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C , Bacteria in the Water Column of Chesapeake Bay, USA. 111 MARINE ECOLOGY PROGRESS SERIES Vol. 95: 91-102, 1993 Published May 19 Mar. Ecol. Prog. Ser. C,bacteria in the water column of Chesapeake Bay, USA. 111. Immunologic relationships of the type species of marine monomethylamine- and methane-oxidizing bacteria to wild estuarine and oceanic cultures John McN. Sieburthl, Paul W. ~ohnson~,Vernon M. Church3, David C. ~aux~ ' Graduate School of Oceanography. University of Rhode Island. Bay Campus. Narragansett, Rhode Island 02882-1197 Department of Microbiology. University of Rhode Island. Kingston, Rhode Island 02881. USA Newsweek. 444 Madison Avenue, New York. New York 10022, USA ABSTRACT: C, bacteria oxldlzing methane and monomethylamine (MMA) are readily enriched from seawater and associated manne materials, but only Methylomonas pelagica oxidizing methane and Methylophaga marina oxlhzing MMA have been adequately characterized. Antisera with good specificity were prepared against these taxonomically and trophlcally different oceanic methylotrophs as well as against methanol dehydrogenase, a key enzyme presumably present in all aerobic methylotrophs. These antisera were used in an indrect imrnunofluorescence procedure to compare the relationship of these type oceanic species to wild-type cultures from the estuarine waters of Chesapeake Bay (USA) and the open sea. Of the 32 methanotrophic enrichments attempted from the Sargasso Sea, 23 (72 %) yielded methane-oxidizers. Of the 27 isolates obtained in pure culture, 25 (96%) were identical to Methlyomonas pelagica. By contrast, none of the 54 estuarine methane- oxidizers from Chesapeake Bay were identical to M. pelagica. 13 % were related and 87 % were unrelated. The anti-MDH (methanol dehydrogenase) serum reacted to 23 O/a of the wild methanotroph cultures. Of the 18 oceanic methylaminotroph enrichrnents, 44% were indistinguishable from Methylophaga marina, 6 % were related, and 50 % were unrelated. In contrast, of the 41 Chesapeake Bay MMA-oxidizers, 12 % were indistinguishable from M. manna, 17 % were related, while 71 % were unrelated. The anti-MDH antiserum reacted with only 15 % of the wild MMA-oxidizing bacteria. The implications of the taxonomic affinities and trophic requirements of the methylotrophs to their estuar- ine and oceanic distribution and to their anaerobic methanogenic bacterial consorts are discussed. INTRODUCTION oxidizers grow on methanol, apparently formaldehyde, and usually dirnethylamine and trimethylamine in The aerobic obligate methylotrophs belong to dis- addition to MMA. They lack the distinctive cyto- tinct C, bacterial taxa that have a unique Cl meta- membranes of the methane- oxidizers and in thin bolism, while the facultative methylotrophs that can section are indistinguishable from C-C utilizing also utilize C-C bonded organic matter belong to heterotrophs. Two very similar marine species that are diverse and unrelated bacterial taxa (Anthony 1982, serologically indistinguishable, Methylophaga marina Large 1983, Green 1992). The methane-oxidizers grow and Methylophaga thallassica (Janvier et al. 1985), on methane and methanol and have distinctive cyto- have been recognized (Green 1992).The MMA-oxidiz- membranes in thin section (Whittenbury & Dalton ers are ubiquitous, algal associated, and apparently 1981, Green 1992). Only 1 oceanic species, Methylo- concentrate with the nanoalgae in the pycnocline monas pelagica (Sieburth et al. 1987),has been recog- (Sieburth & Keller 1988/89). The role of these aerobes nized (Green 1992). The monomethylamine (MMA)- in creating reduced microniches for their methyl- C3 Inter-Research 1993 Mar. Ecol. Prog. Ser. 95: 91-102,1993 otrophic methanogens has been reported in a prelimi- the nanaoalgae whose osmoprotectant glycine betaine nary way (Sieburth 1987, 1988a) and has been more hydrolyzes to release the methylated amines. The de- fully described for Chesapeake Bay, USA, enrichments tection of methane-oxidizers appears to be sporadic by Sieburth (1993). Studies on the occurrence of since stratification is necessary for pycnocline-unique methylotrophs in oceanic waters have been conducted methanogens (Sieburth 1993, Sieburth et al. 1993) to in this laboratory over the past decade. They have grow, and accumulate to populations that would pro- resulted in the enrichment and attempted mainte- duce enough methane to support cultivable popula- nance of hundreds of methane- and MMA-oxidizing tions of methane-oxidizers. Only 30 % of the estuarine cultures. Characterizing the bacteria obtained in these enrichments were related to the test oceanic species, enrichment cultures includes isolation, examination of indicating that estuarine C, bacteria may be quite dif- ultrastructure using electron microscopy, determina- ferent and deserve further study. This is in contrast to tion of DNA base pair composition and the identi- the oceanic cultures of which 96% of the methano- fication of enzymatic pathways (Janvier et al. 1985, trophs were related to Methylomonas pelagica, while Sieburth et al. 1987, Green 1992). These procedures 44 % of the methylotrophs were related to Methylo- are tedious and prohibitive when large numbers of phaga marina. cultures are involved. When such large numbers of isolates have been obtained, the tendency has been to leave them minimally characterized and unnamed MATERIALS AND METHODS (Whittenbury et al. 1970). A method which can dis- criminate between similar and dissimilar species of Bacterial strains. The species of methylotrophs methylotrophs with a high degree of specificity was tested and their culture collection sources and required in order to describe the estuarine and oceanic numbers are listed in Table 1. The methanotrophs distribution of the type marine species, and to detect were cultured in an atmosphere of 50: 50 methane in unrelated cultures requiring further characterization. air on NMS (nitrate mineral salts) medium (Whitten- The indirect immunofluorescence procedure (Kawa- bury et al. 1970), except for Methylomonas pelagica mura 1977), is a proven technique which has been which was grown on NEM-10 (nitrifying enrichment used to investigate the distribution of diverse trophic medium) agarose (Sieburth et al. 1987). The MMA- groups of bacteria (Reed & Dugan 1978, Ward & Perry oxidizers were cultured on 0.1 % methanol with the 1980, Campbell et al. 1983, Xu et al. 1984). Most of nutrient supplement NEM-l (Sieburth et al. 1987). these studies, however, have concentrated on enumer- Enrichment and isolation procedures. The enrich- ation and distribution of these bacteria in natural ment procedure for the methane-oxidizing bacteria has populations. In order to develop a panel of antibodies been described previously (Sieburth et al. 1987).MMA- that would greatly reduce the effort involved in cate- oxidizing enrichments were supplemented with 0.1 % gorizing our aerobic marine methylotrophic bacteria, 3 (w/v)MMA (Sigma Chemical Co., St. Louis, MO, USA), different antisera were prepared. One antiserum was and 2nd spike, a nutrient supplement identical to NEM- to Methylomonaspelagica, the first methane-oxidizing 1 but lacking ammonia, then tightly capped. Pure cul- bacterium isolated and characterized from the open tures of methane-oxidizers were obtained by repeated Sargasso Sea (Sieburth et al. 1987). A second anti- streaking onto NEM-10 plates gelled with 1.2 % agarose serum was also to an open Sargasso Sea culture of (Type I, Sigma Chemical Co.) and incubation in an ours, B3P, that is indistinguishable from Methylophaga atmosphere of 50:50 methane in air, until only 1 marina (Janvier et al. 1985), at least serologically. A morphological type of colony was observed. MMA-oxi- third antiserum was against methanol dehydrogenase dizers were isolated on 0.1 % MMA/2nd spike agarose (MDH), the key enzyme reported to be present in all plates incubated in air. Pure cultures of heterotrophs methylotrophic bacteria (Anthony 1982, Large 1983).A utilizing C-C bonded organic matter were isolated number of different pure cultures of C, bacteria that from methane- and MMA-oxidizing liquid enrichment included methane-oxidizers and MMA-oxidizers, as cultures on Oppenheimer-Zobell agar plates with well as C-C utilizing heterotrophs, were first used to reduced peptone content (the OZR agar of Sieburth test the specificity of the antisera. The main body of 1967).All cultures were incubated at room temperature. this study was a comparison of methane-oxidizing and Sources of enrichments. Xenic algal cultures used as MMA-oxidizing enrichment cultures from Chesa- an inoculum during the fall of 1984 were from the peake Bay with oceanic forms, and is based on the sources noted. The CB cultures were obtained from thesis of Church (1987). samples taken in upper Chesapeake Bay in May 1986, This is a first serological look at both trophic forms of and along the length of Chesapeake Bay in May 1987, aerobic marine methylotrophs. The MMA-oxidizers as described in Sieburth (1993).Stratified tank No. 9 at appear to be ubiquitous due to the ubiquitous nature of the Marine Ecosystems Research Laboratory (MERL, Sieburth et al.: Immunologic study of water-column methanotrophs and rnethylotrophs 93 Table 1. Observed reactions of type cultures of methane- and monomethyl- obtained with vertical profiles during the amine (MMA)-oxidizersto the 3 test antisera. M.p. h4ethylomonaspelagica, VERTEX 5 sedimentation experiments in MDH: methanol dehydrogenase; B3P. an open Sargasso Sea culture which is indistinguishable from A4ethylophaga marina the northeast Pacific in June 1984 (Martin
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