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1.  Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight 
The ISME Journal  2013;7(12):2259-2273.
Time-series are critical to understanding long-term natural variability in the oceans. Bacterial communities in the euphotic zone were investigated for over a decade at the San Pedro Ocean Time-series station (SPOT) off southern California. Community composition was assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and coupled with measurements of oceanographic parameters for the surface ocean (0–5 m) and deep chlorophyll maximum (DCM, average depth ∼30 m). SAR11 and cyanobacterial ecotypes comprised typically more than one-third of the measured community; diversity within both was temporally variable, although a few operational taxonomic units (OTUs) were consistently more abundant. Persistent OTUs, mostly Alphaproteobacteria (SAR11 clade), Actinobacteria and Flavobacteria, tended to be abundant, in contrast to many rarer yet intermittent and ephemeral OTUs. Association networks revealed potential niches for key OTUs from SAR11, cyanobacteria, SAR86 and other common clades on the basis of robust correlations. Resilience was evident by the average communities drifting only slightly as years passed. Average Bray-Curtis similarity between any pair of dates was ∼40%, with a slight decrease over the decade and obvious near-surface seasonality; communities 8–10 years apart were slightly more different than those 1–4 years apart with the highest rate of change at 0–5 m between communities <4 years apart. The surface exhibited more pronounced seasonality than the DCM. Inter-depth Bray-Curtis similarities repeatedly decreased as the water column stratified each summer. Environmental factors were better predictors of shifts in community composition than months or elapsed time alone; yet, the best predictor was community composition at the other depth (that is, 0–5 m versus DCM).
doi:10.1038/ismej.2013.122
PMCID: PMC3834854  PMID: 23864126
marine bacterioplankton; ARISA; community ecology; microbe–microbe interactions; time series
2.  Short-term observations of marine bacterial and viral communities: patterns, connections and resilience 
The ISME Journal  2013;7(7):1274-1285.
Observation of short-term temporal variation in bacterial and viral communities is important for understanding patterns of aquatic microbial diversity. We collected surface seawater once daily for 38 consecutive days with seven more samples interspersed over 40 more days at one location ∼2 km from Santa Catalina Island, California. Bacterial communities were analyzed by automated ribosomal intergenic spacer analysis (ARISA) and viral communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of the conserved T4-like myoviral gene encoding the major capsid protein (g23). Common bacterial and viral taxa were consistently dominant, and relatively few displayed dramatic increases/decreases or ‘boom/bust' patterns that might be expected from dynamic predator-prey interactions. Association network analysis showed most significant covariations (associations) occurred among bacterial taxa or among viral taxa and there were several modular (highly-interconnected) associations (P⩽0.005). Associations observed between bacteria and viruses (P⩽0.005) occurred with a median time lag of 2 days. Regression of all pairwise Bray-Curtis similarities between samples indicated a rate of bacterial community change that slows from 2.1%–0.18% per day over a week to 2 months; the rate stays around 0.4% per day for viruses. Our interpretation is that, over the scale of days, individual bacterial and viral OTUs can be dynamic and patterned; resulting in statistical associations regarded as potential ecological interactions. However, over the scale of weeks, average bacterial community variation is slower, suggesting that there is strong community-level ecological resilience, that is, a tendency to converge towards a ‘mean' microbial community set by longer-term controlling factors.
doi:10.1038/ismej.2013.19
PMCID: PMC3695287  PMID: 23446831
ARISA; bacteria; marine; T4-like myovirus; network; time-series
3.  The founding charter of the Genomic Observatories Network 
GigaScience  2014;3:2.
The co-authors of this paper hereby state their intention to work together to launch the Genomic Observatories Network (GOs Network) for which this document will serve as its Founding Charter. We define a Genomic Observatory as an ecosystem and/or site subject to long-term scientific research, including (but not limited to) the sustained study of genomic biodiversity from single-celled microbes to multicellular organisms.
An international group of 64 scientists first published the call for a global network of Genomic Observatories in January 2012. The vision for such a network was expanded in a subsequent paper and developed over a series of meetings in Bremen (Germany), Shenzhen (China), Moorea (French Polynesia), Oxford (UK), Pacific Grove (California, USA), Washington (DC, USA), and London (UK). While this community-building process continues, here we express our mutual intent to establish the GOs Network formally, and to describe our shared vision for its future. The views expressed here are ours alone as individual scientists, and do not necessarily represent those of the institutions with which we are affiliated.
doi:10.1186/2047-217X-3-2
PMCID: PMC3995929  PMID: 24606731
Biodiversity; Genomics; Biocode; Earth observations
4.  Global distribution and diversity of marine Verrucomicrobia 
The ISME Journal  2012;6(8):1499-1505.
Verrucomicrobia is a bacterial phylum that is commonly detected in soil, but little is known about the distribution and diversity of this phylum in the marine environment. To address this, we analyzed the marine microbial community composition in 506 samples from the International Census of Marine Microbes as well as 11 coastal samples taken from the California Current. These samples from both the water column and sediments covered a wide range of environmental conditions. Verrucomicrobia were present in 98% of the analyzed samples, and thus appeared nearly ubiquitous in the ocean. Based on the occurrence of amplified 16S ribosomal RNA sequences, Verrucomicrobia constituted on average 2% of the water column and 1.4% of the sediment bacterial communities. The diversity of Verrucomicrobia displayed a biogeography at multiple taxonomic levels and thus, specific lineages appeared to have clear habitat preference. We found that subdivision 1 and 4 generally dominated marine bacterial communities, whereas subdivision 2 was more frequent in low salinity waters. Within the subdivisions, Verrucomicrobia community composition were significantly different in the water column compared with sediment as well as within the water column along gradients of salinity, temperature, nitrate, depth and overall water column depth. Although we still know little about the ecophysiology of Verrucomicrobia lineages, the ubiquity of this phylum suggests that it may be important for the biogeochemical cycle of carbon in the ocean.
doi:10.1038/ismej.2012.3
PMCID: PMC3400412  PMID: 22318305
Verrucomicrobia; bacterial communities; ICoMM
5.  Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein 
PLoS ONE  2013;8(3):e57487.
Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.
doi:10.1371/journal.pone.0057487
PMCID: PMC3587595  PMID: 23526944
6.  Marine bacterial, archaeal and protistan association networks reveal ecological linkages 
The ISME Journal  2011;5(9):1414-1425.
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.
doi:10.1038/ismej.2011.24
PMCID: PMC3160682  PMID: 21430787
co-occurrence patterns; stramenopiles; dinoflagellates; SAR11; cyanobacteria; time series
7.  Metagenomics and its connection to microbial community organization 
Microbes dominate most global biogeochemical cycles, and microbial metagenomics (studying the collective microbial genomes) provides invaluable new insights into microbial systems, independent of cultivation. Metagenomic approaches targeting specific genes, e.g. small subunit (ssu) ribosomal RNA (rRNA), can be used to investigate microbial community organization by efficiently showing which taxa of organisms are present, while shotgun approaches show all genes and can indicate what functions the organisms are capable of. But collecting and organizing comprehensive shotgun data is extremely challenging and costly, and, in theory, predicting functionalities from microbial identities alone would save immense effort. However, we don’t yet know to what extent such predictions are applicable.
doi:10.3410/B4-15
PMCID: PMC3410722  PMID: 22912649
8.  Global biogeography of SAR11 marine bacteria 
Metagenomic samples from oceans around the globe were used to examine the biogeography of the dominant marine heterotrophic bacterial clade, SAR11. Analysis uncovers evidence of adaptive radiation in response to environmental parameters, particularly temperature.
By generating 37 new Antarctic metagenomes and analysing the internal transcribed spacer (ITS) regions of the SAR11 clade in a total of 128 surface marine metagenomes, we identified phylotype distributions that strongly correlated with temperature and latitude.By assembling SAR11 genomes from Antarctic metagenome data, we identified specific genes, biases in gene functions and signatures of positive selection in the genomes of the polar SAR11—genomic signatures of adaptive radiation.Our data demonstrate the importance of adaptive radiation in an organism's ability to proliferate throughout the world's oceans, and describe genomic traits characteristic of different phylotypes in specific marine biomes.These bacteria are important marine heterotrophs and have a fundamental role in oceanic nutrient cycling. These findings, therefore, have important implications for our ability to predict how changes in ocean temperature may affect bacterial ecology.
The ubiquitous SAR11 bacterial clade is the most abundant type of organism in the world's oceans, but the reasons for its success are not fully elucidated. We analysed 128 surface marine metagenomes, including 37 new Antarctic metagenomes. The large size of the data set enabled internal transcribed spacer (ITS) regions to be obtained from the Southern polar region, enabling the first global characterization of the distribution of SAR11, from waters spanning temperatures −2 to 30°C. Our data show a stable co-occurrence of phylotypes within both ‘tropical' (>20°C) and ‘polar' (<10°C) biomes, highlighting ecological niche differentiation between major SAR11 subgroups. All phylotypes display transitions in abundance that are strongly correlated with temperature and latitude. By assembling SAR11 genomes from Antarctic metagenome data, we identified specific genes, biases in gene functions and signatures of positive selection in the genomes of the polar SAR11—genomic signatures of adaptive radiation. Our data demonstrate the importance of adaptive radiation in the organism's ability to proliferate throughout the world's oceans, and describe genomic traits characteristic of different phylotypes in specific marine biomes.
doi:10.1038/msb.2012.28
PMCID: PMC3421443  PMID: 22806143
adaptive radiation; Antarctica; metagenome; Pelagibacter; phylotype distribution
9.  Co-occurrence patterns for abundant marine archaeal and bacterial lineages in the deep chlorophyll maximum of coastal California 
The ISME Journal  2011;5(7):1077-1085.
Microorganisms remineralize and respire half of marine primary production, yet the niches occupied by specific microbial groups, and how these different groups may interact, are poorly understood. In this study, we identify co-occurrence patterns for marine Archaea and specific bacterial groups in the chlorophyll maximum of the Southern California Bight. Quantitative PCR time series of marine group 1 (MG1) Crenarchaeota 16S rRNA genes varied substantially over time but were well-correlated (r2=0.94, P<0.001) with ammonia monooxygenase subunit A (amoA) genes, and were more weakly related to 16S rRNA genes for all Archaea (r2=0.39), indicating that other archaeal groups (for example, Euryarchaeota) were numerically important. These data sets were compared with variability in bacterial community composition based on automated ribosomal intergenic spacer analysis (ARISA). We found that archaeal amoA gene copies and a SAR11 (or Pelagibacter) group Ib operational taxonomic unit (OTU) displayed strong co-variation through time (r2=0.55, P<0.05), and archaeal amoA and MG1 16S rRNA genes also co-occurred with two SAR86 and two Bacteroidetes OTUs. The relative abundance of these groups increased and decreased in synchrony over the course of the time series, and peaked during periods of seasonal transition. By using a combination of quantitative and relative abundance estimates, our findings show that abundant microbial OTUs—including the marine Crenarchaeota, SAR11, SAR86 and the Bacteroidetes—co-occur non-randomly; they consequently have important implications for our understanding of microbial community ecology in the sea.
doi:10.1038/ismej.2010.204
PMCID: PMC3146281  PMID: 21228895
Crenarchaeota; SAR11; SAR86; Bacteroidetes; community assembly; time-series
10.  Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications 
Yilmaz, Pelin | Kottmann, Renzo | Field, Dawn | Knight, Rob | Cole, James R | Amaral-Zettler, Linda | Gilbert, Jack A | Karsch-Mizrachi, Ilene | Johnston, Anjanette | Cochrane, Guy | Vaughan, Robert | Hunter, Christopher | Park, Joonhong | Morrison, Norman | Rocca-Serra, Philippe | Sterk, Peter | Arumugam, Manimozhiyan | Bailey, Mark | Baumgartner, Laura | Birren, Bruce W | Blaser, Martin J | Bonazzi, Vivien | Booth, Tim | Bork, Peer | Bushman, Frederic D | Buttigieg, Pier Luigi | Chain, Patrick S G | Charlson, Emily | Costello, Elizabeth K | Huot-Creasy, Heather | Dawyndt, Peter | DeSantis, Todd | Fierer, Noah | Fuhrman, Jed A | Gallery, Rachel E | Gevers, Dirk | Gibbs, Richard A | Gil, Inigo San | Gonzalez, Antonio | Gordon, Jeffrey I | Guralnick, Robert | Hankeln, Wolfgang | Highlander, Sarah | Hugenholtz, Philip | Jansson, Janet | Kau, Andrew L | Kelley, Scott T | Kennedy, Jerry | Knights, Dan | Koren, Omry | Kuczynski, Justin | Kyrpides, Nikos | Larsen, Robert | Lauber, Christian L | Legg, Teresa | Ley, Ruth E | Lozupone, Catherine A | Ludwig, Wolfgang | Lyons, Donna | Maguire, Eamonn | Methé, Barbara A | Meyer, Folker | Muegge, Brian | Nakielny, Sara | Nelson, Karen E | Nemergut, Diana | Neufeld, Josh D | Newbold, Lindsay K | Oliver, Anna E | Pace, Norman R | Palanisamy, Giriprakash | Peplies, Jörg | Petrosino, Joseph | Proctor, Lita | Pruesse, Elmar | Quast, Christian | Raes, Jeroen | Ratnasingham, Sujeevan | Ravel, Jacques | Relman, David A | Assunta-Sansone, Susanna | Schloss, Patrick D | Schriml, Lynn | Sinha, Rohini | Smith, Michelle I | Sodergren, Erica | Spor, Aymé | Stombaugh, Jesse | Tiedje, James M | Ward, Doyle V | Weinstock, George M | Wendel, Doug | White, Owen | Whiteley, Andrew | Wilke, Andreas | Wortman, Jennifer R | Yatsunenko, Tanya | Glöckner, Frank Oliver
Nature Biotechnology  2011;29(5):415-420.
Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences—the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The ‘environmental packages’ apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.
doi:10.1038/nbt.1823
PMCID: PMC3367316  PMID: 21552244
11.  Defining seasonal marine microbial community dynamics 
The ISME Journal  2011;6(2):298-308.
Here we describe, the longest microbial time-series analyzed to date using high-resolution 16S rRNA tag pyrosequencing of samples taken monthly over 6 years at a temperate marine coastal site off Plymouth, UK. Data treatment effected the estimation of community richness over a 6-year period, whereby 8794 operational taxonomic units (OTUs) were identified using single-linkage preclustering and 21 130 OTUs were identified by denoising the data. The Alphaproteobacteria were the most abundant Class, and the most frequently recorded OTUs were members of the Rickettsiales (SAR 11) and Rhodobacteriales. This near-surface ocean bacterial community showed strong repeatable seasonal patterns, which were defined by winter peaks in diversity across all years. Environmental variables explained far more variation in seasonally predictable bacteria than did data on protists or metazoan biomass. Change in day length alone explains >65% of the variance in community diversity. The results suggested that seasonal changes in environmental variables are more important than trophic interactions. Interestingly, microbial association network analysis showed that correlations in abundance were stronger within bacterial taxa rather than between bacteria and eukaryotes, or between bacteria and environmental variables.
doi:10.1038/ismej.2011.107
PMCID: PMC3260500  PMID: 21850055
16S rRNA; microbial; bacteria; community; diversity; model
12.  Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates 
BMC Systems Biology  2011;5(Suppl 2):S15.
Background
The increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval.
Results
We extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified.
Conclusions
The extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.
doi:10.1186/1752-0509-5-S2-S15
PMCID: PMC3287481  PMID: 22784572
13.  Accurate Genome Relative Abundance Estimation Based on Shotgun Metagenomic Reads 
PLoS ONE  2011;6(12):e27992.
Accurate estimation of microbial community composition based on metagenomic sequencing data is fundamental for subsequent metagenomics analysis. Prevalent estimation methods are mainly based on directly summarizing alignment results or its variants; often result in biased and/or unstable estimates. We have developed a unified probabilistic framework (named GRAMMy) by explicitly modeling read assignment ambiguities, genome size biases and read distributions along the genomes. Maximum likelihood method is employed to compute Genome Relative Abundance of microbial communities using the Mixture Model theory (GRAMMy). GRAMMy has been demonstrated to give estimates that are accurate and robust across both simulated and real read benchmark datasets. We applied GRAMMy to a collection of 34 metagenomic read sets from four metagenomics projects and identified 99 frequent species (minimally 0.5% abundant in at least 50% of the data- sets) in the human gut samples. Our results show substantial improvements over previous studies, such as adjusting the over-estimated abundance for Bacteroides species for human gut samples, by providing a new reference-based strategy for metagenomic sample comparisons. GRAMMy can be used flexibly with many read assignment tools (mapping, alignment or composition-based) even with low-sensitivity mapping results from huge short-read datasets. It will be increasingly useful as an accurate and robust tool for abundance estimation with the growing size of read sets and the expanding database of reference genomes.
doi:10.1371/journal.pone.0027992
PMCID: PMC3232206  PMID: 22162995
14.  The Earth Microbiome Project: The Meeting Report for the 1st International Earth Microbiome Project Conference, Shenzhen, China, June 13th-15th 2011 
Standards in Genomic Sciences  2011;5(2):243-247.
This report details the outcome of the 1st International Earth Microbiome Project Conference. The 2-day conference was held at the Kingkey Palace Hotel, Shenzhen, China, on the 14th-15th June 2011, and was hosted by BGI (formally the Beijing Genomics Institute). The conference was arranged as a formal launch for the Earth Microbiome Project, to highlight some of the exciting research projects, results of the preliminary pilot studies, and to provide a discussion forum for the types of technology and experimental approaches that will come to define the standard operating procedures of this project.
doi:10.4056/sigs.2134923
PMCID: PMC3235519
15.  Global Patterns of Bacterial Beta-Diversity in Seafloor and Seawater Ecosystems 
PLoS ONE  2011;6(9):e24570.
Background
Marine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.
Methodology/Principal Findings
The synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean's surface to the deep-sea floor shows that pelagic and benthic communities greatly differ, at all taxonomic levels, and share <10% bacterial types defined at 3% sequence similarity level. Surface and deep water, coastal and open ocean, and anoxic and oxic ecosystems host distinct communities that reflect productivity, land influences and other environmental constraints such as oxygen availability. The high variability of bacterial community composition specific to vent and coastal ecosystems reflects the heterogeneity and dynamic nature of these habitats. Both pelagic and benthic bacterial community distributions correlate with surface water productivity, reflecting the coupling between both realms by particle export. Also, differences in physical mixing may play a fundamental role in the distribution patterns of marine bacteria, as benthic communities showed a higher dissimilarity with increasing distance than pelagic communities.
Conclusions/Significance
This first synthesis of global bacterial distribution across different ecosystems of the World's oceans shows remarkable horizontal and vertical large-scale patterns in bacterial communities. This opens interesting perspectives for the definition of biogeographical biomes for bacteria of ocean waters and the seabed.
doi:10.1371/journal.pone.0024570
PMCID: PMC3169623  PMID: 21931760
16.  Multitiered Approach Using Quantitative PCR To Track Sources of Fecal Pollution Affecting Santa Monica Bay, California 
The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 109 to 1010 cells h−1 and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.
doi:10.1128/AEM.72.2.1604-1612.2006
PMCID: PMC1392893  PMID: 16461716
17.  Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR 
Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2 = 0.99) in fresh water and 23% (r2 = 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <104 enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r2 = 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r2 = 0.93; HA 12%, r2 = 0.87). The optimized method was used with 1-liter field samples from two very different freshwater “creeks” that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.
doi:10.1128/AEM.71.8.4523-4530.2005
PMCID: PMC1183282  PMID: 16085845
18.  Richness and Diversity of Bacterioplankton Species along an Estuarine Gradient in Moreton Bay, Australia 
Bacterioplankton community diversity was investigated in the subtropical Brisbane River-Moreton Bay estuary, Australia (27°25′S, 153°5′E). Bacterial communities were studied using automated rRNA intergenic spacer analysis (ARISA), which amplifies 16S-23S ribosomal DNA internally transcribed spacer regions from mixed-community DNA and detects the separated products on a fragment analyzer. Samples were collected from eight sites throughout the estuary and east to the East Australian Current (Coral Sea). Bacterioplankton communities had the highest operational taxonomic unit (OTU) richness, as measured by ARISA at eastern bay stations (S [total richness] = 84 to 85 OTU) and the lowest richness in the Coral Sea (S = 39 to 59 OTU). Richness correlated positively with bacterial abundance; however, there were no strong correlations between diversity and salinity, NO3− and PO43− concentrations, or chlorophyll a concentration. Bacterioplankton communities at the riverine stations were different from communities in the bay or Coral Sea. The main differences in OTU richness between stations were in taxa that each represented 0.1% (the detection limit) to 0.5% of the total amplified DNA, i.e., the “tail” of the distribution. We found that some bacterioplankton taxa are specific to distinct environments while others have a ubiquitous distribution from river to sea. Bacterioplankton richness and diversity patterns in the estuary are potentially a consequence of greater niche availability, mixing of local and adjacent environment communities, or intermediate disturbance. Furthermore, these results contrast with previous reports of spatially homogeneous bacterioplankton communities in other coastal waters.
doi:10.1128/AEM.70.6.3425-3433.2004
PMCID: PMC427769  PMID: 15184140
20.  Marine Planktonic Archaea Take Up Amino Acids 
Applied and Environmental Microbiology  2000;66(11):4829-4833.
Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.
PMCID: PMC92387  PMID: 11055931
21.  Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers 
Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h−1 and production rates in the dark ranging from 1.9 to 6.1% h−1, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.
PMCID: PMC92222  PMID: 10966392
22.  Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria 
Applied and Environmental Microbiology  1999;65(10):4475-4483.
Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.
PMCID: PMC91596  PMID: 10508078
24.  Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ 
We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria (α-Proteobacteria) and of the Cytophaga-Flavobacterium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.
PMCID: PMC91246  PMID: 10103276
25.  Extraction from Natural Planktonic Microorganisms of DNA Suitable for Molecular Biological Studies 
We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-μm-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100°C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.
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PMCID: PMC202673  PMID: 16347652

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