Non-photosynthetic Bacteria Research Articles

Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non-photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the 'engineering' effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.

Early oxygenation of the terrestrial environment during the Mesoproterozoic

Geochemical data from ancient sedimentary successions provide evidence for the progressive evolution of Earth's atmosphere and oceans. Key stages in increasing oxygenation are postulated for the Palaeoproterozoic era (∼2.3 billion years ago, Gyr ago) and the late Proterozoic eon (about 0.8 Gyr ago), with the latter implicated in the subsequent metazoan evolutionary expansion. In support of this rise in oxygen concentrations, a large database shows a marked change in the bacterially mediated fractionation of seawater sulphate to sulphide of Δ(34)S < 25‰ before 1 Gyr to ≥50‰ after 0.64 Gyr. This change in Δ(34)S has been interpreted to represent the evolution from single-step bacterial sulphate reduction to a combination of bacterial sulphate reduction and sulphide oxidation, largely bacterially mediated. This evolution is seen as marking the rise in atmospheric oxygen concentrations and the evolution of non-photosynthetic sulphide-oxidizing bacteria. Here we report Δ(34)S values exceeding 50‰ from a terrestrial Mesoproterozoic (1.18 Gyr old) succession in Scotland, a time period that is at present poorly characterized. This level of fractionation implies disproportionation in the sulphur cycle, probably involving sulphide-oxidizing bacteria, that is not evident from Δ(34)S data in the marine record. Disproportionation in both red beds and lacustrine black shales at our study site suggests that the Mesoproterozoic terrestrial environment was sufficiently oxygenated to support a biota that was adapted to an oxygen-rich atmosphere, but had also penetrated into subsurface sediment.

Evidence of circadian rhythms in non-photosynthetic bacteria?

Examples of circadian rhythms have been described in eukaryotic organisms and in photosynthetic bacteria, but direct proof of their existence in other prokaryotes is limited and has been largely ignored. The aim of this article is to review existing evidence and to present preliminary results that suggest that the heterotrophic bacterium Pseudomonas putida shows regular variations in its growth pattern synchronized with light/darkness cycles. We put forward the hypothesis that circadian regulation of certain processes can take place in non-photosynthetic prokaryotes and may represent an adaptative advantage in specific environments.

Phylogenetic Substitution Models for Detecting Heterotachy during Plastid Evolution

There is widespread evidence of lineage-specific rate variation, known as heterotachy, during protein evolution. Changes in the structural and functional constraints acting on a protein can lead to heterotachy, and it is plausible that such changes, known as covarion shifts, may affect many amino acids at once. Several previous attempts to model heterotachy have used covarion models, where the sequence undergoes covarion drift, whereby each site may switch independently among a set of discrete classes having different substitution rates. However, such independent switching may not capture biologically important events where the selective forces acting on a protein affect many sites at once. We describe a new class of models that allow the rates of substitution and switching to vary among branches of a phylogenetic tree. Such models are better able to handle covarion shifts. We apply these models to a set of genes occurring in nonphotosynthetic bacteria, cyanobacteria, and the plastids of green and red algae. We find that 4/5 genes show evidence of some form of rate switching and that 3/5 genes show evidence that the relative switching rate differs among taxonomic groups. We conclude that covarion shifts may be frequent during the deep evolution of plastid genes and that our methodology may provide a powerful new tool for investigating such shifts in other systems.

Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria

The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO 2 . A less appreciated function of CO 2 fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO 2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13 C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO 2 and then fixed 68% of this CO 2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO 2 fixation plays a major role in cofactor recycling. When H 2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO 2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N 2 fixation and CO 2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO 2 fixation to recycle reduced cofactors deserves closer scrutiny.

A Brief History of Phytochromes

Photosensory proteins enable living things to detect the quantity and quality of the light environment and to transduce that physical signal into biochemical outputs which entrain their metabolism with the ambient light environment. Phytochromes, which photoconvert between red-absorbing P(r) and far-red-absorbing P(fr) states, are the most extensively studied of these interesting proteins. Critical regulators of a number of key adaptive processes in higher plants, including photomorphogenesis and shade avoidance, phytochromes are widespread in photosynthetic and nonphotosynthetic bacteria, and even in fungi. Cyanobacterial genomes also possess a plethora of more distant relatives of phytochromes known as cyanobacteriochromes (CBCRs). Biochemical characterization of representative CBCRs has demonstrated that this class of photosensors exhibits a broad range of wavelength sensitivities, spanning the entire visible spectrum. Distinct protein-bilin interactions are responsible for this astonishing array of wavelength sensitivities. Despite this spectral diversity, all members of the extended family of phytochrome photosensors appear to share a common photochemical mechanism for light sensing: photoisomerization of the 15/16 double bond of the bilin chromophore.

In situ evidence for pre-capture qualitative selection in the tropical bivalve Lithophaga simplex

Few feeding studies have been performed on tropical bivalves, and in situ feeding studies are lacking altogether. We investigated retention efficiencies for natural particles in the coral-boring tropical mytilid Lithophaga simplex. Using the in situ InEx technique (Yahel et at. 2005; Limnol Oceanogr Methods 3:46-58) SCUBA divers collected samples from the water inhaled and exhaled by undisturbed bivalves at the coral reef of Eilat (Gulf of Aqaba). Particle retention efficiencies were determined using flow cytometry analysis of the paired water samples. The photosynthetic bacterium Synechococcus (0.9 +/- 0.1 mu m) and larger eukaryotic algae (1 to 10 mu m) were preferentially retained by the bivalve with removal efficiencies of up to 90 % (1996 to 2000: averages of 69 +/- 14 % and 60 +/- 17 %, respectively, n = 74 individual bivalves). The minute photosynthetic bacterium Prochlorococcus (0.4 +/- 0.1 mu m) was also moderately retained (41 +/- 19%). Only a small proportion of the non-photosynthetic bacteria (0.3 +/- 0.1 mu m) were retained (5 +/- 18%, median of 9%), despite their numerical dominance in the plankton and considerable size overlap with Prochlorococcus. Size-independent preferential retention was also observed within particle types: (1) L. simplex more efficiently retained Prochlorococcus and picoeukaryotic algal cells with higher chlorophyll content and (2) the small fraction of non-photo synthetic bacteria retained did not differ in size, but bad higher nucleic acid content (compared to the inhaled population) an indicator for viable and active bacteria. We conclude that particle retention is not strictly size-dependent in L. simplex, and probably involves other cell attributes such as cell surface properties and/or motility.

Identification of novel genes putatively involved in the photosystem synthesis of Bradyrhizobium sp. ORS 278

In aerobic anoxygenic phototrophs, oxygen is required for both the formation of the photosynthetic apparatus and an efficient cyclic electron transfer. Mutants of Bradyrhizobium sp. ORS278 affected in photosystem synthesis were selected by a bacteriochlorophyll fluorescence-based screening. Out of the 9,600 mutants of a random Tn5 insertion library, 50 clones, corresponding to insertions in 28 different genes, present a difference in fluorescence intensity compared to the WT. Besides enzymes and regulators known to be involved in photosystem synthesis, 14 novel components of the photosynthesis control are identified. Among them, two genes, hsIU and hsIV, encode components of a protein degradation complex, probably linked to the renewal of photosystem, an important issue in Bradyrhizobia which have to deal with harmful reactive oxygen species. The presence of homologs in non-photosynthetic bacteria for most of the regulatory genes identified during study suggests that they could be global regulators, as the RegA-RegB system.

Carotenoid Biosynthesis in Intraerythrocytic Stages of Plasmodium falciparum

Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. All carotenoids are derived from the C40 isoprenoid precursor geranylgeranyl pyrophosphate, and their chemical and physical properties are associated with light absorption, free radical scavenging, and antioxidant activity. Carotenoids are generally synthesized in well defined subcellular organelles, the plastids, which are also present in the phylum Apicomplexa, which comprises a number of important human parasites, such as Plasmodium and Toxoplasma. Recently, it was demonstrated that Toxoplasma gondii synthesizes abscisic acid. We therefore asked if Plasmodium falciparum is also capable of synthesizing carotenoids. Herein, biochemical findings demonstrated the presence of carotenoid biosynthesis in the intraerythrocytic stages of the apicomplexan parasite P. falciparum. Using metabolic labeling with radioisotopes, in vitro inhibition tests with norflurazon, a specific inhibitor of plant carotenoid biosynthesis, the results showed that intraerythrocytic stages of P. falciparum synthesize carotenoid compounds. A plasmodial enzyme that presented phytoene synthase activity was also identified and characterized. These findings not only contribute to the current understanding of P. falciparum evolution but shed light on a pathway that could serve as a chemotherapeutic target.

Composition and significance of picophytoplankton in Antarctic waters

Filter fractionated picophytoplankton from Antarctic coastal waters (summer 2001) represented only 7–33% of total phytoplankton, even though total stocks were low (average Chl a = 0.32 μg l−1, range = 0.13–1.03 μg l−1). Though all cells passed a 2 μm filter, electron microscopy revealed most cells were over 2 μm, principally Parmales, Phaeocystis sp., and small diatoms. CHEMTAX analysis of HPLC pigment data suggested type 8 haptophytes (e.g. Phaeocystis sp. plus Parmales and pelagophytes) contributed 7–58% of picoplanktonic chlorophyll a, type 6 haptophytes (e.g. coccolithophorids) 18–59%, diatoms 0–18% (mostly type 2 diatoms, e.g. Pseudonitzschia sp., 0–15%), prasinophytes 0–17%, with cell fragments of cryptophytes 0–40%, and dinoflagellates 0–11%. Only stocks of type 8 haptophytes and prasinophytes differed significantly due to successional changes. Zeaxanthin concentrations exceeded estimates from previous cyanobacterial counts and may derive from non-photosynthetic bacteria.

Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana

Homologs of a bacterial β-barrel protein, Omp85, ubiquitously exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Those in non-photosynthetic bacteria and mitochondria are responsible for β-barrel protein sorting to the outer membranes, and thus are essential for viability of the organisms. There are two distinct Omp85 homologs in chloroplasts of the model plant, Arabidopsis thaliana. One of them, Toc75, functions as the main protein import translocation channel, and was shown to be indispensable from a very early stage of embryogenesis. By contrast, the role of another homolog, OEP80, remains elusive. Recently, we showed that disruption of the OEP80 gene causes embryo abortion in A. thaliana at a stage later than that affected by TOC75 knockout. This indicates that the two chloroplastic Omp85 homologs are both essential for viability of plants from very early stages of development, but may have distinct functions. Defining the functional and evolutionary relationships of Toc75 and OEP80 by further studies should advance our understanding of the importance of plastids during embryogenesis, as well as that of the molecular details of plastid biogenesis.Addendum to: Patel R, Hsu SC, Bédard J, Inoue K, Jarvis P. The Omp85-related chloroplast outer envelope protein, OEP80, is essential for viability in Arabidopsis. Plant Physiol 2008; 148:235-45; DOI: 10.1104/pp.108.122754.

“Density equilibrium” method for the quantitative and rapid in situ determination of lipid, hydrocarbon, or biopolymer content in microorganisms

The work provides a simple method, based on a direct density equilibrium measurement, for the rapid in situ estimation of total lipid, hydrocarbon or biopolymer content in a variety of prokaryotic and eukaryotic samples. The method can be readily applied to live microalgae and photosynthetic bacteria, single-celled or colonial microorganisms, as well as cellular fractions and isolated subcellular compartments or components. In this approach, the absolute lipid, hydrocarbon, or biopolymer content of the cells can be readily calculated. This method is especially useful for tracking the oil or polymer content of strains of microalgae and other microorganisms, whose lipid, hydrocarbon or biopolymer content may change with cultivation conditions and/or time, as the case would be in microorganism lipid-induction industrial processes. The method is also useful for the direct in situ measurement of storage polymer accumulation in live cells, such as starch in microalgae and polyhydroxybutyrate, or other polyhydroxyalkanoates, in photosynthetic and non-photosynthetic bacteria.

Identification of Six New Photoactive Yellow Proteins—Diversity and Structure–Function Relationships in a Bacterial Blue Light Photoreceptor†

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per-Arnt-Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP-containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil-dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair-wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly--the absorbance maximum extends from 432 to 465 nm, the pK(a) of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure-function relationships in the PYP family.

Not so simple after all

Biologists have long thought that the internal workings of prokaryotes—the smallest and simplest organisms, including bacteria—are well understood, and have accordingly considered eukaryotic organisms and their cells to be more fascinating objects for study. During the past decade, however, the focus of some research has begun to turn back to prokaryotes—particularly because genomic analyses have shown the enormous flexibility and adaptability of these seemingly simple organisms. Moreover, recent structural research has shown that the cell components of prokaryotes might also be more complex than previously thought. It has long been held that prokaryotic cells lack the internal structure and organization of eukaryotic cells, which have various membrane‐bound organelles, such as mitochondria, chloroplasts and the nucleus. Indeed, prokaryotes are assumed to be the predecessors of some eukaryotic organelles—permanently captured after a period of endosymbiosis. > …there is no rule that says an organelle must be enclosed by a membrane The view that bacterial cells are structurally simpler than those of ‘higher’ organisms is reflected in many text books and web sites with comments such as: “the ribosome is the only prokaryotic organelle”—if, indeed, this highly complex and sophisticated protein‐synthesizing machinery is an organelle in its own right. These types of statement are correct in the sense that prokaryotes contain no membrane‐bound organelles; but there is no rule that says an organelle must be enclosed by a membrane. Many eukaryotic ribosomes are freely suspended in the cytoplasm where they manufacture cytosolic proteins. Furthermore, the word ‘organelle’ itself is being replaced by the terms ‘functional unit’ or ‘compartment’, which are increasingly used to describe coherent structures within a cell that perform clearly defined sets of tasks—such as mitochondria, which create energy by aerobic respiration, or chloroplasts, which do so by photosynthesis. By using this definition, a series of recent discoveries show that prokaryotes also …

An Initial Inventory of Bacteria Found within the Soils and Waters of Great Smoky Mountains National Park

Abstract Soils and waters were collected from Great Smoky Mountains National Park (GSMNP), and bacteria were cultured or DNA cloned from the samples. Oconaluftee Visitors Center and Kephart Prong Trail were sampled to examine distributions of heterotrophs inhabiting streams and riparian soil. Soil from All Taxa Biodiversity Inventory plots at Albright Grove, Cataloochee, and Purchase Knob were also sampled. A total of eleven phyla were detected, of which six were only found via culture-independent techniques. Overall, 69 genera were documented, with differences in their detection in soil and water and by methodology. Firmicutes dominated cultures from soil, while Acidobacteria dominated clone libraries; Bacteroidetes was the dominant phylum in water. Three classes of the phylum Proteobacteria were commonly seen as isolates or clones. Prokaryotic diversity is extraordinary; this is the first inventory examining non-photosynthetic bacteria inhabiting GSMNP and it lays the groundwork for investigations explo...

Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

BackgroundSo far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis.ResultsWe have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i) an L-arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase.ConclusionThe evaluation of 24 cyanobacterial genomes revealed that five different L-arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L-arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways were constitutively expressed with the exception of the transcript for the carbamate kinase, which was substantially up-regulated in cells grown with L-arginine.

Enzymic and mechanistic studies on the conversion of glutamate to 5-aminolaevulinate.

Higher plants, algae, cyanobacteria and several other photosynthetic and non-photosynthetic bacteria synthesize 5-aminolaevulinate by a tRNA(Glu)-mediated pathway. Glutamate is activated at the alpha-carboxyl by ligation to tRNA(Glu) with an aminoacyl-tRNA synthetase. An NADPH-dependent reductase converts glutamyl-tRNA(Glu) to glutamate 1-semialdehyde, which is finally converted to 5-aminolaevulinate by an aminotransferase. These components are soluble and in plants and algae are located in the chloroplast stroma. In plants and algae the tRNA(Glu) is encoded in chloroplast DNA whereas the enzymes are encoded in nuclear DNA. The tRNA(Glu) has a hypermodified 5-methylaminomethyl-2-thiouridine-pseudouridine-C anticodon and probably plays a role in the light-dark regulation of 5-aminolaevulinate synthesis. Ligation of glutamate to tRNA(Glu) requires ATP and Mg2+ and proceeds via a ternary intermediate. Glutamyl-tRNA(Glu) reduction appears to involve formation of a complex. Glutamate 1-semialdehyde non-enzymically synthesized by reductive ozonolysis from gamma-vinyl GABA is used as substrate by the last enzyme. Glutamate-1-semialdehyde aminotransferase contains pyridoxal phosphate as a prosthetic group. The enzyme is converted to spectrally different forms by treatment with 4,5-diaminovalerate or 4,5-dioxovalerate. The pyridoxamine 5'-phosphate form of the enzyme converts (S)-glutamate 1-semialdehyde to 5-aminolaevulinate via 4,5-diaminovalerate through a bi-bi ping-pong mechanism.

SELECTIVE RECOVERY OF MICROALGAE FROM DIVERSE HABITATS USING “PHYTO‐SPECIFIC” 16S rDNA PRIMERS1

Environmental PCR is a common tool for surveying aquatic microalgae; however, universal primers generally employed are not specific to phytoplankton and typically recover nonphotosynthetic bacteria at high frequencies. Using a 16S rDNA “phyto‐specific” primer, we were able to selectively amplify sequences of photosynthetic species from several mixed aquatic samples, even when large numbers of nonphotosynthetic microorganisms were present. We identified 21 microalgal sequences from three different habitats: salt marshes in Virginia, river basins in North Carolina, and sea ice in Alaska. In contrast, universal 16S primers recovered a majority of nonphotosynthetic organisms from some of the same samples. Our results indicate that phytoplankton‐specific primers are efficient in selectively amplifying a broad diversity of microalgae in mixed environmental samples and, therefore, can reduce the noise from extraneous species that often dominates molecular surveys of aquatic samples.

Size independent selective filtration of ultraplankton by hexactinellid glass sponges

Selective feeding by flagellate and ciliate protists is important in shaping microbial communities in pelagic habitats. While less is known of predation on microbial communities in benthic habitats, the abundance and high filtering capacity of sponges suggests that they are key grazers. We studied the feeding preferences of two of the most common glass sponges of North-East Pacific fjords, Rhabdocalyptus dawsoni and Aphrocallistes vastus. Sponges were maintained in large darkened tanks supplied with running seawater from the nearby fjord. The water inhaled and exhaled by the sponges was simultaneously sampled and analyzed using a flow cytometer. Both sponges showed a similar (but not identical) feeding pattern, efficiently removing up to 99 % of the most abundant bacterial cells, whereas clays, silt, and 'debris' particles were expelled into the exhaled water. Filtration efficiencies were maximal for the relatively large and rare eukaryotic algae (3 to 5 μm, 86±9%) and for small non-photosynthetic bacteria (<0.4 pm, 89 ± 10%), while intermediate sized non-photosynthetic bacteria characterized by higher nucleic acid content were efficiently removed in February (92 ± 3%) when overall plankton concentration was low, but not in July (28 ± 16%). The intermediate sized photosynthetic prokaryote Synechococcus (1.1 to 1.5 pm) was also less preferred. Detailed analysis of the ultrastructure of the glass sponge filtration apparatus argues against possible 'by-pass' routes. We suggest that selective filtration involves individual processing, recognition, sorting, and transport of each particle through the sponges' syncytial tissue. Selective grazing by glass sponges, like their pelagic protozoan counterparts, could be an important mechanism shaping microbial communities in the detrital food web of North-East Pacific fjords.

Cyclohexa-1,5-Diene-1-Carbonyl-Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus : Evidence for a Common Benzoyl-CoA Degradation Pathway in Facultative and Strict Anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.