Freshwater Strains Research Articles

Function and evolution of the psbA gene family in marine Synechococcus: Synechococcus sp. WH7803 as a case study

In cyanobacteria, the D1 protein of photosystem II (PSII) is encoded by the psbA multigene family. In most freshwater strains, a D1:1 isoform of this protein is exchanged for a D1:2 isoform in response to various stresses, thereby altering PSII photochemistry. To investigate PSII responses to stress in marine Synechococcus, we acclimated cultures of the WH7803 strain to different growth irradiances and then exposed them to high light (HL) or ultraviolet (UV) radiation. Measurement of PSII quantum yield and quantitation of the D1 protein pool showed that HL-acclimated cells were more resistant to UV light than were low light- (LL) or medium light- (ML) acclimated cells. Both UV and HL induced the expression of psbA genes encoding D1:2 and the repression of the psbA gene encoding D1:1. Although three psbA genes encode identical D1:2 isoforms in Synechococcus sp. WH7803, only one was strongly stress responsive in our treatment conditions. Examination of 11 marine Synechococcus genomic sequences identified up to six psbA copies per genome, with always a single gene encoding D1:1. In phylogenetic analyses, marine Synechococcus genes encoding D1:1 clustered together, while the genes encoding D1:2 grouped by genome into subclusters. Moreover, examination of the genomic environment of psbA genes suggests that the D1:2 genes are hotspots for DNA recombination. Collectively, our observations suggest that while all psbA genes follow a concerted evolution within each genome, D1:2 coding genes are subject to intragenome homogenization most probably mediated by gene conversion.

Sources for sedimentary bacteriohopanepolyols as revealed by 16S rDNA stratigraphy

Bacteriohopanoids are widespread lipid biomarkers in the sedimentary record. Many aerobic and anaerobic bacteria are potential sources of these lipids which sometimes complicates the use of these biomarkers as proxies for ecological and environmental changes. Therefore, we applied preserved 16S ribosomal RNA genes to identify likely Holocene biological sources of bacteriohopanepolyols (BHPs) in the sulfidic sediments of the permanently stratified postglacial Ace Lake, Antarctica. A suite of intact BHPs were identified, which revealed a variety of structural forms whose composition differed through the sediment core reflecting changes in bacterial populations induced by large changes in lake salinity. Stable isotopic compositions of the hopanols formed from periodic acid-cleaved BHPs, showed that some were substantially depleted in (13)C, indicative of their methanotrophic origin. Using sensitive molecular tools, we found that Type I and II methanotrophic bacteria (respectively Methylomonas and Methylocystis) were unique to the oldest lacustrine sediments (> 9400 years BP), but quantification of fossil DNA revealed that the Type I methanotrophs, including methanotrophs related to methanotrophic gill symbionts of deep-sea cold-seep mussels, were the main precursors of the 35-amino BHPs (i.e. aminopentol, -tetrol and -triols). After isolation of the lake approximately 3000 years ago, one Type I methanotroph of the 'methanotrophic gill symbionts cluster' remained the most obvious source of aminotetrol and -triol. We, furthermore, identified a Synechococcus phylotype related to pelagic freshwater strains in the oldest lacustrine sediments as a putative source of 2-methylbacteriohopanetetrol (2-Me BHT). This combined application of advanced geochemical and paleogenomical tools further refined our knowledge about Holocene biogeochemical processes in Ace Lake.

Chronic effect of NaCl salinity on a freshwater strain of Daphnia magna Straus (Crustacea: Cladocera): A demographic study

Daphnia magna is mainly recognized as a freshwater cladoceran, but there are some strains that grow in brackish waters. The tolerance to salinity of a freshwater strain was assessed at NaCl concentrations of 0, 2, 4, 6, and 7 g L −1. The green microalga Ankistrodesmus falcatus was fed at optimal concentration (4×10 5 cells mL −1). Reproduction and survival were recorded in two experimental series: in the first one, 20 female neonates were individually studied for each treatment. In the second, cohorts of 10 female neonates were distributed in each of five replicates per treatment. In both cases, experiments were conducted over a full life-cycle. The determined 48-h LC 50 for NaCl was 5.48 g L −1, but we recorded reproduction at up to 7 g NaCl L −1. The average clutch size, total progeny, number of clutches, and longevity were significantly reduced by the NaCl concentration ( P < 0.01 ); total progeny ranged from 467 to 25 neonates as edge values for NaCl concentrations of 0–7 g L −1. Inter-brood time was significantly higher for females grown at 7 g NaCl L −1 (3.9 days). The Life Table analysis demonstrates that average lifespan, life expectancy at birth, net reproductive rate and intrinsic rate of growth were also significantly reduced according to NaCl concentration. Based on the results for the two highest NaCl concentrations (6 and 7 g L −1), we conclude that the used D. magna strain was acclimated to develop satisfactorily under concentrations of up to 6 g NaCl L −1; however, the established salinity conditions reduced significantly reproduction and survival in this strain.

Microbial corrosion of galvanized steel by a freshwater strain of sulphate reducing bacteria ( Desulfovibrio sp.)

Microbially influenced corrosion of galvanized steel was investigated exposing coupons (2.0 × 2.0 cm) to culture of sulphate reducing bacteria strain Desulfovibrio sp. The coupons were exposed to the SRB culture for 4, 8, 24, 72, 96, 168, 360 and 744 h along with a control set in uninoculated medium. The results from the present study suggest that SRB were responsible for the corrosion of the galvanized steel. The corrosion rate of galvanized steel was not be correlated with the number of sessile SRB cells. Also Desulfovibrio sp. had an ability to produce EPS (Extracellular Polimeric Substances) and biodegrade the carbohydrate, that is the predominant component of the EPS produced by them. When Desulfovibrio sp. cells were in logarithmic phase, the concentration of dissolved zinc in the bulk solution was very toxic for Desulfovibrio desulfuricans.

Influence of Primary Producers on Bioavailability of Desorption Resistant Organic Pollutants in the Sediments

The experiments were conducted on three freshwater microalgal strains, which were grown in the top chambers of retrofitted filter assemblies. The bottom receivers contained control sediments and two types of 14C-labeled, phenanthrene contaminated sediments. The first contaminated sediment had both the reversible and desorption resistant fractions (referred to as partially washed sediment), while the second contaminated sediment had the desorption resistant fraction only (completely washed sediment). A second set of controls was added to isolate the effect of microalgae. Despite minor variations in toxicity, there was no significant difference (P > 0.05) between the growth curves of the three algal strains. From a bioavailability perspective, there was a significant difference (P < 0.05) in desorption rates in chambers containing microalgae. Pooled data from the three cultures indicated that, for assemblies with desorption resistant sediment (completely washed) and microalgae, the top chamber phenanthrene concentrations were approximately 90–100% of the theoretical maximum concentrations. However, filter assemblies without microalgae had only 5–6% of the theoretical maximum. Results also indicated that approximately 91.7–92.4% of the desorbed phenanthrene is directly sequestered by the microalgae in assemblies with the completely washed sediment. These results indicated that the term “desorption-resistant” fraction from a purely physical perspective may not be truly desorption-resistant in presence of microalgae.

Changes in the community structure and activity of betaproteobacterial ammonia‐oxidizing sediment bacteria along a freshwater–marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.

Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis

Bodonid flagellates (class Kinetoplastea) are abundant, free-living protozoa in freshwater, soil and marine habitats, with undersampled global biodiversity. To investigate overall bodonid diversity, kinetoplastid-specific PCR primers were used to amplify and sequence 18S rRNA genes from DNA extracted from 16 diverse environmental samples; of 39 different kinetoplastid sequences, 35 belong to the subclass Metakinetoplastina, where most group with the genus Neobodo or the species Bodo saltans, whilst four group with the subclass Prokinetoplastina (Ichthyobodo). To study divergence between freshwater and marine members of the genus Neobodo, 26 new Neobodo designis strains were cultured and their 18S rRNA genes were sequenced. It is shown that the morphospecies N. designis is a remarkably ancient species complex with a major marine clade nested among older freshwater clades, suggesting that these lineages were constrained physiologically from moving between these environments for most of their long history. Other major bodonid clades show less-deep separation between marine and freshwater strains, but have extensive genetic diversity within all lineages and an apparently biogeographically distinct distribution of B. saltans subclades. Clade-specific 18S rRNA gene primers were used for two N. designis subclades to test their global distribution and genetic diversity. The non-overlap between environmental DNA sequences and those from cultures suggests that there are hundreds, possibly thousands, of different rRNA gene sequences of free-living bodonids globally.

Crystal-size and shape distributions of magnetite from uncultured magnetotactic bacteria as a potential biomarker

We studied the sizes and shapes of magnetite nanocrystals produced by several types of uncultured magnetotactic bacteria to understand whether their size distributions can be used for identifying the biogenic origin of magnetite crystals in geological samples. The two-dimensional projections of the crystals were measured on transmission electron microscope (TEM) images, and features of crystal-size and shape-factor distributions (CSD and SFD, respectively) were analyzed. In agreement with previous results, most magnetite CSD curves are asymmetric and negatively skewed; however, one magnetotactic strain produced particles that have a normal size distribution. A statistical analysis of CSDs and SFDs (both from this and previous studies) reveals similarities among magnetite from magnetotactic strains from various locations. In particular, crystals in a cultured marine strain (MC-2) were indistinguishable from magnetite from a freshwater strain. We tested whether CSDs of distinct magnetosome types can be recovered from the shape and size data of all particles combined in samples that contain several types of magnetosomes; such samples can be used as models for rocks that contain magnetite nanocrystals of unknown and, presumably, various origins. If the SFDs of the distinct magnetosome types occurring in the same sample differ, the CSDs of individual magnetosome types can be retrieved from bulk data. In such cases the characteristic shape of the size distribution can be used for identifying magnetite as originating from magnetotactic bacteria.

Living under an atomic force microscope

ABSTRACTAn approach for long‐term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution by Atomic Force Microscopy (AFM) was developed in this study.Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response to environmental changes and mineral nucleation at the cell's surface. Continuously cultured cyanobacteria of the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immobilized on poly‐l‐lysine‐coated glass slides. Both immobilization and environmental conditions were optimized in order to facilitate long‐term experiments (&gt; 100 h) with living cells. AFM samples were investigated in situ in two different solutions: Culture medium was used for cultivation experiments and nutrient‐free NaHCO3/CaCl2 solutions (supersaturated with respect to calcite) for long‐term characterizations of the changes in cell surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence microscopy, independently.No indications for extended starvation were found within the relevant timescales. Analysing the influence of Ca2+ on the surface of S. leopoliensis, we found significant changes compared to a Ca‐free solution. Few hours after CaCl2 was added to the circumfluent solution, small protuberances were observed on the cell surface.These are promising results to environmental scientists for a wide range of applications, as cell response to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit from this new approach.

Genetic diversity of goniomonads: an ancient divergence between marine and freshwater species

Goniomonas is a ubiquitous free-living, phagotrophic zooflagellate genus related to the photosynthetic cryptophytes. Only one freshwater and two marine species have been described. Although the two marine species (G. pacifica and G. amphinema) are morphologically quite distinct from each other, it was unclear until recently whether one of them (G. pacifica) is really separate from the freshwater G. truncata because their morphology and size are so similar. We have isolated six new Goniomonas strains and amplified and sequenced their small subunit ribosomal RNA genes. Our results reveal remarkable genetic diversity within all three nominal species and confirm that G. pacifica is genetically very distinct from G. truncata. Within each morphospecies there is more divergence than between many cryptophyte genera and there are many more substantially different genotypes of Goniomonas than established ‘species’. This implies that there are numerous undescribed ‘species’ of Goniomonas and suggests that goniomonad genetic diversity might equal that of the cryptophytes. Phylogenetic analysis of 10 Goniomonas sequences shows two very robust clades, one consisting only of ‘G. truncata’ and the other comprising G. pacifica-related sequences and a separate cluster of genotypes that probably represent G. amphinema. Thus, one clade comprises five freshwater strains, which have markedly longer genes, and the other clade comprises five marine strains. The divergence between all freshwater and all marine sequences is several times greater than that within one morphospecies. This deep genetic divergence implies that for several hundred million years, freshwater goniomonads have not been able to colonize marine habitats effectively, and vice versa. Our trees show that the 18S rRNA gene has evolved in freshwater strains faster than in the marine strains and robustly support the holophyly of all goniomonads and their sister relationship with cryptophytes.

Pitting corrosion of titanium by a freshwater strain of sulphate reducing bacteria ( Desulfovibrio vulgaris)

Pitting corrosion of titanium (ASTM Grade 2) was investigated by exposing coupons (2.0 × 1.5 cm) to a semi-continuous culture of sulphate reducing bacteria (SRB) strain Desulfovibrio vulgaris. The coupons were exposed to the SRB culture for 90 days along with a control set in uninoculated medium. During the course of the experiment sulphide was estimated at intervals of 4 days and the concentrations ranged from 0.2 to 0.4 mM in the bulk medium. Epifluorescence microscopy revealed extensive colonization by the SRB and the cell count ranged from 10 4 to 10 5 cells cm −2. Optical microscopy revealed the presence of two types of pits; large hemispherical pits of ∼2 mm diameter and many micropits. Pitting was not observed in control coupons exposed to SRB free medium. SEM and CSLM pictures showed the corroded titanium surface with several micropits, along with typical rod shaped SRB cells in the pitted regions. EDAX analysis revealed peaks for Ti, O, N, C, Fe and P in the pitted region. XPS data showed clear peaks for titanium sulphur (three states) and phosphorous. The study reveals that at room temperature and in the presence of 0.2–0.4 mM of sulphide, as well as the putative phosphine, SRB can promote pitting corrosion of titanium by formation of titanium sulphide.

Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313.

Cyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low- to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 μmol photon m −2 s −1 and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean ( Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light ( Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. { Nostoc} strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.

Analysis of the hli gene family in marine and freshwater cyanobacteria.

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.

The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella.

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.

Structure, function and regulation of the cyanobacterial high-affinity bicarbonate transporter, BCT1.

The cmpABCD operon of the cyanobacterium Synechococcus sp. strain PCC7942, which is transcribed specifically under CO2-limited growth conditions, encodes an ATP-binding cassette (ABC) transporter involved in HCO3-uptake (designated BCT1). The product of the cmpA gene is a 42-kDa protein anchored to the plasma membrane, which binds HCO3- with high affinity (Kd= 5 µM) and acts as the substrate-binding protein of the transporter. The apparent Km(HCO3-) of BCT1 is 15 µM. BCT1 has the highest affinity for HCO3- among the HCO3- transporters of the Synechococcus strain and is essential for competitive utilization of HCO3- under CO2-limited conditions. BCT1 is closely related to the cyanobacterial nitrate/nitrite transporter (NRT) encoded by the nrtABCD genes. The BCT1 and NRT transporters, together with the putative cyanate transporter of cyanobacteria, comprise a monoanion transporter subfamily in the family of ABC importers. BCT1 and NRT are present in most fresh-water strains of cyanobacteria but seem to be absent in marine cyanobacterial strains. The low CO2-responsive induction of thecmp operon requires a LysR family protein CmpR, which is similar to CbbR (RbcR), the activator of the CO2 fixation operons of chemoautotrophic and purple photosynthetic bacteria.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions.

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O2 l−1. High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO2 fixation above 112 nmol min−1 (mg protein)−1, an about 90% increase in the protein carbon at the expense of CO2 carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)−1 indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with Mr 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb3-type oxidase. A cytochrome c with Mr 12 kDa was detected in the soluble fraction.

Sulfur isotope fractionation during microbial sulfate reduction by toluene-degrading bacteria

Sulfate-reducing bacteria contribute considerably to the mineralization of petroleum hydrocarbons (PHC) in contaminated environments. Stable sulfur isotope fractionation during microbial sulfate reduction was investigated in microcosm experiments with different cultures of sulfate-reducing bacteria for various initial sulfate concentrations using toluene as the sole carbon source. Experiments were conducted with the marine strain Desulfobacula toluolica, the fresh water strain PRTOL1, and an enrichment culture from a PHC-contaminated aquifer. Sulfate reduction rates ranged from 7 ± 1 to 494 ± 9 nmol cm−3 d−1, whereas specific sulfate reduction rates (sSRR) ranged from 8.9 × 10−15 to 3.9 × 10−13 ± 9.2 × 10−14 mol cell−1 d−1. Calculated enrichment factors (ϵ) for the fractionation of stable sulfur isotopes during microbial sulfate reduction ranged from 19.8 ± 0.9 to 46.9 ± 2.1‰. In general, values of ϵ and sSRR obtained in our experiments were similar to those reported previously for sulfate-reducing bacteria incubated with readily available carbon sources under optimal growth conditions. Moreover, we found no obvious correlation between ϵ and sSRR values when data from all our microcosm experiments were combined or when we combined our data with several previously published data sets. In contrast, ϵ values determined in our enrichment culture experiments (average 23.5 ± 4.3‰) agreed well with ϵ values determined in a recent field study performed in situ in a PHC-contaminated aquifer. Thus, results from this laboratory study provide valuable information on stable sulfur isotope fractionation during microbial sulfate reduction under conditions that more closely resemble those in PHC-contaminated environments, i.e., for a variety of sulfate concentrations, including low sulfate concentrations, and for a an important PHC-constituent (toluene) used as sole carbon source.