The ecology of pelagic freshwater methylotrophs assessed by a high-resolution monitoring and isolation campaign

Abstract

Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 10(7) cells l(-1) with pronounced peaks in spring and autumn-winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (μmax=0.4 d(-1)), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, 'Candidatus Methylopumilus planktonicus' (LD28) and 'Candidatus Methylopumilus turicensis' (PRD01a001B).

Figure 1

Collapsed maximum likelihood tree of the 16S rDNA of Methylophilaceae. Lineages LD28 (‘Ca. Methylopumilus planktonicus) and PRD01a001B (‘Ca. Methylopumilus turicensis) are shown in gray. The scale bar at the bottom applies to 1% sequence divergence.

Figure 2

Seasonality of abundances (10⁷ cells l⁻¹) of microbes affiliated with LD28 (‘Ca. Methylopumilus planktonicus'. (a) and PRD01a001B (‘Ca. Methylopumilus turicensis'. (b) in Lake Zurich, Switzerland, during the investigation period 2008–2011. Sampling dates and depths are displayed as white circles.

Figure 3

Redundancy analysis of environmental parameters explaining the variability in cell numbers of microbes affiliated with LD28 and PRD01a001B in Lake Zurich. LD28, abundances of LD28; PRD, abundances of PRD01a001B; total chla, total chlorophyll a; P.rub. chla, chlorophyll a associated with Planktothrix rubescens; diat. chla, chlorophyll a associated with diatoms; O₂, oxygen concentrations; NH₄, ammonium concentrations; NO₃, nitrate concentrations; PO₄, phosphate concentrations.

Figure 4

(a) Growth (cells l⁻¹) of isolate MMS-2-53 (‘Ca. Methylopumilus planktonicus') in sterile lake water containing different amounts of methanol. (b, c) Cell volumes (μm³) of strain MMS-2-53 during growth and in situ in Lake Zurich at different depths and during different periods of the year (c, insert). spring incr., LD28 population increase in 5 m depth in spring; spring decl., population decline in 5 m depth in spring; hypolimn., LD28 in the hypolimnion (80 m depth); autumn incr., LD28 population increase in 5 m depth in autumn.

Figure 5

(a) Relative abundance of LD28 in the epilimnion (that is, 0–10 m depth) of Lake Zurich (% of DAPI) and water temperature (°C). (b) Correlation of relative abundances of LD28 to water temperature. (c) Abundance yield (10⁹ cells l⁻¹) of isolate MMS-2-53 grown for 6 weeks at different incubation temperatures and after addition of 100 μM methylamine.

Figure 6

(a, b) Number of protein-coding genes shared between ‘Ca. Methylopumilus planktonicus' MMS-2-53 (a) or ‘Ca. Methylopumilus turicensis' MMS-10A-171 (b) and genome sequenced members of Methylophilaceae and a typical freshwater microbe (Polynucleobacter necessarius subsp. asymbioticus QLW-P1DMWA-1). Protein identity cutoffs of ⩾30% and ⩾70% (minLrap⩾0.8) were applied. (c, d) Reconstruction of methylotrophic pathways of ‘Ca. Methylopumilus planktonicus' MMS-2-53 (c) and ‘Ca. Methylopumilus turicensis' MMS-10A-171 (d). Genes involved in methylotrophy are given in gray (see Supplementary Table S2 for more information). Dashed lines indicate spontaneous reactions, gray lines indicate possible pathways for CH₃X oxidation. unkn. MT, unknown methyltransferase; CH₃OH, methanol; CH₃X, other C1 compounds, for example, methylamine; CH₂O, formaldehyde; HCOOH, formate; H₄F, tetrahydrofolate; H₄MPT, tetrahydromethanopterin; H6P; hexulose-6-phosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone; 6PG, 6-phospho-gluconate; Ru5P, ribulose-5-phosphate; RuMP cycle, ribulose monophosphate cycle.