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> Section III. Tools, methodologies, instrumentation, approaches

> Chapter 5. Culturing the “Uncultur ables” Oceanography

New Cultivation Strategies Bring More , Volume 2, a quarterly 20, Number The O journal of Microbial Plankton into the Laboratory

By Stephen J. Giovannoni, R achel A . Foster , Michael S. R appé, and Sl ava Epstein

The Importance of Cultures from pure cultures, and cultures often had been described, but there was virtu- in the Age of Genomics offer the best possibilities to test hypoth- ally no certainty that any of them were ceanography Society. Copyright 2007 by The O It is difficult to describe the properties eses that emerge from genome sequences abundant in the ecosystem. The problem of a microorganism without first cul- or ecological studies. Figure 1 illustrates was the plate count anomaly—micro- tivating it. There has been a great deal some of these relationships. scopically counted cells in seawater out- of effort and considerable success in predicting microbial metabolic activi- ties from genome sequences, (i.e., func- ...the most efficient way to gather tional genes), assembling genomes from

information about a microorganism is ceanography Society. A metagenomic data, testing the activities ceanography Society. Send all correspondence to:ceanography Society. Send [email protected] Th e O or of cells in situ, and from studying com- to study it in culture and relate that plex microbial assemblages in the labora- knowledge to field observations tory. But, the fact remains that the most ll rights reserved. Permission is reserved. ll rights granted to in teaching copy this and research. for use article Republication, systemmatic reproduction, efficient way to gather information about a microorganism is to study it in culture The concept of “the assay” is an numbered colonies on plates by four and relate that knowledge to field obser- important part of microbial cultivation. orders of magnitude, leaving scientists in vations. Genomes are easily determined It is a rare microbial cultivation experi- a quandary as to whether their isolates ment that does not succeed in growing were important, but rarely formed colo- Stephen J. Giovannoni (steve. something. The question is: what are you nies, or were rare specialists that could [email protected]) is Professor, looking for? For decades, phytoplank- endure the conditions on a Petri dish. Department of Microbiology, Oregon State ton cultivation was a far more successful The solution to this problem came University, Corvallis, OR, USA. Rachel a. endeavor than the cultivation of marine from ribosomal RNA technology, ceanography Society, P Foster is Postdoctoral Research Scientist, simply because phytoplankton which was applied to measure micro- Lamont-Doherty Earth Observatory of could be identified by their morphology bial diversity in most major ecosystems, Columbia University, Palisades, NY, USA. or fluorescent properties. Bacteria are including the ocean (Olsen et al., 1986). Michael S. Rappé is Assistant Research hard to tell apart by their appearances, Ribosomal RNA sequences gave lists of Box 1931, Rockville, M D 20849-1931, U 1931, Rockville, O Box Professor, Hawaii Institute of Marine so being handy with a microscope wasn’t , a code for their identifica- Biology, SOEST, University of Hawaii, very helpful for determining whether a tion, and their placement in phyloge- Kaneohe, HI, USA. Slava Epstein is heterotrophic microbial isolate growing netic trees. As it turned out, most of the Associate Professor, Department of Biology on a plate was a rare community mem- organisms detected by this approach and Marine Science Center, Northeastern ber or an abundant species. By 1990, had no binomial name and had never University, Boston, MA, USA. lots of heterotrophic marine bacteria been described, ostensibly because S A .

62 Oceanography Vol. 20, No. 2 they were “uncultivatable” (Rappé and Geochemistry Giovannoni, 2003). For marine bacteria, the one excep- tion to the rule above was the oxygenic phototrophs—unicellular Metagenomics Pre-Genomics and prochlorophytes—which were first described in 1979 and 1988 (Chisholm et al., 1988; Waterbury et al., 1979). These very abundant organisms were not easy Genome Physiology to culture. But, they became the targets Sequence In Culture of focused cultivation efforts because Post-Genomics their abundance could be confirmed by Figure 1. The role of microbial cultivation and genomics in oceanographic science. optical methods that detected their auto- Genomics augments laboratory studies of microbial physiology that have the goal of predicting the geochemical activities of cells in nature. Metagenomics circum- fluorescence. This example illustrates the vents the need for cultures, but without cultures, hypotheses that arise from the importance of cultivation approaches scrutiny of genome sequences are more difficult to test. that are initiated with prior knowledge of the target organisms. Recent success in the cultivation of abundant heterotrophic phology and color; ability to perform and frequently able to use nitrate as a bacterioplankton taxa could not have fermentative growth; and overall base terminal electron acceptor for facultative been successful without the large data- composition, among others. In hind- anaerobic growth. bases of 16S ribosomal RNA sequences sight, it is now generally recognized that The earliest reports to describe and that direct attention to important species. only a select few name microorganisms isolated from present in any given seawater sample seawater placed the more commonly Cultivated Marine possess the ability to replicate and form recovered groups within the genera Microorganisms Before the colonies on solid media containing high Pseudomonas, Vibrio, and Spirillum Ribosomal RNA Revolution levels of organic nutrients, which was the within the family Pseudomonadaceae; Since late in the nineteenth century, predominant medium and mechanism the genera Achromobacter and microbial cells in seawater have been by which marine microbes were tradi- Flavobacterium within the family studied, scrutinized, and enumerated based primarily on their ability to repli- cate and form colonies on solid growth ...cultures often offer the best possibilities media, as well as morphological charac- teristics of cells observed under a micro- to test hypotheses that emerge from scope. For as long as these cells have genome sequences or ecological studies. been domesticated in the laboratory, microbiologists have also sought to pro- vide them names via the development of tionally domesticated. This highly selec- Achromobacteriaceae; and the genus identification, description, and classifica- tive methodology resulted in the recov- Bacillus within the family Bacillaceae tion systems (i.e., a ) appropri- ery of isolates with fairly uniform gross (see ZoBell, 1946, for a review). As addi- ate for marine microbes. Early systems traits: the vast majority were Gram- tional morphological, physiological, relied upon such characteristics as cell negative chemoorganotrophs, motile via and molecular (i.e., DNA base com- morphology; motility; presence, number, polar or peritrichous (uniformly distrib- position) traits were investigated over and positioning of flagella; colony mor- uted) flagella, straight or curved rods, the ensuing 30 years, many genera and

Oceanography June 2007 63 species of marine origin were redistrib- related clades (groups of organisms New Approaches Provide uted, renamed, or created de novo to with a common ancestor, or a mono- Novel Isolates accommodate newly discovered isolates phyletic group) of the gamma subclass New methods are leading to the isola- and groupings. For example, in 1957, of the . One clade con- tion of many new microbial species, Campbell created the genus Beneckea sists of the closely related genera Vibrio including strains that are important to to include marine microorganisms pos- and Photobacterium (Baumann and oceanographers. These methods include sessing a straight-rod cellular morphol- Schubert, 1984), while the second con- simple modifications to traditional cul- ogy with peritrichous flagellation, fac- sists of the original members of the tivation strategies, development of high- throughput culturing (HTC), combining cultivation with culture-independent methods for quick identification of iso- New methods are leading to the isolation lates, design and use of novel growth of many new microbial species, including chambers, and application of single-cell isolation procedures (Figure 2). strains that are important to oceanographers. The simplest modifications to tradi- tional media recipes, such as lowering the nutrient concentrations, amending ultatively anaerobic metabolism, and genus Alteromonas, plus newly cultivated nontraditional sources of nutrients, and the ability to utilize chitin and grow strains that have been subdivided into increasing incubation times, led to the fermentatively on glucose with the pro- several different genera based in part on successful isolation of previously uncul- duction of acid but no gas (Baumann 16S rRNA gene sequence comparisons. tured bacteria (Connon and Giovannoni, et al., 1971; Campbell, 1957), though Genera in this clade include the original 2002; Eilers et al., 2001; Joseph et al., in 1980, it was subsequently abolished Alteromonas plus Pseudoalteromonas, 2003; Stevenson et al., 2004). Ultimately, and its species included within the Marinomonas, Shewanella, Glaciecola, replicating the natural conditions (e.g., genus Vibrio (Baumann et al., 1980; Idiomarina, Colwellia, Thalassomonas, temperature, light, nutrients) as much Campbell, 1957). The end result of the Oceanimonas, Ferrimonas, and as possible should lead to a higher prob- era in marine microbiology preced- Psychromonas, among others (Ivanova ability of isolation. Furthermore, a good ing the widespread use of ribosomal et al., 2004). A second noteworthy dis- understanding of a target’s metabolism RNA sequences in systematic stud- covery was that the characteristic helical is useful and a prerequisite for medium ies was that most marine bacterial cellular morphology considered to be modifications; however, for the major- species were included within a fairly the main delineating trait of members of ity of uncultured representatives, only a limited number of genera, including the genus Oceanospirillum proved to be 16S rRNA sequence is known. Vibrio, Pseudomonas, Oceanospirillum, polyphyletic: species originally assigned The isolation of two novel pelagic Aeromonas, Deleya, Flavobacterium, to the genus Oceanospirillum have been gamma-Proteobacteria strains, KT71 and Alteromonas, and Marinomonas. shown to form several separate lineages NOR5, from the North Sea was achieved The ribosomal RNA revolution in the gamma and alpha subclasses of by reducing the inorganic N and P brought with it a gradual refinement the Proteobacteria based on 16S rRNA sources in the growth media to the ambi- to the inter- and intra-generic relation- gene sequence comparisons, and have ent levels in coastal waters (Eilers et al., ships of cultivated marine microorgan- since been placed into a variety of new 2001). In addition to reducing nutrient isms that continues to the present day. genera, including Marinospirillum, concentrations, prolonging the incuba- One noteworthy discovery was that a Pseudospirillum, Oceanobacter, and tion period while continually removing majority of the named species of cul- Terasakiella (Satomi et al., 2002; newly formed colonies from the growth turable marine bacteria falls within two Satomi et al., 1998). plates was instrumental in the isola-

64 Oceanography Vol. 20, No. 2 tion of KT71 and NOR5. The removal and other mutually beneficial interac- The better-known examples of tight of colonies allows the slower-growing tions have been known for at least a cen- synergies between groups of differ- targets to compete with faster-growing tury (Schink, 2002; Stams et al., 2006). ent, often unrelated microorganisms (“weed”) populations. The ability of many microorganisms are methanogenic (Chauhan et al., Amendments of nontraditional to synchronize activities by intra- and 2004; Conrad and Klose, 1999; Stams sources of nutrients, signaling molecules, interspecific signaling is a more recently et al., 2005) and anaerobic methane- or inhibitory compounds (for unwanted discovered class of interactions (Bassler, oxidizing consortia (Boetius et al., microorganisms) also show promise. For 2002; Waters and Bassler, 2005). 2000). Concerted biodegradation of example, isolation of several heterotro- phic bacterioplankton strains from the Central Baltic Sea was greatly enhanced by the addition of signal molecules (cyclic AMP and acyl hormoserine lac- tone) (Bruns et al., 2002), and dormant Micrococcus luteus could be resuscitated by a cytokine-like growth-promoting factor from a spent medium of an actively growing culture of the same spe- cies (Mukamolova et al., 1998). In the isolation of the pathogenic bacterium, Mycobacterium tuberculosis (H37Ra), the addition of an aged culture superna- 1 µm 1 µm tant increased the viability and initiated growth of M. tuberculosis in liquid cul- ture (Sun and Zhang, 1999). These examples clearly show that inter- and intraspecific interactions can influence the growth of microbial spe- cies. Microbial communities are viewed 0.25 µm by many as complex and highly struc- tured assemblies of interdependent populations connected to each other through a multitude of interactions that have evolved over billions of years of natural selection. The diverse metabolic capabilities of microbial species create 1 mm 500 µm essentially infinite possibilities for two (or more) microorganisms to perform Figure 2. Examples of recent successful cultured isolates. a–b. DIC (a) and SEM (b) views of a marine isolate uncultivable on standard media but successfully isolated reactions that neither partner alone is fit in diffusion chambers incubated in situ. The strain has 97% 16S rRNA gene similar- to do. Synergies based on commensal- ity to Arcobacter nitrofigilis. Photos by S. Epstein and W. Fowle, Northeastern University isms (symbiotic relationships where one c. SAR11 cells negatively stained alongside a 0.514 micron latex bead. Photo from Rappé et al., 2002 d–e. Tuft and puff morphologies of cultured diazotrophic cyanobacterium, benefits and the other is not Trichodesmium erythraeum (d) and T. thiebautii (e). Photos courtesy of Dr. H.W. Paerl, harmed), cross-feeding, co-metabolism, University of North Carolina at Chapel Hill, Institute of Marine Sciences

Oceanography June 2007 65 complex organic compounds by con- (1 to 10 cells of inoculum per culture) phylogenetic surveys of ribosomal RNA sortia of two and more members is also and examine the cultures for microbial genes cloned from environmental DNA. a well-established fact (Guevara and growth by means of flow cytometry, Among the microorganisms that were Zambrano, 2006; Pettigrew et al., 1990; which is effective for counting very cultured, many were unique cell lineages Sanchez et al., 2005). Often, the interac- dilute populations of cells. Two to sixty and included cultures related to the tions within consortia are based on one percent of cells from marine waters clones SAR11 (alpha-Proteobacteria), partner, or group of partners, stimulat- around Alaska and the Netherlands OM43 (beta-Proteobacteria, related ing the growth and activities of the other were reported to be culturable by this to the methylotroph Methylophilus member(s) of the consortium by provid- method when filtered, autoclaved sea- methylotrophus), SAR92 (gamma- ing them with limiting electron donors, water was used as the medium (Button Proteobacteria), and OM60/OM241 acceptors, or growth factors, or modify- et al., 1993; Schut et al., 1993). This (gamma-Proteobacteria). ing the environment. However, this is work resulted in the description of Some newly described taxa that were unlikely to be a full list of possibilities as two new , Sphingomonas isolated by high throughput dilution- the nature of synergistic interactions in alaskensis and Cycloclasticus oligotrophus to-extinction methods are: Candidatus many other exciting consortia remains (Button et al., 1998; Button et al., 1993; Pelagibacter ubique (SAR11 clade), to be elucidated (e.g., apparently obli- Schut et al., 1993). Croceibacter atlanticus, Parvularcula gate phototrophic consortia) (Overmann S.A. Connon and coworkers reported bermudensis, , and Schubert, 2002). a HTC method with dilution-to- , Oceanicola granu- It follows that at least some micro- extinction procedures in natural sea- losus, Robiginitalea biformata, Pelagibaca bial species may be incapable of grow- water media, and also applied a new bermudensis, Maricola koreensis, Maricola ing in pure culture unless provided with array technology for rapidly screening bermudensis and Litoribacter rhodopsini- metabolites of their natural partners. isolates by epifluorescence microscopy cus (SAR92 clade) (Button, 1998; Cho Because the natural milieu likely con- (Connon and Giovannoni, 2002). This and Giovannoni, 2003a, 2003b, 2003c, tains at least some such metabolites, approach makes use of dilution to isolate 2004; Cho et al., 2004; Rappé et al., 2002; growth media based on naturally occur- the cells into microtiter dish wells. The Schut et al., 1993; Vancanneyt et al., ring components show considerable new arraying procedures are a modifica- 2001; Wang et al., 1996). Similar to a HTC dilution approach, Bruns et al. (2003) developed the Populations that are easily identifiable MicroDrop micro dispenser system, which significantly accelerates extinc- and collected are not always easily tion culturing practices. This system maintained in culture. inoculates 96-well microtiter plates with a highly reproducible droplet vol- ume in less than a minute. When the promise. The use of very small inocula tion of standard methods for counting system was compared with the conven- into these media further enhances cells on polycarbonate membranes, but tional most probable number (MPN) microbial recovery. D.K. Button pio- because of the small dimensions of the approach, culturable efficiencies were neered the development of meth- filtration device, the procedure is effec- systematically lower for the MicroDrop ods for isolating bacterial cultures in tive with small volumes of very dilute technique; however, there is more rep- media made from natural seawater or cultures. Using the arrays and fluorescent lication in the MicroDrop method, lake water (Button et al., 1993). His in situ hybridization (FISH) enabled and therefore higher statistical signifi- approach was to dilute natural commu- experimentalists to focus on high- cance (10 times lower standard devia- nities of microorganisms to extinction priority organisms that emerged from tion) (Bruns et al., 2003). It should be

66 Oceanography Vol. 20, No. 2 noted that both approaches (MPN and A similar approach was adopted by from the original environmental sample. MicroDrop) cultivated similar phylo- Ferrari et al. (2005), where a sediment This suggested the successful isola- genetic groups, including a numerically slurry was filtered onto a polycarbonate tion of subpopulations; however, one dominant, but not-yet-cultured beta- membrane filter and placed on top of a caveat regarding cultivation by the FACS Proteobacterium strain. soil extract. In both systems, exchange approach is the requirement for the use Zengler et al. (2002) describe a of nutrients and other chemicals with of a cell-surface molecule (lipid, protein, novel strategy that separates individual the environment are allowed without carbohydrate) to instill viability of the microbes into microcapsules, then cul- the loss of cells; both led to successful population after sorting. tivates the capsules “together” in a chro- matography column filled with amended growth media at a controlled flow rate. Information, such as a microbe’s Growth is identified by microscopy, and nutritional requirements, enzyme microcapsules indicating growth are sorted by flow cytometry into microtiter expression in situ, and genetic potential, dishes containing organic-rich media. all can be used to predict optimal This type of system allows interactions conditions for cultivation. (i.e., cross-feeding of metabolites or signaling) to occur between the differ- ent microbes during the growth phase cultivation of a significant fraction of Others have sorted populations of in the column while each remains sepa- inoculated cells that did not grow on microbes for cultivation simply by pick- rated in its “caged micro colony” and conventional media. Feasible alternatives ing individuals. For example, the cyano- provides more than 10,000 bacterial iso- for bacterioplankton would be to incu- bacterial symbiont (Calothrix sp. SC01) lates per environmental sample. Zengler bate in situ or to concentrate cells onto a of the diatom Chaetoceros compressus et al. (2002) reported high cultivation membrane filter, then place the filter in was identified by green and blue epifluo- efficiencies for bacterioplankton (and a bottle with growth media. For exam- rescence microscopy, then successfully soil microbes), but no novel cultures ple, planktonic iron-oxidizing bacteria isolated into culture using a micropipette were propagated. were successfully isolated by floating a (unpublished work of author Foster). The continuous search for cultiva- polycarbonate filter on modified media Ziegler et al. (1990) gathered filamentous tion methods that mimic the natural (De Bruyn et al., 1990). bacteria (Gordona, Microthrix) by micro- environment led to moving the cultiva- The innovations described below manipulation with special tools (Blackall tion effort into the environment itself are difficult to classify because they use et al., 1995; Tandoi et al., 1992), and and to developing devices to achieve this “tricks” that involve neither natural others have used laser micro-dissection goal. The majority of these devices have media nor natural environment. Flow technology (Frohlich and Konig, 2000). been successfully applied to non-pelagic cytometry combined with fluorescent Populations that are easily identifi- systems (i.e., sediments). Kaeberlein activated cell sorting (FACS) was used able and collected are not always eas- et al. (2002) designed a simple diffu- to quantify, fractionate, and isolate indi- ily maintained in culture. For example, sion growth chamber with a stainless vidual bacterial communities (Park et al., the colonial diazotroph, Trichodesmium steel washer and two 0.03 µm-pore-size 2005). When this technique was applied spp., evaded pure isolation for decades. polycarbonate membranes. The growth to activated sludge and hydrothermal Its eventual successful isolation required chamber can be incubated in situ, where vent samples, subpopulations of cells frequent monitoring, a specialized diffusion provides the microorgansims obtained by FACS yielded reproducible growth medium, chemical treatment inside the chamber to interact with natu- denaturing gradient gel electrophore- (cycloheximide) during initial isola- rally occurring growth components. sis (DGGE) patterns that were different tion, and subsequent special handling

Oceanography June 2007 67 and culture flasks for maintenance function, and distribution. Information, Williams & Wilkins Co., Baltimore, MD. (Prufert-Bebout et al., 1993). Another such as a microbe’s nutritional require- Blackall, L.L., E.M. Seviour, M.A. Cunningham, R.J. Seviour, and P. Hugenholtz. 1995. ‘Microthrix par- interesting example is populations of the ments, enzyme expression in situ, and vicella’ is a novel deep branching member of the unicellular diazotrophs, Crocosphaera genetic potential, all can be used to pre- Actinomycetes subphylum. Systematic and Applied Microbiology 17:513–518. watsonii (WH8501), formerly known as dict optimal conditions for cultivation. Boetius, A., K. Ravenschlag, C.J. Schubert, D. Rickert, F. Widdel, A. Gieseke, R. Amann, B.B. Jorgensen, U. Witte, and O. Pfannkuche. 2000. A marine micro- bial consortium apparently mediating anaerobic oxidation of methane [see comments]. Nature New technologies specific for cultivation... 407:623–626. Bruns, A., H. Cypionka, and J. Overmann. 2002. hold promise for targeting the isolation Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bac- of microorganisms... teria from the central Baltic Sea. Applied and Environmental Microbiology 68:3,978–3,987. Bruns, A., H. Hoffelner, and J. Overmann. 2003. A novel approach for high throughput cultivation Synechocystis sp. (WH8501), which were In comparison, attention to and focus on assays and the isolation of planktonic bacteria. FEMS Microbiology Ecology 45:161–171. first isolated offshore of the western trop- developing novel techniques in cultiva- Button, D.K. 1998. Nutrient uptake by microorgan- ical Atlantic Ocean in 1985 (Waterbury tion has been disproportionately low. isms according to kinetic parameters from theory as related to cytoarchitecture. Microbiology and and Rippka, 1989); however, they were New technologies specific for cultiva- Molecular Biology Reviews 62:636–645. not recognized as important contribu- tion, including single-cell manipula- Button, D.K., B.R. Robertson, P. Lepp, and T. Schmidt. 1998. A small, dilute cytoplasm, high-affinity, tors in nitrogen fixation until a decade tion devices, high-throughput screening novel bacterium isolated by extinction culture later (Zehr et al., 2001). Still other rel- techniques, and simple modifications to and having kinetics compatible with growth at ambient concentrations of dissolved nutrients in evant examples are the many strains of traditional approaches, hold promise for seawater. Applied and Environmental Microbiology Synechococcus and Prochlorococcus species targeting the isolation of microorgan- 64:4,467–4,476. Button, D.K., F. Schut, P. Quang, R. Martin, and B.R. that have been brought into pure culture. isms, but still require discrete attention Robertson. 1993. Viability and isolation of marine Currently, genomes for 13 Synechococcus to the study of new isolates. bacteria by dilution culture: Theory, procedures, and initial results. Applied and Environmental (WH8102, CC9605, CC9902, PCC7942, Microbiology 59:881–891. PCC9311, WH5701, RS9916, RS9917, Acknowledgements Campbell, L.L. 1957. Genus V. Beneckea. Pp. 328–332 in Bergey’s Manual of Determinative Bacteriology, WH7805, BL107, MIT9220, M11.1, This paper was supported by National R.S. Breed, E.G.D. Murray, and N.R. Smith, M16.17) isolates and 11 Prochlorococcus Science Foundation grants EF-0424599, eds, Seventh Edition. Williams & Wilkins Co., Baltimore, MD. (CCMP1986, MED4, MIT9313, DEB-0207085, OCE-0102248, OCE- Chauhan, A., A. Ogram, and K.R. Reddy. 2004. MIT9312, MIT9211, AS9601, MIT9303, 0221267, and OPP-0214231, and by Syntrophic-methanogenic associations along a nutrient gradient in the Florida Everglades. 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