Protozoa as Hosts for Endosymbioses and the Conversion of Symbionts into Organelles1,2

ABSTRACT. Protozoa may be thought of as preadapted to serve as hosts for cellular endosymbionts by virtue of their widespread ability to take up particles by endocytosis. The absence of the cell wall so characteristic of plants and fungi and the commonly large size of most protozoa are additional factors favoring protozoan cells for endosymbioses. The conversion of symbiont into a cellular organelle (e.g. a mitochondrion or chloroplast) is more complicated, especially since the latter do not code for all of their own proteins. Thus, such conversions are held to be rare. Among protozoa, numerous foraminifera appear to have characteristics making them very favorable as hosts for certain algae. Such adaptations, both physiological and morphological in nature, are discussed. Also discussed in this paper are the ways by which (present-day) chloroplasts and mitochondria may have been derived from early endosymbionts: a single ancestral cyanobacterium, in the first case, and a single ancestral purple-nonsulfur bacterium, in the second. Mechanisms for insertion of proteins into and across the organellar membranes had to be evolved for all genes transferred from the symbionts into the host nucleus.     

Bacterial Endosymbioses of Gutless Tube-Dwelling Worms in Nonhydrothermal Vent Habitats

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to γ-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of α-, β-, γ-, and ε-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to β-proteobacteria. An unidentified pogonophoran from the world’s deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.     

Protozoa in Planktonic Food Webs1,2

Protozoa are now being recognized as important members of planktonic food webs. This is due to the inclusion of microbial links in our paradigm of trophic relationships. Heterotrophic microflagellates and ciliates are major grazers of bacteria. They can stimulate production through nutrient recycling and can transform microbial production into larger particles, which are then available for macroconsumers. In this paper we add new groups, the small (< 20 μm) ciliates and myxotrophic flagellates, to the planktonic food web.     

Macroinvertebrate Responses to Algal and Bacterial Manipulations in Streams

Our study was designed to assess the relative importance of algae and bacteria as sources of energy for stream macroinvertebrates. In one experiment, we manipulated algae by artificially shading six sections in each of two streams, one stream with an open canopy (clear-cut drainage basin) and the other with a closed canopy (forested drainage basin); both streams were in Hubbard Brook Experimental Forest, New Hampshire, USA. Chlorophyll a concentrations were reduced from 0.2 to 0.05 μg/cm² in artificially shaded sections of both streams. However, macroinvertebrates showed no response to these algal manipulations in either the clear-cut or forested stream. Nutrient concentrations (N and P) were low and limiting to primary production in both the clear-cut and forested streams. Additionally, both streams had relatively low macroinvertebrate densities suggesting bottom-up controls were important in macroinvertebrate abundance. However, the forested stream did have higher macroinvertebrate densities presumably because of higher inputs of coarse particulate organic matter from the riparian vegetation. In a second experiment, in Augusta Creek, Michigan, we manipulated both algae and bacteria. To reduce algae, we artificially shaded experimental stream channels so that chlorophyll a was reduced from natural levels of 3.0–5.6 to 0.4–0.7 μg/cm². Half of the shaded channels had dissolved organic carbon (DOC – sucrose) dripped into them to raise DOC levels by 2–3 mg/l and thus stimulate bacterial abundance. Open channels, with higher algal abundance, had higher densities of Ephemerella, but only in November when nymphs were larger. Channels with increased DOC had higher bacterial abundances, higher densities of Chironomidae and lower densities of Heptageniidae. Several other macroinvertebrate taxa that were at relatively low abundance in our samples showed no significant response to these manipulations. Our results suggest that early instar Ephemerella may not rely as heavily on algae as later instars. Also, certain taxa were able to use the heterotrophic microbial community, especially chironomids which increased in numbers when bacterial density increased; thus, the bacterial carbon source may be more important to some stream macroinvertebrates than previous studies have suggested.     

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.     

Functional Groups in the Protozoa: Roles in Differing Ecosystems1,2

Feeding habits of freshwater protozoa were used to group species into functional, trophic groups. Community structure in differing ecosystems was examined in relation to the number of species occurring in the functional group categories. Six wetland ecosystems and a large river ecosystem were studied. Changes in community structure during the colonization of artificial substrates were also examined. Changes during colonization were studied in a mesotrophic lake, in low-order streams, and in laboratory microecosystems. In the latter case, the response of colonizing communities to a heavy metal toxicant was studied. All communities studied were dominated by bactivorous-detritivorous species and, to a lesser extent, by photosynthetic species. The chief functional role of substrate-associated protozoans appears to be the processing of dead organic matter and its associated bacterial flora. Functional groups utilizing resources other than detrital or mineral nutrients (saprotrophs, algivores, omnivores, and predators) were always minor community components. Colonizing communities were often dominated by photosynthetic species during early colonization stages but were again dominated by bactivorous-detritivorous species at species equilibrium. Low levels of toxicant (Cd) reduced numbers of both photosynthetic and bactivorous-detritivorous species. Higher toxicant levels virtually eliminated photosynthetic species and reduced bacterial detritivores by over one-half. Roles of protozoan species in ecosystems are closely tied to the processing of detritus and the recycling of mineral nutrients. Enumeration of individuals in functional categories is proposed as a simplified method for studying the abundance and activity of protozoa in ecosystems. Examination of changes in functional group composition and the relationship of functional group abundances to rates of carbon processing are suggested for studies of the importance of protozoa to the flow of energy and materials in ecosystems.     

Effect of Protozoa on Bacterial Degradation of an Aromatic Compound

Prototrophic and growth factor-requiring strains of Alcaligenes spp. were used to study the effect of a protozoan, Tetrahymena pyriformis, on the degradation of p-aminobenzoate. The protozoan inhibited activity of the prototrophic bacterium by reducing its population size. For the growth factor-requiring strain of Alcaligenes, T. pyriformis provided the required growth factors so that the predator permitted the bacteria to grow and to continue p-aminobenzoate degradation. T. pyriformis inhibited bacterial activity when the amino acid supply was in excess, but activity of the auxotrophic strain of Alcaligenes was stimulated by the protozoan when the amino acid supply was limiting, although the bacterial population size was reduced by the protozoan.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO₂ by the tricarboxylic acid cyle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO2 by the tricarboxylic acid cycle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Stimulation of Bacterial DNA Synthesis by Algal Exudates in Attached Algal-Bacterial Consortia

Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus [W. E. C. Moore, E. P. Cato, and L. V. H. Moore, Int. J. Syst. Bacteriol. 35:382-407, 1985]). The organisms were attached to the surfaces at cell densities of approximately 5 × 10⁴ cells cm^-2 (diatoms) and 5 × 10⁶ cells cm^-2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [³H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [³H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 × 10^-21 to 17.9 × 10^-21 mol of [³H]thymidine incorporated cell^-1 h^-1) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon.     

Algal and Bacterial Activities in Acidic (pH 3) Strip Mine Lakes

Reservoir 29 and Lake B are extremely acid lakes (epilimnion pHs of 2.7 and 3.2, respectively), because they receive acidic discharges from coal refuse piles. They differ in that the pH of profundal sediments in Reservoir 29 increased from 2.7 to 3.8 during the period of thermal stratification, whereas permanently anoxic sediments in Lake B had a pH of 6.2. The pH rise in Reservoir 29 sediments was correlated with a temporal increase in H₂S concentration in the anaerobic hypolimnion from 0 to >1 mM. The chlorophyll a levels in the epilimnion of Reservoir 29 were low, and the rate of primary production was typical of an oligotrophic system. However, there was a dense 10-cm layer of algal biomass at the bottom of the metalimnion. Production by this layer was low owing to light limitation and possibly H₂S toxicity. The specific photosynthetic rates of epilimnetic algae were low, which suggests that nutrient availability is more important than pH in limiting production. The highest photosynthetic rates were obtained in water samples incubated at pH 2.7 to 4. Heterotrophic bacterial activity (measured by [¹⁴C]glucose metabolism) was greatest at the sediment/water interface. Bacterial production (assayed by thymidine incorporation) was as high in Reservoir 29 as in a nonacid mesotrophic Indiana lake.     

Influence of Algal Photosynthesis on Biofilm Bacterial Production and Associated Glucosidase and Xylosidase Activities

Natural photosynthetic biofilms were incubated under light (100 mmol m-2 s-1) and dark conditions to elucidate the impact of photosynthesis on bacterial production, abundance, biovolume, biomass, and enzyme activities over 24 h. Use of organic carbon-free media limited carbon sources to algal photosynthesis and possibly the polysaccharides of the biofilm matrix. Bacterial production of biofilm communities was significantly higher in light incubations (p <0.001). The greatest differences in production rates between light and dark incubations occurred between 8 and 24 h. Biomass-specific a- and b-glucosidase and b-xylosidase activities were stimulated by photosynthesis, with significantly greater activities occurring at hours 16 and 24 in the light treatment (p <0.01). The results indicate that algal photosynthesis can have a significant impact on bacterial productivity, biomass, biovolume, and enzyme production over longer time periods at low photon flux densities (?100 mmol m-2 s-1).     

藻华爆发期太湖不同区域沉积物细菌多样性

本研究以藻华爆发期的太湖不同区域沉积物为对象, 测定氮、磷和生物量碳等理化性质, 并通过末端限制性酶切片断长度多态性分析(T-RFLP)的分子生态学手段, 鉴定不同富营养化湖区7个标准采样位点沉积物中细菌群落结构差异。结果表明, 总氮、总磷含量以6#、16#样品最高, 且各形态氮和各形态磷的趋势也与总氮、总磷趋势基本一致; 生物量碳为6#和16#明显高于其他位点, 其余各位点相近。T-RFLP结果显示, 各位点细菌多样性间存在差异, 其中16#样品的多样性指数最低, 6#、7#和1#较高; 在种群组成上各个位点无论是组分还是比例均有显著差异。另外, 所取7个沉积物样品中与氮、磷循环相关的α、β和γ-变形菌纲含量非常丰富, 且与硫、铁循环相关的δ-变形菌纲含量也很丰富。     

Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.     

Algal Exudates and Stream Organic Matter Influence the Structure and Function of Denitrifying Bacterial Communities

Within aquatic ecosystems, periphytic biofilms can be hot spots of denitrification, and previous work has suggested that algal taxa within periphyton can influence the species composition and activity of resident denitrifying bacteria. This study tested the hypothesis that algal species composition within biofilms influences the structure and function of associated denitrifying bacterial communities through the composition of organic exudates. A mixed population of bacteria was incubated with organic carbon isolated from one of seven algal species or from one of two streams that differed in anthropogenic inputs. Pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) revealed differences in the organic composition of algal exudates and stream waters, which, in turn, selected for distinct bacterial communities. Organic carbon source had a significant effect on potential denitrification rates (DNP) of the communities, with organics isolated from a stream with high anthropogenic inputs resulting in a bacterial community with the highest DNP. There was no correlation between DNP and numbers of denitrifiers (based on nirS copy numbers), but there was a strong relationship between the species composition of denitrifier communities (as indicated by tag pyrosequencing of nosZ genes) and DNP. Specifically, the relative abundance of Pseudomonas stutzeri-like nosZ sequences across treatments correlated significantly with DNP, and bacterial communities incubated with organic carbon from the stream with high anthropogenic inputs had the highest relative abundance of P. stutzeri-like nosZ sequences. These results demonstrate a significant relationship between bacterial community composition and function and provide evidence of the potential impacts of anthropogenic inputs on the structure and function of stream microbial communities.