Sub-seafloor biogeochemical processes and microbial life in the Baltic Sea

The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.     

Microbial activity in biogeochemical gradients – new aspects of research

In April 2004, the German Research Foundation (DFG) and the National Science Foundation (NSF) co‐organized a meeting for US and German junior geoscientists in Washington DC. As an outcome of this Research Conference titled ‘Earth, Fire, Water, Air and Life’, a follow‐up workshop took place in May 2005 at the Eberhard‐Karls University of Tuebingen in Germany. This workshop covered new aspects of research to improve the understanding of steep biogeochemical gradients covering pH changes, redox zones, as well as solute and particulate concentration variations in aqueous systems. Detailed understanding of biogeochemistry in this context delivers new fundamental aspects in interdisciplinary research. Such work is also urgently needed to control ever‐increasing scarcity of water that is to large parts driven by decreasing water quality. Research ideas on gradients in a biogeochemical context that were discussed by a subgroup of biogeochemists during that workshop are summarized and presented here.     

Fibrillarin-like proteins occur in the domain Archaea.

Fibrillarin is found in the nucleolus of Eucarya and associated with small nucleolar RNAs. It is involved in the processing of precursor rRNA. Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanococcus vannielii, have been isolated. The genes were named flpA (fibrillarin-like protein).     

Glycosyltransferases and oligosaccharyltransferases in Archaea: putative components of the N-glycosylation pathway in the third domain of life

The ability of Eukarya, Bacteria and Archaea to perform N -glycosylation underlies the importance and possible antiquity of this post-translational protein modification. However, in contrast to the relatively well-studied eukaryal and bacterial pathways, the archaeal N -glycosylation process is less understood. To remedy this disparity, the following study has examined 56 available archaeal genomes with the aim of identifying glycosyltransferases and oligosaccharyltransferases, including those putatively catalyzing this post-translational processing event. This analysis reveals that while oligosaccharyltransferases, central components of the N -glycosylation pathway, are found across the range of archaeal phenotypes, the N -glycosylation machinery of hyperthermophilic Archaea may well rely on fewer components than do the parallel systems of nonhyperthermophilic Archaea. Moreover, genes encoding predicted glycosyltransferases of hyperthermophilic Archaea tend to be far more scattered within the genome than is the case with nonhyperthermophilic species, where putative glycosyltransferase genes are often clustered around identified oligosaccharyltransferase-encoding sequences.     

Flavin mononucleotide-binding flavoprotein family in the domain Archaea

The protein (AfpA, for archaeoflavoprotein) encoded by AF1518 in the genome of Archaeoglobus fulgidus was produced in Escherichia coli and characterized. AfpA was found to be a homodimer with a native molecular mass of 43 kDa and containing two noncovalently bound flavin mononucleotides (FMNs). The cell extract of A. fulgidus catalyzed the CO-dependent reduction of AfpA that was stimulated by the addition of ferredoxin. Ferredoxin was found to be a direct electron donor to purified AfpA, whereas rubredoxin was unable to substitute. Neither NADH nor NADPH was an electron donor. Ferricyanide, 2,6-dichlorophenolindophenol, several quinones, ferric citrate, bovine cytochrome c, and O(2) accepted electrons from reduced AfpA, whereas coenzyme F(420) did not. The rate of cytochrome c reduction was enhanced in the presence of O(2) suggesting that superoxide is a product of the interaction of reduced AfpA with O(2). Although AF1518 was previously annotated as encoding a decarboxylase involved in coenzyme A biosynthesis, the results establish that AfpA is an electron carrier protein with ferredoxin as the physiological electron donor. The genomes of several diverse Archaea contained afpA homologs clustered with open reading frames annotated as homologs of genes encoding reductases involved in the oxidative stress response of anaerobes from the domain BACTERIA: A potential role for AfpA in coupling electron flow from ferredoxin to the putative reductases is discussed. A search of the databases suggests that AfpA is the prototype of a previously unrecognized flavoprotein family unique to the domain Archaea for which the name archaeoflavoprotein is proposed.     

Biosynthetic pathways, gene replacement and the antiquity of life

The appearance of oxygen in the Earth's atmosphere, a by‐product of oxygenic photosynthesis invented by primitive cyanobacteria, stands as one of the major events in the history of life on Earth. While independent lines of geological data suggest that oxygen first began to accumulate in the atmosphere c. 2.2 billion years ago, a growing body of biomarker data purports to push this date back fully 500 million years, based on the presumption that an oxygen‐dependent biochemistry was functional at this time. Here, we present a cautionary tale in the extension of modern biochemistry into Archean biota, identifying a suite of examples of evolutionary convergence where an enzyme catalysing a highly specific, O₂‐requiring reaction has an oxygen‐independent counterpart, able to carry out the same reaction under anoxic conditions. The anaerobic enzyme has almost certainly been replaced in many reactions by the more efficient and irreversible aerobic version that uses O₂. We suggest that the unambiguous interpretation of Archean biomarkers demands a rigorous understanding of modern biochemistry and its extensibility into ancient organisms.     

RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

Effects of benthivorous fish on biogeochemical processes in lake sediments

1. Studies of aquatic environments have shown that community organisation may strongly affect ecosystem functioning. One common phenomenon is a change in nutrient level following a shift in the fish community composition. Although several hypotheses have been suggested, there is no consensus on which mechanisms are involved. Our study evaluated indirect effects of benthivorous fish on the biogeochemical processes at the sediment–water interface separately from direct effects caused by nutrient excretion or sediment resuspension. 2. We assigned field enclosures to three treatments representing typical pond communities; without fish, addition of approximately 10 small tench or addition of one large bream. After one summer, we monitored the water chemistry, benthic invertebrates and periphyton in the enclosures and sampled sediment cores for laboratory analysis of biochemical process rates (oxygen, phosphorus and nitrogen exchange between sediment and water, and denitrification rate). 3. Fish had strong negative effects on benthic invertebrates, but weaker effects on periphyton, organic content and porosity of the sediment. Moreover, there were significant positive fish effects on both phosphorus and nitrogen concentrations in the water. However, there were no general treatment effects on sediment processes that could explain the treatment effects on water chemistry in the enclosures. 4. Hence, overall treatment effects attenuated along the chain of interactions. We conclude that the observed effect of benthic fish on water chemistry was probably because of direct effects on nutrient excretion or resuspension of sediment. The similarity between bream and tench treatments suggests large niche complementarity despite their different habitat preferences.     

Purine biosynthesis in the domain Archaea without folates or modified folates.

The established pathway for the last two steps in purine biosynthesis, the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) to IMP, is known to utilize 10-formyl-tetrahydrofolate as the required C1 donor cofactor. The biosynthetic conversion of ZMP to IMP in three members of the domain Archaea, Methanobacterium thermoautotrophicum deltaH, M. thermoautotrophicum Marburg, and Sulfolobus solfataricus, however, has been demonstrated to occur with only formate and ATP serving as cofactors. Thus, in these archaea, which use methanopterin (MPT) or another modified folate in place of folate as the C1 carrier coenzyme, neither folate nor a modified folate serves as a cofactor for this biosynthetic transformation. It is concluded that archaea, which function with modified folates such as MPT, are able to carry out purine biosynthesis without the involvement of folates or modified folates.     

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.     

A modulator domain controlling thermal stability in the Group II chaperonins of Archaea

Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122 °C. The C-terminal 15–25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif (⁵²⁸EKEKEKEGEK5³⁷) proved to be a key domain for stabilization at or near 100 °C. Mutations “tuned” the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca²⁺, especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca²⁺. The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.     

C-terminal domain of archaeal O-phosphoseryl-tRNA kinase displays large-scale motion to bind the 7-bp D-stem of archaeal tRNASec

O-Phosphoseryl-tRNA kinase (PSTK) is the key enzyme in recruiting selenocysteine (Sec) to the genetic code of archaea and eukaryotes. The enzyme phosphorylates Ser-tRNA^Sec to produce O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) that is then converted to Sec-tRNA^Sec by Sep-tRNA:Sec-tRNA synthase. Earlier we reported the structure of the Methanocaldococcus jannaschii PSTK (MjPSTK) complexed with AMPPNP. This study presents the crystal structure (at 2.4-Å resolution) of MjPSTK complexed with an anticodon-stem/loop truncated tRNA^Sec (Mj*tRNA^Sec), a good enzyme substrate. Mj*tRNA^Sec is bound between the enzyme’s C-terminal domain (CTD) and N-terminal kinase domain (NTD) that are connected by a flexible 11 amino acid linker. Upon Mj*tRNA^Sec recognition the CTD undergoes a 62-Å movement to allow proper binding of the 7-bp D-stem. This large reorganization of the PSTK quaternary structure likely provides a means by which the unique tRNA^Sec species can be accurately recognized with high affinity by the translation machinery. However, while the NTD recognizes the tRNA acceptor helix, shortened versions of MjPSTK (representing only 60% of the original size, in which the entire CTD, linker loop and an adjacent NTD helix are missing) are still active in vivo and in vitro, albeit with reduced activity compared to the full-length enzyme.     

Not so innocent: Impact of fluorophore chemistry on the in vivo properties of bioconjugates

The combination of targeting ligands and fluorescent dyes is a powerful strategy to observe cell types and tissues of interest. Conjugates of peptides, proteins, and, in particular, monoclonal antibodies (mAbs) exhibit excellent tumor targeting in various contexts. This approach has been translated to a clinical setting to provide real-time molecular insights during the surgical resection of solid tumors. A critical element of this approach is the generation of highly fluorescent bioconjugates that maintain the properties of the parent targeting ligand. A number of studies have found that fluorophores can dramatically impact the pharmacokinetic and tumor-targeting properties of the bioconjugates they are meant to only innocently observe. In this review, we summarize several examples of these effects and highlight strategies that have been used to mitigate them. These include the application of site-specific labeling chemistries, modulating label density, and altering the structure of the fluorescent probe itself. In particular, we point out the significant potential of fluorophores with hydrophilic but net-neutral structures. Overall, this review highlights recent progress in refining the in vivo properties of fluorescent bioconjugates, and we hope, will inform future efforts in this area.     

Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.     

Effect of nutrient loading on biogeochemical processes in tropical tidal creeks

The effect of increased nutrient loads on biogeochemical processes in macrotidal, mangrove-lined creeks was studied in tropical Darwin Harbour, Australia. This study uses an integrative approach involving multiple benthic and pelagic processes as measures of ecosystem function, and provides a comparison of these processes in three tidal creeks receiving different loads of treated sewage effluent. There were significant differences in process rates between Buffalo Creek (BC) (hypereutrophic), which receives the largest sewage loads; Myrmidon Creek (MC) (oligotrophic–mesotrophic) which receives smaller sewage inputs; and Reference Creek (RC) (oligotrophic) which is comparatively pristine. Benthic nutrient fluxes and denitrification were more than an order of magnitude higher and lower, respectively, in BC and denitrification efficiency (DE) was <10%. Pelagic primary production rates were also much higher in BC but respiration exceeded primary production resulting in severe drawdown of O₂ concentrations at night. Hypoxic conditions released oxide-bound phosphorus and inhibited coupled nitrification–denitrification, enhancing benthic nitrogen and phosphorus fluxes, leading to a build-up of excess nutrients in the water column. Poor water quality in BC was exacerbated by limited tidal flushing imposed by a narrow meandering channel and sandbar across the mouth. In contrast to BC, the effect of the sewage load in MC was confined to the water column, and the impact was temporary and highly localized. This is attributed to the effective flushing of the sewage plume with each tidal cycle. Denitrification rates in MC and RC were high (up to 6.83 mmol N m^?2 day^?1) and DE was approximately 90%. This study has identified denitrification, benthic nutrient fluxes and pelagic primary production as the biogeochemical processes most affected by nutrient loading in these tidal creek systems. Physical process play a key role and the combined influence of nutrient loading and poor tidal flushing can have serious consequences for ecosystem functioning.     

A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.