Abundant SAR11 viruses in the ocean.

Several reports proposed that the extraordinary dominance of the SAR11 bacterial clade in ocean ecosystems could be a consequence of unusual mechanisms of resistance to bacteriophage infection, including 'cryptic escape' through reduced cell size and/or K-strategist defence specialism. Alternatively, the evolution of high surface-to-volume ratios coupled with minimal genomes containing high-affinity transporters enables unusually efficient metabolism for oxidizing dissolved organic matter in the world's oceans that could support vast population sizes despite phage susceptibility. These ideas are important for understanding plankton ecology because they emphasize the potentially important role of top-down mechanisms in predation, thus determining the size of SAR11 populations and their concomitant role in biogeochemical cycling. Here we report the isolation of diverse SAR11 viruses belonging to two virus families in culture, for which we propose the name 'pelagiphage', after their host. Notably, the pelagiphage genomes were highly represented in marine viral metagenomes, demonstrating their importance in nature. One of the new phages, HTVC010P, represents a new podovirus subfamily more abundant than any seen previously, in all data sets tested, and may represent one of the most abundant virus subfamilies in the biosphere. This discovery disproves the theory that SAR11 cells are immune to viral predation and is consistent with the interpretation that the success of this highly abundant microbial clade is the result of successfully evolved adaptation to resource competition.

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

SAR11 marine bacteria require exogenous reduced sulphur for growth.

Sulphur is a universally required cell nutrient found in two amino acids and other small organic molecules. All aerobic marine bacteria are known to use assimilatory sulphate reduction to supply sulphur for biosynthesis, although many can assimilate sulphur from organic compounds that contain reduced sulphur atoms. An analysis of three complete 'Candidatus Pelagibacter ubique' genomes, and public ocean metagenomic data sets, suggested that members of the ubiquitous and abundant SAR11 alphaproteobacterial clade are deficient in assimilatory sulphate reduction genes. Here we show that SAR11 requires exogenous sources of reduced sulphur, such as methionine or 3-dimethylsulphoniopropionate (DMSP) for growth. Titrations of the algal osmolyte DMSP in seawater medium containing all other macronutrients in excess showed that 1.5 x 10(8) SAR11 cells are produced per nanomole of DMSP. Although it has been shown that other marine alphaproteobacteria use sulphur from DMSP in preference to sulphate, our results indicate that 'Cand. P. ubique' relies exclusively on reduced sulphur compounds that originate from other plankton.

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen.

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

A latitudinal diversity gradient in planktonic marine bacteria.

For two centuries, biologists have documented a gradient of animal and plant biodiversity from the tropics to the poles but have been unable to agree whether it is controlled primarily by productivity, temperature, or historical factors. Recent reports that find latitudinal diversity gradients to be reduced or absent in some unicellular organisms and attribute this to their high abundance and dispersal capabilities would suggest that bacteria, the smallest and most abundant organisms, should exhibit no latitudinal pattern of diversity. We used amplified ribosomal intergenic spacer analysis (ARISA) whole-assemblage genetic fingerprinting to quantify species richness in 103 near-surface samples of marine bacterial plankton, taken from tropical to polar in both hemispheres. We found a significant latitudinal gradient in richness. The data can help to evaluate hypotheses about the cause of the gradient. The correlations of richness with latitude and temperature were similarly strong, whereas correlations with parameters relating to productivity (chlorophyll, annual primary productivity, bacterial abundance) and other variables (salinity and distance to shore) were much weaker. Despite the high abundance and potentially high dispersal of bacteria, they exhibit geographic patterns of species diversity that are similar to those seen in other organisms. The latitudinal gradient in marine bacteria supports the hypothesis that the kinetics of metabolism, setting the pace for life, has strong influence on diversity.

Identification of candidate structured RNAs in the marine organism 'Candidatus Pelagibacter ubique'.

BACKGROUND: Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. RESULTS: To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. CONCLUSION: This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task.

Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11.

The genome sequence of the marine bacterium 'Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that 'Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 microM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 x 10(6) cells ml(-1) to 8.16 x 10(6) cells ml(-1) (R(2) = 0.992). Serine was capable of substituting for glycine at 1.5 microM. 'Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that 'Cand. P. ubique' is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.

Local similarity analysis reveals unique associations among marine bacterioplankton species and environmental factors.

MOTIVATION: Characterizing the diversity of microbial communities and understanding the environmental factors that influence community diversity are central tenets of microbial ecology. The development and application of cultivation independent molecular tools has allowed for rapid surveying of microbial community composition at unprecedented resolutions and frequencies. There is a growing need to discern robust patterns and relationships within these datasets which provide insight into microbial ecology. Pearson correlation coefficient (PCC) analysis is commonly used for identifying the linear relationship between two species, or species and environmental factors. However, this approach may not be able to capture more complex interactions which occur in situ; thus, alternative analyses were explored. RESULTS: In this paper we introduced local similarity analysis (LSA), which is a technique that can identify more complex dependence associations among species as well as associations between species and environmental factors without requiring significant data reduction. To illustrate its capability of identifying relationships that may not otherwise be identified by PCC, we first applied LSA to simulated data. We then applied LSA to a marine microbial observatory dataset and identified unique, significant associations that were not detected by PCC analysis. LSA results, combined with results from PCC analysis were used to construct a theoretical ecological network which allows for easy visualization of the most significant associations. Biological implications of the significant associations detected by LSA were discussed. We also identified additional applications where LSA would be beneficial. AVAILABILITY: The algorithms are implemented in Splus/R and they are available upon request from the corresponding author.

Energy starved Candidatus Pelagibacter ubique substitutes light-mediated ATP production for endogenous carbon respiration.

Previous studies have demonstrated that Candidatus Pelagibacter ubique, a member of the SAR11 clade, constitutively expresses proteorhodopsin (PR) proteins that can function as light-dependent proton pumps. However, exposure to light did not significantly improve the growth rate or final cell densities of SAR11 isolates in a wide range of conditions. Thus, the ecophysiological role of PR in SAR11 remained unresolved. We investigated a range of cellular properties and here show that light causes dramatic changes in physiology and gene expression in Cand. P. ubique cells that are starved for carbon, but provides little or no advantage during active growth on organic carbon substrates. During logarithmic growth there was no difference in oxygen consumption by cells in light versus dark. Energy starved cells respired endogenous carbon in the dark, becoming spheres that approached the minimum predicted size for cells, and produced abundant pili. In the light, energy starved cells maintained size, ATP content, and higher substrate transport rates, and differentially expressed nearly 10% of their genome. These findings show that PR is a vital adaptation that supports Cand. P. ubique metabolism during carbon starvation, a condition that is likely to occur in the extreme conditions of ocean environments.

Marine bacterial, archaeal and protistan association networks reveal ecological linkages.

Microbes have central roles in ocean food webs and global biogeochemical processes, yet specific ecological relationships among these taxa are largely unknown. This is in part due to the dilute, microscopic nature of the planktonic microbial community, which prevents direct observation of their interactions. Here, we use a holistic (that is, microbial system-wide) approach to investigate time-dependent variations among taxa from all three domains of life in a marine microbial community. We investigated the community composition of bacteria, archaea and protists through cultivation-independent methods, along with total bacterial and viral abundance, and physico-chemical observations. Samples and observations were collected monthly over 3 years at a well-described ocean time-series site of southern California. To find associations among these organisms, we calculated time-dependent rank correlations (that is, local similarity correlations) among relative abundances of bacteria, archaea, protists, total abundance of bacteria and viruses and physico-chemical parameters. We used a network generated from these statistical correlations to visualize and identify time-dependent associations among ecologically important taxa, for example, the SAR11 cluster, stramenopiles, alveolates, cyanobacteria and ammonia-oxidizing archaea. Negative correlations, perhaps suggesting competition or predation, were also common. The analysis revealed a progression of microbial communities through time, and also a group of unknown eukaryotes that were highly correlated with dinoflagellates, indicating possible symbioses or parasitism. Possible 'keystone' species were evident. The network has statistical features similar to previously described ecological networks, and in network parlance has non-random, small world properties (that is, highly interconnected nodes). This approach provides new insights into the natural history of microbes.

Transcriptional and translational regulatory responses to iron limitation in the globally distributed marine bacterium Candidatus pelagibacter ubique.

Iron is recognized as an important micronutrient that limits microbial plankton productivity over vast regions of the oceans. We investigated the gene expression responses of Candidatus Pelagibacter ubique cultures to iron limitation in natural seawater media supplemented with a siderophore to chelate iron. Microarray data indicated transcription of the periplasmic iron binding protein sfuC increased by 16-fold, and iron transporter subunits, iron-sulfur center assembly genes, and the putative ferroxidase rubrerythrin transcripts increased to a lesser extent. Quantitative peptide mass spectrometry revealed that sfuC protein abundance increased 27-fold, despite an average decrease of 59% across the global proteome. Thus, we propose sfuC as a marker gene for indicating iron limitation in marine metatranscriptomic and metaproteomic ecological surveys. The marked proteome reduction was not directly correlated to changes in the transcriptome, implicating post-transcriptional regulatory mechanisms as modulators of protein expression. Two RNA-binding proteins, CspE and CspL, correlated well with iron availability, suggesting that they may contribute to the observed differences between the transcriptome and proteome. We propose a model in which the RNA-binding activity of CspE and CspL selectively enables protein synthesis of the iron acquisition protein SfuC during transient growth-limiting episodes of iron scarcity.

Proteorhodopsins: an array of physiological roles?

Metagenomic analyses have revealed widespread and diverse retinal-binding rhodopsin proteins (named proteorhodopsins) among numerous marine bacteria and archaea, which has challenged the notion that solar energy can only enter marine ecosystems by chlorophyll-based photosynthesis. Most marine proteorhodopsins share structural and functional similarities with archaeal bacteriorhodopsins, which generate proton motive force via light-activated proton pumping, thereby ultimately powering ATP production. This suggests an energetic role for proteorhodopsins. However, results from a growing number of investigations do not readily fit this model, which indicates that proteorhodopsins could have a range of physiological functions.

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I.

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.

Polyphyletic photosynthetic reaction centre genes in oligotrophic marine Gammaproteobacteria.

Ecological studies indicate that aerobic anoxygenic phototrophic bacteria (AAP) that use bacteriochlorophyll to support phototrophic electron transport are widely distributed in the oceans. All cultivated marine AAP are alpha-3 and alpha-4 Proteobacteria, but metagenomic evidence indicates that uncultured AAP Gammaproteobacteria are important members of ocean surface microbial communities. Here we report the description of obligately oligotrophic, marine Gammaproteobacteria that have genes for aerobic anoxygenic photosynthesis. Three strains belonging to the OM60 clade were isolated in autoclaved seawater media. Polymerase chain reaction assays for the pufM gene show that these strains contain photosynthetic reaction centre genes. DNA sequencing and phylogenetic analysis indicate that the pufM genes are polyphyletic, suggesting multiple instances of lateral gene transfer. Peptide sequences from six photosynthesis genes (pufL, pufM, pufC, pufB, pufA and puhA) were detected by proteomic analyses of strain HTCC2080 cells grown aerobically in seawater. They closely match predicted peptides from an environmental seawater bacterial artificial chromosome clone of gammaproteobacterial origin, thus identifying the OM60 clade as a significant source of gammaproteobacterial AAP genes in marine systems. The cell yield and rate of growth of HTCC2080 in autoclaved, aerobic seawater increased in the light. These findings identify the OM60 clade as a source of Gammaproteobacteria AAP genes in coastal oceans, and demonstrate that aerobic, anoxygenic photosynthetic metabolism can enhance the productivity of marine oligotrophic bacteria that also grow heterotrophically in darkness.

Annually reoccurring bacterial communities are predictable from ocean conditions.

Factors influencing patterns in the distribution and abundance of plant and animal taxa modulate ecosystem function and ecosystem response to environmental change, which is often taken to infer low functional redundancy among such species, but such relationships are poorly known for microbial communities. Using high-resolution molecular fingerprinting, we demonstrate the existence of extraordinarily repeatable temporal patterns in the community composition of 171 operational taxonomic units of marine bacterioplankton over 4.5 years at our Microbial Observatory site, 20 km off the southern California coast. These patterns in distribution and abundance of microbial taxa were highly predictable and significantly influenced by a broad range of both abiotic and biotic factors. These findings provide statistically robust demonstration of temporal patterning in marine bacterial distribution and abundance, which suggests that the distribution and abundance of bacterial taxa may modulate ecosystem function and response and that a significant subset of the bacteria exhibit low levels of functional redundancy as documented for many plant and animal communities.

A dynamic programming algorithm for binning microbial community profiles.

MOTIVATION: A number of community profiling approaches have been widely used to study the microbial community composition and its variations in environmental ecology. Automated Ribosomal Intergenic Spacer Analysis (ARISA) is one such technique. ARISA has been used to study microbial communities using 16S-23S rRNA intergenic spacer length heterogeneity at different times and places. Owing to errors in sampling, random mutations in PCR amplification, and probably mostly variations in readings from the equipment used to analyze fragment sizes, the data read directly from the fragment analyzer should not be used for down stream statistical analysis. No optimal data preprocessing methods are available. A commonly used approach is to bin the reading lengths of the 16S-23S intergenic spacer. We have developed a dynamic programming algorithm based binning method for ARISA data analysis which minimizes the overall differences between replicates from the same sampling location and time. RESULTS: In a test example from an ocean time series sampling program, data preprocessing identified several outliers which upon re-examination were found to be because of systematic errors. Clustering analysis of the ARISA from different times based on the dynamic programming algorithm binned data revealed important features of the biodiversity of the microbial communities.

Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series.

We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising >1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities <98%, most taxa displayed differences in ARISA fragment lengths >10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r2=0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.