Assimilation of polysaccharides and glucose by major bacterial groups in the Delaware Estuary

The contribution of major bacterial groups to the assimilation of extracellular polymeric substances (EPS) and glucose in the Delaware Estuary was assessed using microautoradiography and fluorescence in situ hybridization. Bacterial groups contributed to EPS and glucose assimilation in part according to their distribution in the estuary. Abundance of the phylogenetic groups explained 35% and 55% of the variation in EPS and glucose assimilation, respectively. Actinobacteria contributed 70% to glucose assimilation in freshwater, while Alphaproteobacteria assimilated 60% of this compound in saline water. In contrast, various bacterial groups dominated the assimilation of EPS. Actinobacteria and Betaproteobacteria contributed the most in the freshwater section, whereas Cytophaga-like bacteria and Alpha- and Gammaproteobacteria participated in EPS assimilation in the lower part of the estuary. In addition, we examined the fraction of bacteria in each group that assimilated glucose or EPS. Overall, the fraction of bacteria in all groups that assimilated glucose was higher than the fraction that assimilated EPS (15 to 30% versus 5 to 20%, respectively). We found no correlation between the relative abundance of a group in the estuary and the fraction of bacteria actively assimilating glucose or EPS; the more active groups were often less abundant. Our results imply that the bacterial community in the Delaware Estuary is not controlled solely by "bottom-up" factors such as dissolved organic matter.     

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. ¹³C-labeled EPS and ¹³C-labeled cellulose were purified from bacterial cultures grown on [¹³C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from ¹³C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.     

Diversity of 16S rRNA genes in metagenomic community of the freshwater sponge Lubomirskia baicalensis

Phylogenetic analysis of the nucleotide sequences of 16S rRNA genes in the metagenomic community of Lubomirskia baicalensis has revealed taxonomic diversity of bacteria associated with the endemic freshwater sponge. Fifty-four operational taxonomic units (OTUs) belonging to six bacterial phyla (Actinobacteria, Proteobacteria (class ??-Proteobacteria and ??-Proteobacteria) Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospira) have been identified. Actinobacteria, whose representatives are known as antibiotic producers, is the dominant phylum of the community (37%, 20 OTUs). All sequences detected shared the maximal homology with unculturable microorganisms from freshwater habitats. The wide diversity of bacteria closely coexisting with the Baikal sponge indicate the complex ecological relationships in the community formed under the unique conditions of Lake Baikal.     

Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([¹⁴C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and Actinobacteria. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

The feeding biology of Hydrobia ventrosa (Montagu). II. Allocation of the components of the carbon-budget and the significance of the secretion of dissolved organic material

The deposit-feeding prosobranch Hydrobia ventrosa (Montagu) was fed ¹⁴C-labelled food for a short period. As food, sterile detritus (homogenously ¹⁴C-labelled, dried barley hay), detritus with attached bacteria, and pure bacteria were used. The distribution of the ingested ¹⁴C was followed for a 24-h period. It was found that the assimilation efficiencies of sterile hay, hay with bacteria, and pure bacteria were 34, 56, and 70 %, respectively. This indicates a significance of bacteria for deposit-feeders. There is a considerable loss of dissolved organic material, in part due to leakage from faecal pellets (13 % of the ingested C in the case of a pure bacterial meal and 7 % of the ingested C in the case of sterile hay). The animals also excrete about 30 % of the assimilated carbon. Excretion of mucus constitutes about 9 % of the assimilated carbon. The fraction of assimilated carbon respired depends on the nature of the food. For sterile hay, hay with bacteria, and pure bacteria the percentage respired was 53, 30, and 38 %, respectively. Growth efficiency is, therefore, higher when protein-rich bacteria are included in the diet.     

Increased Light Availability Reduces the Importance of Bacterial Carbon in Headwater Stream Food Webs

Many ecosystems rely on subsidies of carbon and nutrients from surrounding environments. In headwater streams that are heavily shaded by riparian forests, allochthonous inputs from terrestrial systems often comprise a major part of the organic matter budget. Bacteria play a key role in organic matter cycling in streams, but there is limited evidence about how much bacterial carbon is actually assimilated by invertebrate and fish consumers, and how bacterial carbon assimilation varies among streams. We conducted stable isotope tracer additions of ¹³C-acetate, that is assimilated only by bacteria, and ¹⁵N-ammonium, that is assimilated by both bacteria and algae, in two small, shaded streams in the Adirondack region of New York State, USA. Our goal was to determine whether there is an important trophic link between bacteria and macroconsumers, and whether the link changes when the light environment is experimentally altered. In 2009, we evaluated bacterial carbon use in both streams with natural canopy cover using 10-day dual-isotope tracer releases. The canopy was then thinned in one stream to increase light availability and primary production and tracer experiments were repeated in 2010. As part of the tracer experiments, we developed a respiration assay to measure the δ ¹³C content of live bacteria, which provided critical information for determining how much of the carbon assimilated by invertebrate consumers is from bacterial sources. Some invertebrate taxa, including scraper mayflies (Heptagenia spp.) that feed largely on biofilms assimilated over 70% of their carbon from bacterial sources, whereas shredder caddisflies (Pycnopsyche spp.) that feed on decomposing leaves assimilated less than 1% of their carbon from bacteria. Increased light availability led to strong declines in the magnitude of bacterial carbon fluxes to different consumers (varying from ?17 to ?91% decrease across invertebrate taxa), suggesting that bacterial energy assimilation differs not only among consumer taxa but also within the same consumer taxa in streams with different ecological contexts. Our results demonstrate that fluxes of bacterial carbon to higher trophic levels in streams can be substantial, that is over 70% for some taxa, but that invertebrate taxa vary considerably in their reliance on bacterial carbon, and that local variation in carbon sources controls how much bacterial carbon invertebrates use.     

Measurement of Glucose Utilization by Pseudomonas fluorescens That Are Free-Living and That Are Attached to Surfaces

The assimilation and respiration of glucose by attached and free-living Pseudomonas fluorescens were compared. The attachment surfaces were polyvinylidene fluoride, polyethylene, and glass. Specific uptake of [¹⁴C]glucose was determined after bacterial biomass was measured by (i) microscopic counts or (ii) prelabeling of cells by providing [³H]leucine as substrate, followed by dual-labeling scintillation counting. The glucose concentration was 1.4, 3.5, 5.5, 7.6, or 9.7 μM. Glucose assimilation by cells which became detached from the surfaces during incubation with glucose was also measured after the detached cells were collected by filtration. The composition of the substratum had no effect on the amount of glucose assimilated by attached cells. Glucose assimilation by attached cells exceeded that by free-living cells by a factor of between 2 and 5 or more, and respiration of glucose by surface-associated cells was greater than that by free-living bacteria. Glucose assimilation by detached cells was greater than that by attached bacteria. Measurements of biomass by microscopic counts gave more consistent results that those obtained with dual-labeling, but in general, results obtained by both methods were corroborative.     

The Native Bacterioplankton Community in the Central Baltic Sea Is Influenced by Freshwater Bacterial Species

The Baltic Sea is one of the largest brackish environments on Earth. Despite extensive knowledge about food web interactions and pelagic ecosystem functioning, information about the bacterial community composition in the Baltic Sea is scarce. We hypothesized that due to the eutrophic low-salinity environment and the long water residence time (>5 years), the bacterioplankton community from the Baltic proper shows a native “brackish” composition influenced by both freshwater and marine phylotypes. The bacterial community composition in surface water (3-m depth) was examined at a single station throughout a full year. Denaturing gradient gel electrophoresis (DGGE) showed that the community composition changed over the year. Further, it indicated that at the four extensive samplings (16S rRNA gene clone libraries and bacterial isolates from low- and high-nutrient agar plates and seawater cultures), different bacterial assemblages associated with different environmental conditions were present. Overall, the sequencing of 26 DGGE bands, 160 clones, 209 plate isolates, and 9 dilution culture isolates showed that the bacterial assemblage in surface waters of the central Baltic Sea was dominated by Bacteroidetes but exhibited a pronounced influence of typical freshwater phylogenetic groups within Actinobacteria, Verrucomicrobia, and Betaproteobacteria and a lack of typical marine taxa. This first comprehensive analysis of bacterial community composition in the central Baltic Sea points to the existence of an autochthonous estuarine community uniquely adapted to the environmental conditions prevailing in this brackish environment.     

“Endomicrobia” and Other Bacteria Associated with the Hindgut of Dermolepida albohirtum Larvae

Symbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individual Dermolepida albohirtum third-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal and Actinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in all D. albohirtum larvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the “Endomicrobia,” Firmicutes, Proteobacteria, and Actinobacteria and were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects.     

Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary

The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.     

Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell ³H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect ³H-leucine incorporation in light–dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance.     

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment,Stem Colonization,and Virulence

Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium''s gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum''s sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.     

Molecular Fingerprint and Dominant Environmental Factors of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Sediments from the Yellow River Estuary,China

Nitrite-dependent anaerobic methane oxidation (n-damo) is performed by “Candidatus Methylomirabilis oxyfera” (M. oxyfera), which connects the carbon and nitrogen global nutrient cycles. In the present study, M. oxyfera-like bacteria sequences were successfully recovered from Yellow River Estuary sediments using specific primers for 16S rRNA and pmoA genes. A M. oxyfera-like sequences analysis based on the 16S rRNA gene revealed greater diversity compared with the pmoA gene; the 16S rRNA gene sequences retrieved from the Yellow River Estuary sediments belong to groups A as well as B and were mainly found in freshwater habitats. Quantitative PCR showed that 16S rRNA gene abundance varied from 9.28±0.11×10³ to 2.10±0.13×10⁵ copies g^-1 (dry weight), and the pmoA gene abundance ranged from 8.63±0.50×10³ to 1.83±0.18×10⁵ copies g^-1 (dry weight). A correlation analysis showed that the total organic carbon (TOC) and ammonium (NH₄ ⁺) as well as the ratio of total phosphorus to total nitrogen (TP/TN) influenced the M. oxyfera-like bacteria distribution in the Yellow River Estuary sediments. These findings will aid in understanding the n-damo bacterial distribution pattern as well as their correlation with surrounding environmental factors in temperate estuarine ecosystems.     

Yield and physicochemical properties of EPS from Halomonas sp. strain TG39 identifies a role for protein and anionic residues (sulfate and phosphate) in emulsification of n‐hexadecane

In this study, we investigated the yield and physicochemical properties of the high molecular weight extracellular polymeric substance (HMW–EPS) produced by Halomonas sp. strain TG39 when grown on different types and ratios of substrates. Glucose (1% w/v) and a peptone/yeast extract ratio of 5.1 (0.6% w/v final concentration) yielded an EPS fraction (HMW‐glucose) exhibiting the highest anionic activity (20.5) and specific emulsifying activity (EI₂₄ = 100%) compared to EPS produced by cells grown on mannitol, sucrose, malt extract or no carbon source. The HMW–EPS fractions were capable of binding ≈255–464 mg of methylene blue (MB) per gram of EPS, which represents the highest reported binding of MB by a bacterial EPS. A comparative evaluation of these properties to those of commercial hydrocolloids indicated that the combined effect of protein and anionic residues of the HMW–EPS contributed to its ability to emulsify n‐hexadecane. Liquid chromatography revealed the HMW‐glucose EPS to be a heterogeneous polymer with a polydispersity index of 1.8. This work presents evidence of a correlation between the anionic nature and protein content of bacterial EPS with its emulsifying qualities, and identifies EPS produced by strain TG39 as a high MB‐binding bacterial sorbant with potential biotechnological application. Biotechnol. Bioeng. 2009;103: 207–216. © 2008 Wiley Periodicals, Inc.     

Methylovorus sp. MP688 exopolysaccharides contribute to oxidative defense and bacterial survival under adverse condition

Methylovorus sp. MP688 is an aerobic bacterium that can grow on reduced C1 compounds such as methanol, being regarded as an attractive producer for many commercial materials including polysaccharides. The aim of the study was to learn more information about the biochemical and physiological functions of extracellular polysaccharides (EPS) produced by Methylovorus sp. MP688. Firstly, gene clusters involved in EPS synthesis were identified by whole genome sequence analysis. Then EPS produced by Methylovorus sp. MP688 were isolated and purified by centrifugation, precipitation and deproteinization. Purified EPS displayed antioxidant activity towards DPPH free radical, hydroxyl radical and superoxide anion radical. Glucose, galactose and mannose were identified to be main component monosaccharides in EPS. One mutant with defect in EPS production was obtained by knocking out epsA gene within EPS synthesis cluster. Strain with deletion of epsA exhibited compromised growth ability in the presence of oxidative stress due to the sharp reduction in EPS synthesis. Meanwhile, the intracellular antioxidant scavengers were activated to a higher level in order to counteract with the excess harmful radicals. In addition, EPS were assimilated by Methylovorus sp. MP688 to survive under disadvantage condition when the preferred carbon source was exhausted. It was reasonable to conclude that EPS produced by Methylovorus sp. MP688 contributed to oxidative defense and bacterial survival under adverse condition.     

Bacterial Community Responses to Soils along a Latitudinal and Vegetation Gradient on the Loess Plateau,China

Soil bacterial communities play an important role in nutrient recycling and storage in terrestrial ecosystems. Loess soils are one of the most important soil resources for maintaining the stability of vegetation ecosystems and are mainly distributed in northwest China. Estimating the distributions and affecting factors of soil bacterial communities associated with various types of vegetation will inform our understanding of the effect of vegetation restoration and climate change on these processes. In this study, we collected soil samples from 15 sites from north to south on the Loess Plateau of China that represent different ecosystem types and analyzed the distributions of soil bacterial communities by high-throughput 454 pyrosequencing. The results showed that the 142444 sequences were grouped into 36816 operational taxonomic units (OTUs) based on 97% similarity. The results of the analysis showed that the dominant taxonomic phyla observed in all samples were Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria and Planctomycetes. Actinobacteria and Proteobacteria were the two most abundant groups in all samples. The relative abundance of Actinobacteria increased from 14.73% to 40.22% as the ecosystem changed from forest to sandy, while the relative abundance of Proteobacteria decreased from 35.35% to 21.40%. Actinobacteria and Proteobacteria had significant correlations with mean annual precipitation (MAP), pH, and soil moisture and nutrients. MAP was significantly correlated with soil chemical and physical properties. The relative abundance of Actinobacteria, Proteobacteria and Planctomycetes correlated significantly with MAP, suggesting that MAP was a key factor that affected the soil bacterial community composition. However, along with the MAP gradient, Chloroflexi, Bacteroidetes and Cyanobacteria had narrow ranges that did not significantly vary with the soil and environmental factors. Overall, we conclude that the edaphic properties and/or vegetation types are driving bacterial community composition. MAP was a key factor that affects the composition of the soil bacteria on the Loess Plateau of China.     

Compositions isotopiques naturelles des bactéries hétérotrophes et détermination de l'origine du carbone organique dissous biodisponible

Several studies of salt marsh systems have attempted to quantify the flow of organic matter between the land and coastal waters. However, the techniques used could not identify sources of dissolved organic carbon (DOC) rapidly assimilated by heterothrophic bacteria. Recently, the assay of carbon isotope ratios has allowed characterization of the different sources of organic matter in salt marshes. In this study, we wanted to find out if the natural isotopic composition assayed in heterotrophic bacteria distinguished the origin of bioavailable DOC. We determined the δ¹³C values for 1) three bacterial strains and their nucleic acids cultured on glucose and tryptose substrates, respectively, and 2) naturally occurring bacteria recovered from seawater in which salt marsh vegetation had been immersed. First, we showed that the isotopic fractionation was the same for the three bacterial strains cultured on the same synthetic substrate, but could vary depending on the nature of DOC. There was no significant difference between the δ¹³ C values of bacteria and their nucleic acids. Second, natural bacteria were grown in a medium enriched in DOC from halophytic plants. The δ¹³C values of this community were close to those of dissolved organic carbon from plant leachates. The comparison between the isotopic ratios of natural bacteria in Vibrio alginolyticus showed that the heterogeneity of the bacterial community averaged the isotopic fractionation from the preferential assimilation of organic compounds in the medium by each bacterial strain. The δ¹³ C values recorded for the bacterial community in the field and their nucleic acids made it possible to identify the source of organic matter readily accessible to microorganisms in a coastal ecosystem.     

Microbial community of the water column of the Selenga River-Lake Baikal biogeochemical barrier

The microbial communities of the estuarine zone and the mixing zone of river and lake waters in the Selenga River estuary were studied using the fluorescence in situ hybridization (FISH) method. The microorganisms belonging to the phylogenetic group Gammaproteobacteria were found to predominate in the river estuary, constituting up to 17% of the total bacterial community. Among cultivable microorganisms, organotrophic bacteria were predominant (2040 CFU/ml) in this zone, which results in high rates of microbial production (6.0 μg C/(l day). The microbial community structure changed with distance from the river estuary; representatives of the Alpha-, Beta-, and Gammaproteobacteria were present in equal proportions; psychrotolerant and oligotrophic bacteria were numerous. The rate of heterotrophic carbon dioxide assimilation decreased to 3.8 μg C/(l day). At 5–7 km from the river estuary, where the hydrologic, physical, and chemical conditions are similar to those of lake waters, members of the Betaproteobacteria, which are typical of the open waters of Lake Baikal, are the major representatives of planktonic microorganisms.     

Involvement of Cell Surface Structures in Size-Independent Grazing Resistance of Freshwater Actinobacteria

We compared the influences of grazing by the bacterivorous nanoflagellate Poterioochromonas sp. strain DS on ultramicrobacterial Actinobacteria affiliated with the Luna-2 cluster and ultramicrobacterial Betaproteobacteria of the species Polynucleobacter cosmopolitanus. These bacteria were almost identical in size (<0.1 μm³) and shape. Predation on a Polynucleobacter strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of ≥39 days. The differences in predation mortality between the actinobacterial strains and the Polynucleobacter strain clearly demonstrated size-independent grazing resistance for the investigated Actinobacteria. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait.Predation by phagotrophic flagellates is considered (besides the effect of viruses and sedimentation) one of the major mortality factors affecting planktonic bacteria in freshwater and marine environments (13, 24, 28). A large number of investigations demonstrated that bacteria with small cell sizes are less susceptible to grazing mortality caused by flagellates than medium-sized cells (e.g., references 9 and 34). However, even bacterial cells with ultramicrobacterial (<0.1 μm³) cell sizes can be ingested and probably processed by bacterivorous flagellates (4). Interestingly, an increasing number of observations indicate that planktonic Actinobacteria indigenous to freshwater systems are less vulnerable to protistan predation than other taxa of freshwater bacterioplankton. Pernthaler and colleagues observed in a two-stage continuous cultivation system that freshwater Actinobacteria increased in relative and absolute abundance in the presence of a bacterivorous flagellate (26). Other investigations also indicate at least a low level of vulnerability of freshwater Actinobacteria to protistan grazing (17, 29). Experiments with a single actinobacterial strain affiliated with the Luna-2 cluster indicated complete grazing resistance for this freshwater strain against predation by a bacterivorous flagellate (14). Incubation of the strain in the presence of the flagellate over a period of 20 days resulted only in minor changes of bacterial cell numbers. Addition of heat-killed cells of another bacterial species serving as alternative prey resulted even in simultaneous increases of actinobacterial and flagellate numbers. Thus, even the rapidly growing flagellate population was not able to efficiently reduce the actinobacterial cell numbers. Recently, experiments with natural bacterial and protist communities demonstrated a strong negative selection of planktonic Actinobacteria by bacterivorous flagellates (18). In this investigation, the relative abundances of several phylogenetic groups of bacteria inside food vacuoles of flagellate cells and in the surrounding water have been determined. Actinobacterial cells were strongly underrepresented in the food vacuoles compared to their presence in the surrounding water. Currently, it is not known if the observed strong predation resistance of freshwater Actinobacteria is the result of postingestional protection or digestion protection taking place after ingestion of cells by a predator. Furthermore, it is unknown if this grazing protection is a common trait shared by all planktonic Actinobacteria present in freshwater systems or if only some taxa are protected.Importantly, Actinobacteria constitute large fractions (up to 60%) of bacterioplankton in freshwater systems (8) and seem to present a ubiquitous component of freshwater bacterioplankton (8, 23, 37). Almost all actinobacterial taxa identified by cultivation-independent methods so far represent indigenous taxa exclusively known to occur in freshwater habitats (8, 37, 39), and only some of these indigenous actinobacterial taxa are represented by cultivated strains (7, 14-16).The cultivated strains representing indigenous freshwater Actinobacteria provide excellent opportunities for detailed studies of the grazing resistance mechanisms of this ecologically important group of bacteria. Cultivated strains affiliated with the Luna cluster (14), also known as the acII clade (37), currently represent the best-characterized planktonic Actinobacteria indigenous to freshwater systems. Many of the cultivated Luna strains are characterized by C-shaped (selenoid) cells with ultramicrobacterial sizes of <0.07 μm³. An electron microscopy investigation revealed a so-called S-layer at the surfaces of their cells (14). S-layers are monomolecular arrays composed of identical protein or glycoprotein subunits forming crystalline layers at the cell surfaces of many Bacteria and Archaea (30, 35). We hypothesize that this S-layer is involved in the grazing protection of the previously investigated freshwater Actinobacteria.In the study presented here, we investigated (i) if grazing resistance is a common trait among strains of the Luna-2 cluster, (ii) if the grazing protection previously demonstrated for an actinobacterial strain affiliated with the Luna-2 cluster is independent of the small size and the C shape of the cells, and (iii) if the cell surface structure is involved in the grazing resistance mechanism of Luna-2 strains.     

Comparative Transcriptome Analysis Reveals That Lactose Acts as an Inducer and Provides Proper Carbon Sources for Enhancing Exopolysaccharide Yield in the Deep-Sea Bacterium Zunongwangia profunda SM-A87

Many marine bacteria secrete exopolysaccharides (EPSs) that have important ecological and physiological functions. Numerous nutritional and environmental factors influence bacterial EPS production. However, the regulatory mechanisms of EPS production are poorly understood. The deep-sea Bacteroidetes bacterium Zunongwangia profunda SM-A87 can produce high quantities of EPS, and its EPS production is enhanced significantly by lactose. Here, we studied the reasons behind the significant advantage that lactose has over other carbon sources in EPS production in SM-A87. RNA-seq technologies were used to study lactose-regulated genes in SM-A87. The expression level of genes within the EPS gene cluster was up-regulated when lactose was added. Supplement of lactose also influenced the expression of genes located outside the EPS gene cluster that are also involved in EPS biosynthesis. The major glycosyl components of SM-A87 EPS are mannose, glucose and galactose. Genomic metabolic pathway analyses showed that the EPS precursor GDP-mannose can be synthesized from glucose, while the precursor UDP-glucose must be synthesized from galactose. Lactose can provide glucose and galactose simultaneously and prevent glucose inhibition. Lactose can also greatly stimulate the growth of SM-A87. Taken together, lactose acts not only as an inducer but also as a carbohydrate source for EPS production. This research broadens our knowledge of the regulation of EPS production in marine bacteria.