introduction

The term 'unculturable' used in this communication has been adopted from the literature to indicate that certain organisms, which have been proven viable and/or detectable under the microscope, cannot be cultured. Often, especially in molecular studies on the diversity of natural communities, the term is used to circumscribe those organisms whose existence is unexpected. Unculturable bacteria fall into one of the two following categories.

Obligately symbiotic and parasitic organisms These bacteria thrive under host-provided conditions but fail to grow in or on bacteriological media, e.g. Prochloron didemni (symbiont of ascidians), Cristispira (symbiont of molluscs), members of Holospora, Caedibacter, Lyticum, and Blattabacterium, rickettsiae, and others (endosymbionts of protozoa, insects, lice, fungi and invertebrates other than arthropods), Pasteuria penetrans (parasite of root-knot nematodes), Anaplasma marginale (intracellular parasite of bovine erythrocytes), and

Mycobacterium leprae. *To whom correspondence should be addressed. 0960-3115 (~ 1992 Chapman & Hall

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Viable but non-culturable organisms Cells of mainly native marine bacteria have been tested to be alive by a number of methods described for differentiating living from dead or moribund cells (Colwell et al., 1985; Roszak and Colwell, 1987), but fail to undergo cell division in or on routinely used growth media. Apparently these 'non-recoverable' organisms (Xu et al., 1982) react towards stress conditions in their environment by reducing their metabolic activities and turning into a dormant stage. This phenomenon is connected with an increase in cell number and decrease in cell size (Novitsky and Morita, 1978). Possible mechanisms involved in the formation of the so-called 'ultramicrobacteria', dwarf- or mini-cells which remain culturable, have been discussed by Jannasch (1979). It has been estimated that only 1% or less of cells from terrestial or aquatic habitats can be isolated on agar media (Jannasch and Jones, 1959; Alexander, 1961). However, the question as to whether the failure of the majority of non-culturable cells to grow under laboratory conditions is due to the dormant status of cells, the lack of optimal cultivation conditions (suboptimal temperature, selective atmosphere, growth media and/or supplement, inactivation of cells, or damage during probe sampling), or a combination of both factors, has not been answered as yet. The fact is that, due to the limitations in methodologies, most natural environments are poorly described in terms of the complexity of microbial populations contained therein. As a consequence, concepts could not be offered in order to obtain information about important ecological problems (which often led to omissions of the microbiological part in broad ecological studies), such as the detection (i)

of the genetic variation in a given sample (e.g. novel groups of organisms, information about their phylogeny and, derived therefrom, understanding about their evolution); (ii) of the dominating microflora and monitoring the seasonal variation of a community in time and space; (iii) monitoring the fate of free nucleic acids and organisms (engineered or wild types) released into the environment, as well as the horizontal exchange of genetic material; (iv) how growth occurs in the environment and factors affecting survival.

Molecular microbial ecology Recent progress in molecular techniques and their application to the identification of organisms in environmental samples, irrespective of their growth requirements, have marked a turning-point in microbial ecology. In addition to the ongoing attempts to cultivate organisms, research is concentrating on the identification of strains either by using antibodies (preferably monoclonal antibodies) or, more widely used today, by stretches of nucleic acids proven to be highly specific for a given strain, a species, a group of related species, or for a group of organisms sharing similar metabolic activities. Moreover, in addition to phylogenetic types and diversity of the community members, information can be obtained about community structure, and consequently about metabolic activities. Nucleic acid-based phylogenetic studies (rRNA/DNA sequence analysis), molecular genetic studies of plasmids and genes coding for proteins involved in primary and

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secondary metabolism (including gene cloning and sequence analysis), led to the development of nucleic acid probes, novel labelling procedures and hybridization technologies, used today for the identification of organisms, for example pathogens, via gene amplification by the polymerase chain reaction (PCR), or restriction length fragment polymorphism (RFLP). This communication is a brief summary of recent developments in the field of molecular microbial ecology, with special emphasis on the molecular identification of novel types and (possibly) non-culturable organisms by sequences and probes. Methods

Nucleic acid probes Use of nucleic acid probes requires knowledge about factors affecting hybridization (Sambrook et al., 1989; Stahl and Amann, 1991) and decisions have to be made as to which of the many published strategies to follow in order to guarantee successful application. Whilst outlines of the basic methods and protocols, as well as the citation of the most important original literature, are beyond the scope of this paper, only a few reviews, textbooks and original publications will be included in this section. Excellent and extensive compilations are found in Sayler and Layton (1990) and Ward et al. (1992) for probes targeting DNA and rRNAJrDNA genes, respectively. The main points to consider are:

(i)

Type of probe, e.g. length (oligonucleotide or larger fragments, up to total DNA; Lee and Fuhrman, 1990), random (cloned genomic or cloned fragments with unknown sequence), or specified fragments (sequence known). (ii) Hybridization techniques, e.g. colony- or plaque-hybridization, blot-hybridization of isolated nucleic acids, Southern-hybridization, or direct-hybridization of single cells with fluorescently labelled oligonucleotides. The latter procedure has marked a pioneering step in environmental studies because of the ability of this system to detect directly individual cells in a mixed culture (DeLong et al., 1989a). (iii) Probe labelling and hybrid detection, e.g. radioactive, via 32p, via end labelling (for oligonucleotides), or via Nick translation (for larger fragments); and detection by autoradiography or non-radioactive, using one of several commercially available systems, that basically follow the same principle in that enzyme-linked antibodies specifically detect the DNA- or RNA-probe-hapten complex followed by a colorometric assay, for example the biotin/photobiotin-streptavidin, the digoxigenin, or the antigenic sulfone systems. Labelling of oligonucleotides with fluorescent dyes is achieved by adding an aminolinker during automated synthesis, followed by coupling with a fluorescent marker. Despite progress in the development of nonradioactive detection systems, 3/p-labelled oligonucleotides are still most widely used in environmental studies (Britschgi and Giovannoni, 1991; Tsai and Olsen, 1991). Ribosomal R N A / D N A probes So long as it is carried out selectively, any nucleic acid stretch can, in principle, be used as a molecular probe. Depending upon the goal of the study, probes have been described which are directed against certain genes or fragments thereof (e.g. those coding for

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surface epitopes, virulence factors, toxins, metabolic properties). Studies directed to determine individual organisms and to unravel microbial communities, however, have demonstratd the high potential of probes targeting rRNA or rDNA. Identification is facilitated by the presence of (mostly) multiple and almost identical copies of rRNA genes per chromosome and the high number of ribosomes per active cell, reaching 5 × 10 4 copies. Although cloned rDNA probes have been described, there are several advantages that favour the use of oligonucleotides as probes. This is due to the intrinsic feature of the rRNA to contain stretches of varying degrees of sequence conservation. Whilst probes directed against highly variable regions may be species- or even subspecies-specific, those targeting regions of less variability may hybridize with rRNA of a phylogenetically broader group of organisms. Probes have been designed for almost all levels of relationships and for many higher taxa (Ward et al., 1992). An additional advantage of rRNA based probes is the availability of an extensive rRNA database (the Ribosomal RNA Database Project has recently released more than 450 full 16S rRNA sequences). These pre-aligned sequences not only facilitate the search for appropriate target sites but are extremely valuable for a pre-selection of probe specificity. Oligonucleotide probes can be readily synthesized, purified, and labelled. Extensive protocols have been published which cover single-cell detection with radioactively labelled (Giovannoni et al., 1988) or fluorescent-labelled probes (DeLong et al., 1989a; DeLong and Shah, 1990; Amann et al., 1990b), colony- (Liesack et al., 1991a) and dot-blot hybridization (Stackebrandt et al., 1991) as well as flow cytometry for analysis of certain members of a population (Amann et al., 1990a). As shown at least in dot-blot hybridization, differences of as little as a single mismatch between two related target sites can be detected, provided stringent washing conditions are applied (up to the Tm of the homologous hybrid) (Stackebrandt et al., 1991). Analyses o f naturally occurring r R N A and r D N A

Two strategies have been developed that are based on rRNA and rDNA sequences to infer both phylogenetic relationships among naturally occurring organisms, and identification of organisms within their environment. One approach is based on the recovery of rRNA that is transcribed into cDNA, cloned and sequenced. Although certain shortcomings of this method have been reported (Kemmerling et al., 1990; Weller et al., 1991) this approach was successful in the description of a hot spring community (Ward et al., 1990a, b, 1991). The alternative approach is based on the recovery of high molecular weight DNA directly from the sample or from isolated organisms, followed by the amplification of rDNA, cloning and sequencing. Several methodological variations are in use, mainly referring to: (i) the isolation of nucleic acids; (ii) the selection of primers; and (iii) the detection of organisms (Ward et al., 1992). The availability of taxon-specific PCR primers and oligonucleotide probes (Stahl and Amann, 1991; Weisburg et al., 1991; Giovannoni, 1991; Ward et al., 1992) should allow a selective enrichment of rRNA genes and clones, respectively, from the selected taxa. Sequences obtained from analysis of the partial gene library can be compared to homologous sequences in the database and the phylogenetic neighbours determined (Giovannoni et al., 1990; Ward et al., 1990a; Britschgi and Giovannoni, 1991; also see below). Sequences can be examined for the presence of taxon-specific probe and primer sites for future rapid identification of the relevant organisms (e.g. by the phylogenetic

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stain method, flow cytometry, or colony hybridization), and selective amplification of rDNA isolated from different types of natural samples, respectively. Ribosomal DNA analysis of micro-organisms from a soil sample A soil sample from the south-eastern slope of Mount Coot-tha, Brisbane, Australia was analysed. Cells were mechanically disintegrated directly in the soil sample, using a BeadBeater. Extraction of total genomic DNA was performed as described by Ogram et al. (1988). Slight modifications of the procedure will be described in a subsequent publication (Liesack and Stackebrandt, 1992). In order to construct a bacterial 16S rDNA library, PCR-mediated amplification was carried out using two bacterial universal primers with overhangs containing restriction sites. Stretches of about 1200 nucleotides were cloned into phage vector M13 as described previously (Sambrook et al., 1989; Stackebrandt and Liesack, 1991). Analysis of 30 recombinants was performed by both traditional and automated (ABI, Burwood, Australia) DNA sequencing, using an universal M13 primer. Sequences were initially aligned to 16S rRNA sequences of representatives of all major bacterial lines of descent and in a second step to those of its phylogenetic neighbours. Methods concerning the calculation of evolutionary distance values and the generation of a phylogenetic tree by the neighbourliness method, implemented in the SAGE program (Technoma, Heidelberg), have been described (Moreno et al., 1990).

Results and discussion

Sequence information of rRNA/rDNA from hundreds of pure cultures has been used for the initial designation of primers, in rRNA and rDNA sequence analysis, in the amplification by PCR, in the generation of rcDNA, and in defining numerous, mostly oligonucleotide probes for identification of taxa at various levels (Fig. 1). Sequences obtained from a gene library of a natural sample already provides important information about the phylogenetic position of the organisms from which the sequence originate but it is the probe, that in the end, is needed to screen a gene library for homologous clones (and eventually for enumeration) and to verify the presence (and eventually the physiological status) of the respective organism in the environment. Molecular identification of non-culturable mono-species populations Analysis of 5S rRNA isolated from high resolution polyacrylamide gels was the first straight-forward method in the determination of the phylogenetic complexity of three natural populations with limited species composition. Prokaryotic symbionts detected in marine invertebrates belonging to the gamma sub-class of Proteobacteria; iron-oxidizing bacteria of the beta subgroup of Proteobacteria were isolated from a leaching pond atop a copper recovery dump in New Mexico. Sulfur metabolizing archaeae and Thermus spp. were identified as being members of the Octopus Spring (Yellowstone National Park) community (Olsen et al., 1986). Although the potential for in situ hybridization of cells with taxon-specific oligonucleotides directed against rRNA was indicated in the latter publication, it was not until 1988 that the first protocols became available (Giovannoni et al., 1988; DeLong et al., 1989a).

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Prochloron didemni, a symbiont of didemnid ascidians, was the first pure batch of a non-culturable organism identified by 16S rRNA analysis (Seewaldt and Stackebrandt, 1982). Cells were obtained in sufficient amounts and purity from the host organism to isolate 16S rRNA. Oligonucleotide cataloguing revealed the membership of this species (and of three other species from different hosts) to the cyanobacteria. Certain signature oligonucleotides for members of the genus Prochloron were identified but no attempts were made for in situ studies in this pre-probe era. Chlamydia psittaci was analysed by the reverse transcriptase (RT) sequence technique (Weisburg et al., 1986). Signatures revealed a remote relatedness to members of Planctomycetaceae and this information was later used for the design of a species-specific probe (Gene-Trak, Framingham, MS). Sequence information on M. leprae led to the generation of highly specific rDNA probes (Liesack et al., 1991b), but neither these, nor those developed against a broad spectrum of pathogenic bacteria, including the etiological agent of bacillary angiomatosis (Relman et al., 1990), and A. marginale (Weisburg et al., 1991) were developed for in situ studies. Investigations were also performed on symbionts of the insect pea aphid (Unterman et al., 1989), and current studies involve Wolbachia spp., insect symbionts (H. Robertson, University of Illinois, personal communication) and Cristispira, a symbiont of the American oyster (Pelletier et al., 1991). Environmental studies using probes to determine the presence and abundance of defined species include the rumen tract (Stahl et al., 1988), soil coastal sediments (Devereux et al., 1990), soil (Witt et al., 1989), root nodules of Casuarina (Hahn et al., 1989, 1990), and mycorrhizas (Gardes et al., 1991). Recently, certain non-culturable strains have been analysed by rRNA technologies, and their presence has been verified by fluorescent probes. Halospora obtusa, a symbiont of ciliates, was analysed by gene amplification and subsequent rDNA sequence analysis. Individual cells of the endosymbiont could be detected and differentiated in situ by tetramethylrhodamine-labelled probes (Amann et al., 1991). Similarly, an axenic culture of a bacterial symbiont of shipworms was analysed by RT sequence analysis of a large fragment of the 16S rRNA and the presence of the symbiont within the gills of the host mollusc verified in situ by Texas Red labelled probes (Distel et al., 1991). Molecular identification of communities Initial results of the identification of novel groups of organisms and the complexity of natural microbial communities in environmental samples have already proven the superiority of the molecular approach (mainly 16S rRNA-based studies) over the traditional, microscope- and culture medium-based approach to analyse microbial populations, including their non-culturable and, as yet, not cultivated members. Several groups of organisms, described as non-culturable, have been detected by molecular analysis and their presence in gene libraries verified by molecular probing. Information about their phylogenetic neighbours may, however, provide clues as to how to cultivate them. Although application of the novel techniques described here is still in its infancy, the speed at which methods are adapted to new problems emerging from studies on natural samples is breathtaking (see Methods above, Fig. 1). Analysed communities include the above mentioned thermal environment (e.g. Octopus Spring; Stahl et al., 1985; Ward et al., 1987, 1990a), marine environment (e.g. Sargasso Sea picoplankton; Giovannoni et

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al., 1990), and the Pacific Ocean Aloha site picoplankton (DeLong et al., 1989b). We have recently begun to analyse the bacterial community of an Australian soil sample and part of the data will be discussed in the following section. Towards a molecular analysis of a soil sample

A partial 16S rDNA gene library was generated in phage vector M13 using DNA isolated from Mount Coot-tha soil, Brisbane. Up to now 100 clones have been analysed. Sequences were examined for formation of chimeric gene artifacts (Liesack et al., 1991c) and, in helical regions, for PCR-introduced errors. In an intitial step, about 250 nucleotides downstream from the 5' terminus of the 16S rRNA, containing conserved and highly variable regions, were sequenced and the information compared to the 16S rRNA database. Of these clones, 50% could be assigned to members of the alpha subclass of Proteobacteria, whilst the remaining fall into either known main lines of descent (Woese, 1987), or, constitute new lines (Liesack and Stackebrandt, 1992). A more detailed analysis with members of the alpha proteobacteria revealed the existence of three gene clusters, containing one, two and seven sequences, respectively. Three clones occurred as duplicates (Fig. 2). None of the sequence types is absolutely identical with a 16S rRNA sequence from cultured strains. One cluster (MC 6/MC 23) was found to be related to a group consisting of strain BTAil (a phototrophic symbiont of the legume Aeschynomene; Young et al., 1991), B. japonicum, Rhodopseudomonas palustris, and the cat-scratch disease bacillus. Nucleotide differences ranged between one and four, which is in the same order of magnitude as that found between the cultured strains. The sequence of MC7 is slightly less related, exhibiting five to nine nucleotide differences. The third gene cluster occupies a more isolated position between B. japonicum and relatives and Azorhizobium caulinodans. Intra- and intercluster nucleotide differences ranged from 14 to 21 and 14 to 35, respectively. Consequently, each of the members of gene cluster three appeared to be almost as unrelated to each other as they were to bradyrhizobia, azorhizobia and their relatives. As indicated in previous studies on different natural samples (Britschgi and Giovannoni, 1991; DeLong et al., 1989a) the presence of clusters with closely related genes is confirmed by our data. We can also confirm that the majority of the analysed gene fragments indicate the presence of novel organisms, not isolated so far. This is, most likely, the case for sequences of gene cluster three where variations are too large to account for cistron microheterogeneity. On the other hand, more sequence information is needed from described cultures known to belong to the alpha Proteobacteria. It is worth noting, that alpha Proteobacteria were found to be a major part of the picoplankton communities from the Sargasso Sea (Britschgi and Giovannoni, 1991) and North-Central Pacific Ocean (DeLong et al., 1989b). We have, as yet, neither tried to cultivate the organisms from which the sequences were obtained nor have we developed probes for in situ detection, enumeration, and species/strain identification. Information on the phylogenetic position of the organisms, i.e. their relative closeness to nitrogenfixing and phototrophic strains are, however, important hints for the development of appropriate enrichment cultures. In order to understand the complexity of ecological processes, the successful isolation and verification of an organism from which genetic information has been retrieved is not to be considered a troublesome duty but should have the same priority as the molecular analyses.

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,[

Rhodospi r i ] ] um rubrum Hirschia ba]tica Erythrobacter ] ongus Hyphomicrobi um vu] gare Brucel la abortus Rhi zobi um ] egumi nosarum Rhi zobi um f r e d i i Agrobacteri um tumefaci ens Azorhi zobi um cau] inodans

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MC6/MC23 BTAi ] Figure 2. Phylogenetic relationships of 16S rDNA clones (MC) from Mount Coot-tha soil, Brisbane, Australia, to a-Proteobacteria. Bar represents 3.5% nucleotide substitutions.

Conclusions

(i)

Molecular probes, i.e. antibodies and nucleic acid probes, complement each other in the detection and identification of micro-organisms, including unculturable forms.

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(ii) When sequence information is available, oligonucleotide probes are the tool of choice. Their reaction in hybridization assays is predictable and conditions can be adjusted to guarantee highly selective specificity. Changes in hybridization conditions to lesser stringency may reveal information about the presence of phylogenetic neighbours of the target organism. (iii) Alternatively, stretches of nucleic acids of unknown sequence, amplified by cloning or random PCR, may also serve as probes. Tests for selectivity are generally more laborious. (iv) Short probes are able to penetrate bacterial cell walls to hybridize targets (preferably rRNA) within the cell. This technique allows the identification of single cells in situ e.g. in biopsy, biofilm, glycocalyx and other environmental samples. (v) Probes directed against genes of primary and secondary metabolism allow identification of physiological and biochemical properties in environmental samples and may detect gene transfer and genetic instabilities. (vi) Probes directed against mRNA and rRNA may eventually be able to detect the physiologically active components of a community.

Acknowledgement The experimental part of this communication was supported by a grant from the Australian Research Council (ARC 815-90/91).

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