The Hallam Lab

Investigating microbial communities…


For the most current list of publications, see Steven Hallam's public Google Scholar profile.

56. Hawley AK, Brewer HM, Norbeck AD, Paša-Tolić L, Hallam SJ. 2014. Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes. Proc Natl Acad Sci USA. 111(31):11395-400. doi: 10.1073/pnas.1322132111. (Published online 2014 Jul 22)

Marine oxygen minimum zones (OMZs) are intrinsic water column features arising from respiratory oxygen demand during organic matter degradation in stratified waters. Currently OMZs are expanding due to global climate change with resulting feedback on marine ecosystem function. Here we use metaproteomics to chart spatial and temporal patterns of gene expression along defined redox gradients in a seasonally stratified fjord to better understand microbial community responses to OMZ expansion. The expression of metabolic pathway components for nitrification, anaerobic ammonium oxidation (anammox), denitrification, and inorganic carbon fixation were differentially expressed across the redoxcline and covaried with distribution patterns of ubiquitous OMZ microbes including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and inorganic carbon fixation pathways affiliated with Thaumarchaeota dominated dysoxic waters, and denitrification, sulfur oxidation, and inorganic carbon fixation pathways affiliated with the SUP05 group of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic waters. Nitrifier nitrite oxidation and anammox pathways affiliated with Nirospina, Nitrospira, and Planctomycetes, respectively, also exhibited redox partitioning between dysoxic and suboxic waters. The numerical abundance of SUP05 proteins mediating inorganic carbon fixation under anoxic conditions suggests that SUP05 will become increasingly important in global ocean carbon and nutrient cycling as OMZs expand.
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55. Hanson NW, Konwar KM, Hawley AK, Altman T, Karp PD, Hallam SJ. 2014. Metabolic pathways for the whole community. BMC Genomics. 15(1):619. (Published online: 22 July 2014)

A convergence of high-throughput sequencing and computational power is transforming biology into information science. Despite these technological advances, converting bits and bytes of sequence information into meaningful insights remains a challenging enterprise. Biological systems operate on multiple hierarchical levels from genomes to biomes. Holistic understanding of biological systems requires agile software tools that permit comparative analyses across multiple information levels (DNA, RNA, protein, and metabolites) to identify emergent properties, diagnose system states, or predict responses to environmental change.
Here we adopt the MetaPathways annotation and analysis pipeline and Pathway Tools to construct environmental pathway/genome databases (ePGDBs) that describe microbial community metabolism using MetaCyc, a highly curated database of metabolic pathways and components covering all domains of life. We evaluate Pathway Tools' performance on three datasets with different complexity and coding potential, including simulated metagenomes, a symbiotic system, and the Hawaii Ocean Time-series. We define accuracy and sensitivity relationships between read length, coverage and pathway recovery and evaluate the impact of taxonomic pruning on ePGDB construction and interpretation. Resulting ePGDBs provide interactive metabolic maps, predict emergent metabolic pathways associated with biosynthesis and energy production and differentiate between genomic potential and phenotypic expression across defined environmental gradients.
This multi-tiered analysis provides the user community with specific operating guidelines, performance metrics and prediction hazards for more reliable ePGDB construction and interpretation. Moreover, it demonstrates the power of Pathway Tools in predicting metabolic interactions in natural and engineered ecosystems.
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54. Hanson, N.W., K.M. Konwar, S.-J. Wu, S.J. Hallam. 2014. MetaPathways v2.0: A master-worker model for environmental Pathway/Genome Database construction on grids and clouds. Comput. IEEE Proc. Intell. in Bioinf. and Comput. Biol. Honolulu, Hawaii, May 21-24.

The development of high-throughput sequencing technologies over the past decade has generated a tidal wave of environmental sequence information from a variety of natural and human engineered ecosystems. The resulting flood of information into public databases and archived sequencing projects has exponentially expanded computational resource requirements rendering most local homology-based search methods inefficient. We recently introduced MetaPathways v1.0, a modular annotation and analysis pipeline for constructing environmental Pathway/Genome Databases (ePGDBs) from environmental sequence information capable of using the Sun Grid engine for external resource partitioning. However, a command-line interface and facile task management introduced user activation barriers with concomitant decrease in fault tolerance. Here we present MetaPathways v2.0 incorporating a graphical user interface (GUI) and refined task management methods. The MetaPathways GUI provides an intuitive display for setup and process monitoring and supports interactive data visualization and sub-setting via a custom Knowledge Engine data structure. A master-worker model is adopted for task management allowing users to scavenge computational results from a number of worker grids in an ad hoc, asynchronous, distributed network that dramatically increases fault tolerance. This model facilitates the use of EC2 instances extending ePGDB construction to the Amazon Elastic Cloud.
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53. Strachan, C.R., R. Singh, D. VanInsberghe, K. Ievdokymenko, K. Budwill, W.W. Mohn, L.D. Eltis, S.J. Hallam, 2014. Metagenomic scaffolds enable combinatorial lignin transformation. P. Natl. Acad. Sci. USA.

Strachan PNAS 2014 sm

Engineering the microbial transformation of lignocellulosic biomass is essential to developing modern biorefining processes that alleviate reliance on petroleum-derived energy and chemicals. Many current bioprocess streams depend on the genetic tractability of Escherichia coli with a primary emphasis on engineering cellulose/hemicellulose catabolism, small molecule production, and resistance to product inhibition. Conversely, bioprocess streams for lignin transformation remain embryonic, with relatively few environmental strains or enzymes implicated. Here we develop a biosensor responsive to monoaromatic lignin transformation products compatible with functional screening in E. coli. We use this biosensor to retrieve metagenomic scaffolds sourced from coal bed bacterial communities conferring an array of lignin transformation phenotypes that synergize in combination. Transposon mutagenesis and comparative sequence analysis of active clones identified genes encoding six functional classes mediating lignin transformation phenotypes that appear to be rearrayed in nature via horizontal gene transfer. Lignin transformation activity was then demonstrated for one of the predicted gene products encoding a multicopper oxidase to validate the screen. These results illuminate cellular and community-wide networks acting on aromatic polymers and expand the toolkit for engineering recombinant lignin transformation based on ecological design principles.
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52. Hurwitz, B.L., S.J. Hallam, M.B. Sullivan, 2013. Metabolic reprogramming by viruses in the sunlit and dark ocean. Genome Biology.

Hurwitz GenBio 2013 sm
Background: Marine ecosystem function is largely determined by matter and energy transformations mediated by microbial community interaction networks. Viral infection modulates network properties through mortality, gene transfer and metabolic reprogramming.
Results: Here we explore the nature and extent of viral metabolic reprogramming throughout the Pacific Ocean depth continuum. We describe 35 marine viral gene families with potential to reprogram metabolic flux through central metabolic pathways recovered from Pacific Ocean waters. Four of these families have been previously reported but 31 are novel. These known and new carbon pathway auxiliary metabolic genes were recovered from a total of 22 viral metagenomes in which viral auxiliary metabolic genes were differentiated from low-level cellular DNA inputs based on small subunit ribosomal RNA gene content, taxonomy, fragment recruitment and genomic context information. Auxiliary metabolic gene distribution patterns reveal that marine viruses target overlapping, but relatively distinct pathways in sunlit and dark ocean waters to redirect host carbon flux towards energy production and viral genome replication under low nutrient, niche-differentiated conditions throughout the depth continuum.
Conclusions: Given half of ocean microbes are infected by viruses at any given time, these findings of broad viral metabolic reprogramming suggest the need for renewed consideration of viruses in global ocean carbon models
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51. Buck, K.R., J.P. Barry, S.J. Hallam, 2013. Thioploca spp. sheaths as niches for bacterial and protistan assemblages. Marine Ecology. (published online November 2013)

Buck MarEco 2013 sm
Sulfide oxidizing bacterial mats are common in regions of the continental shelves characterized by high primary production and the resultant oxygen minimum zone. These mats are made up of several species of Beggiatoa and/or Thioploca, which oxidize sulfide that is generated in the sediment. Thioploca spp. inhabit a large polysaccharide sheath that encompasses bundles of 1–20 filaments (trichomes), each ranging from 3 to 60 μm in diameter. This sheath has been shown to be a critical component of the autecology ofThioploca. Analysis of Thioploca from cold seeps in Monterey Bay using light and transmission electron microscopy identified new and diverse microbial assemblages associated with interstitial spaces between trichomes, inside the sheath. Small diameter, non-vacuolate, filamentous prokaryotes were numerous. Amoebae, euglenozoa, ciliates and other protists of unknown affiliation were observed in sheaths. Most of the protists possessed food vacuoles and some protists showed ultrastructural evidence of endosymbionts. These observations suggest that Thioploca sheaths may serve as oases on the sea floor, providing nutritional and detoxification services to previously unrecognized microbial partners.
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50. Mewis, K., Z. Armstrong, Y.C. Song, S.A. Baldwin, S.G Withers, S.J. Hallam, 2013. Biomining active cellulases from a mining bioremediation system. Journal of Biotechnology. (published online September 2013)

Mewis JBioTech 2013 sm
Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-d-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.
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49. Wright, J.J., K. Mewis, N.W. Hanson, K.M. Konwar, K.R. Maas, S.J. Hallam, 2013. Genomic properties of Marine Group A bacteria indicate a role in the marine sulfur cycle. The ISME Journal. (published online September 2013)

Wright ISME 2013 sm
Marine Group A (MGA) is a deeply branching and uncultivated phylum of bacteria. Although their functional roles remain elusive, MGA subgroups are particularly abundant and diverse in oxygen minimum zones and permanent or seasonally stratified anoxic basins, suggesting metabolic adaptation to oxygen-deficiency. Here, we expand a previous survey of MGA diversity in O2-deficient waters of the Northeast subarctic Pacific Ocean (NESAP) to include Saanich Inlet (SI), an anoxic fjord with seasonal O2 gradients and periodic sulfide accumulation. Phylogenetic analysis of small subunit ribosomal RNA (16S rRNA) gene clone libraries recovered five previously described MGA subgroups and defined three novel subgroups (SHBH1141, SHBH391, and SHAN400) in SI. To discern the functional properties of MGA residing along gradients of O2 in the NESAP and SI, we identified and sequenced to completion 14 fosmids harboring MGA-associated 16S RNA genes from a collection of 46 fosmid libraries sourced from NESAP and SI waters. Comparative analysis of these fosmids, in addition to four publicly available MGA-associated large-insert DNA fragments from Hawaii Ocean Time-series and Monterey Bay, revealed widespread genomic differentiation proximal to the ribosomal RNA operon that did not consistently reflect subgroup partitioning patterns observed in 16S rRNA gene clone libraries. Predicted protein-coding genes associated with adaptation to O2-deficiency and sulfur-based energy metabolism were detected on multiple fosmids, including polysulfide reductase (psrABC), implicated in dissimilatory polysulfide reduction to hydrogen sulfide and dissimilatory sulfur oxidation. These results posit a potential role for specific MGA subgroups in the marine sulfur cycle.
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48. An D. et al, 2013. Metagenomics of Hydrocarbon Resource Environments Indicates Aerobic Taxa and Genes to be Unexpectedly Common, Environmental Science and Technology (published online July 2013).

An EnvST 2013
D. An, S.M. Caffrey, J. Soh, A. Agrawal, D. Brown, K. Budwill, X. Dong, P.F. Dunfield, J. Foght, L.M. Gieg, S.J. Hallam, N.W. Hanson, Z. He, T.R. Jack, J. Klassen, K.M. Konwar, E. Kuatsjah, C. Li, S. Larter, V. Leopatra, C.L Nesbø, T.B. Oldenburg, A.P. Page, E. Ramos-Padron, F. Rochman, A. Saidi-Mehrabad, C.W. Sensen, P. Sipahimalani, Y.C. Song, S. Wilson, G. Wolbring, G. Wong, G. Voordouw.

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, the paradigm that HREs are strictly anaerobic and typically methanogenic for much of their history may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.
Link to article >

47. Mewis, K., Z. Armstrong, Y.C. Song, S.A. Baldwin, S.G. Withers, S.J. Hallam, 2013. Biomining active cellulases from a mining bioremediation system, Journal of Biotechnology (published online August 2013).

Mewis JBioTech 2013 sm
Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6,144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyze 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy Database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially-derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.
Link to article >

46. Rink, C., P. Schwientek, A. Sczyrba, N.N. Ivanova, I.J. Anderson, J. Cheng, A. Darling, S. Malfatti, B.K. Swan, E.A. Gies, J.A. Dodsworth, B.P. Hedlund, G. Tsiamis, S.M. Sievert, W. Liu, J.A. Eisen, S.J. Hallam, N.C. Kyrpides, R. Stepanauskas, E.M. Rubin, P. Hugenholtz, T. Woyke, 2013. Insights into the phylogeny and coding potential of microbial dark matter, Nature (published online July 2013).

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

45. Swan, B.K., B. Tupper, A. Sczyrba, F.M. Lauro, M. Martinez-Garcia, J.M. González, H. Luo, J.J. Wright, Z.C. Landry, N.W. Hanson, B.P. Thompson, N.J. Poulton, P. Schwientek, S.G. Acinas, S.J. Giovannoni, M.A. Moran, S.J. Hallam, R. Cavicchioli, T. Woyke, R. Stepanauskas, 2013. Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean, PNAS 110(28):11463-11468

Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.

44. Konwar, K.M., N.W. Hanson, A.P. Pagé, S.J. Hallam, 2013. MetaPathways: a modular pipeline for constructing pathway/genome databases from environmental sequence information, BMC Bioinformatics, 14:202

A central challenge to understanding the ecological and biogeochemical roles of microorganisms in natural and human engineered ecosystems is the reconstruction of metabolic interaction networks from environmental sequence information. The dominant paradigm in metabolic reconstruction is to assign functional annotations using BLAST. Functional annotations are then projected onto symbolic representations of metabolism in the form of KEGG pathways or SEED subsystems.

Here we present MetaPathways, an open source pipeline for pathway inference that uses the PathoLogic algorithm to map functional annotations onto the MetaCyc collection of reactions and pathways, and construct environmental Pathway/Genome Databases (ePGDBs) compatible with the editing and navigation features of Pathway Tools. The pipeline accepts assembled or unassembled nucleotide sequences, performs quality assessment and control, predicts and annotates noncoding genes and open reading frames, and produces inputs to PathoLogic. In addition to constructing ePGDBs, MetaPathways uses MLTreeMap to build phylogenetic trees for selected taxonomic anchor and functional gene markers, converts General Feature Format (GFF) files into concatenated GenBank files for ePGDB construction based on third-party annotations, and generates useful file formats including Sequin files for direct GenBank submission and gene feature tables summarizing annotations, MLTreeMap trees, and ePGDB pathway coverage summaries for statistical comparisons.

MetaPathways provides users with a modular annotation and analysis pipeline for predicting metabolic interaction networks from environmental sequence information using an alternative to KEGG pathways and SEED subsystems mapping. It is extensible to genomic and transcriptomic datasets from a wide range of sequencing platforms, and generates useful data products for microbial community structure and function analysis. The MetaPathways software package, installation instructions, and example data can be obtained from
See poster>

43. Solonenko, S.A., J.C. Ignacio-Espinoza, A. Alberti, C. Cruaud, S.J. Hallam, K. Konstantinidis, G. Tyson, P. Wincker, M.B. Sullivan, 2013. Sequencing platform and library preparation choices impact viral metagenomes, BMC Genomics 14:320

Microbes drive the biogeochemistry that fuels the planet. Microbial viruses modulate their hosts directly through mortality and horizontal gene transfer, and indirectly by re-programming host metabolisms during infection. However, our ability to study these virus-host interactions is limited by methods that are low-throughput and heavily reliant upon the subset of organisms that are in culture. One way forward are culture-independent metagenomic approaches, but these novel methods are rarely rigorously tested, especially for studies of environmental viruses, air microbiomes, extreme environment microbiology and other areas with constrained sample amounts. Here we perform replicated experiments to evaluate Roche 454, Illumina HiSeq, and Ion Torrent PGM sequencing and library preparation protocols on virus metagenomes generated from as little as 10pg of DNA.

Using %G + C content to compare metagenomes, we find that (i) metagenomes are highly replicable, (ii) some treatment effects are minimal, e.g., sequencing technology choice has 6-fold less impact than varying input DNA amount, and (iii) when restricted to a limited DNA concentration (<1μg), changing the amount of amplification produces little variation. These trends were also observed when examining the metagenomes for gene function and assembly performance, although the latter more closely aligned to sequencing effort and read length than preparation steps tested. Among Illumina library preparation options, transposon-based libraries diverged from all others and adaptor ligation was a critical step for optimizing sequencing yields. These data guide researchers in generating systematic, comparative datasets to understand complex ecosystems, and suggest that neither varied amplification nor sequencing platforms will deter such efforts.

42. Ulloa, O., J.J. Wright, L. Belmar, S.J. Hallam, 2013. Pelagic oxygen minimum zone microbial communities, Part 7 in The Prokaryotes - Prokaryotic Communities and Ecophysiology

Oxygen minimum zones (OMZs) are regions of the global ocean in which dissolved oxygen in the water column is reduced or totally absent due to poor ventilation, sluggish circulation, and a high demand of oxygen by microbial aerobic respiration. Open-ocean OMZs are prominent in the eastern tropical and subarctic Pacific and the northern Indian Oceans (Fig. 7.1). The actual concentration of dissolved oxygen varies among recognized OMZs, and determining whether or not they reach total anoxia based exclusively on oxygen measurements has until recently been a problem due to technical limitations (Revsbech et al. 2009; Thamdrup et al. 2012). This is highly relevant because microbial-encoded enzymes mediating aerobic and anaerobic transformations of elements (e.g., nitrogen, sulfur, and carbon) manifest different oxygen sensitivities.

It has long been recognized that many OMZs are hotspots for oxygen-sensitive nitrogen transformations, where nitrate serves as themain terminal electron acceptor for the oxidation of organic matter (Lam and Kuypers 2011). In such cases, denitrification and anaerobic ammonium oxidation (anammox) contribute to the removal of fixed nitrogen as N2, with resulting impacts on global nutrient cycles and the climate system (Codispoti et al. 2001). Geochemical signs of the functioning of these anaerobic processes include the presence of an inorganic fixed nitrogen deficit relative to phosphorus in addition to the accumulation of nitrite and excess N2 in the oxygen-deficient regions of the water column. Processes occurring in the boundary regions of OMZs also contribute to the production of the potent greenhouse gas nitrous oxide (N2O), due primarily to the activity of nitrifiers at low oxygen levels. Thus, continued OMZ expansion is an emerging environmental concern, as it will likely exacerbate the loss of fixed nitrogen from the ocean in addition to increasing N2O production (Keeling et al. 2010; Codispoti 2010).

41. Durno, E.W., N.W. Hanson, K.M. Konwar, S.J. Hallam, 2013. Expanding the boundaries of local similarity analysis, BMC Genomics 14(Suppl 1):S3

Pairwise comparison of time series data for both local and time-lagged relationships is a computationally challenging problem relevant to many fields of inquiry. The Local Similarity Analysis (LSA) statistic identifies the existence of local and lagged relationships, but determining significance through a p-value has been algorithmically cumbersome due to an intensive permutation test, shuffling rows and columns and repeatedly calculating the statistic. Furthermore, this p-value is calculated with the assumption of normality -- a statistical luxury dissociated from most real world datasets.

To improve the performance of LSA on big datasets, an asymptotic upper bound on the p-value calculation was derived without the assumption of normality. This change in the bound calculation markedly improved computational speed from O(pm2n) to O(m2n), where p is the number of permutations in a permutation test, m is the number of time series, and n is the length of each time series. The bounding process is implemented as a computationally efficient software package, FASTLSA, written in C and optimized for threading on multi-core computers, improving its practical computation time. We computationally compare our approach to previous implementations of LSA, demonstrate broad applicability by analyzing time series data from public health, microbial ecology, and social media, and visualize resulting networks using the Cytoscape software.

The FASTLSA software package expands the boundaries of LSA allowing analysis on datasets with millions of co-varying time series. Mapping metadata onto force-directed graphs derived from FASTLSA allows investigators to view correlated cliques and explore previously unrecognized network relationships. The software is freely available for download at:

40. Allers, E., J.J. Wright, K.M. Konwar, C.G. Howes, E. Beneze, S.J. Hallam, M.B. Sullivan, 2012. Diversity and population structure of Marine Group A bacteria in the Northern subarctic Pacific Ocean, ISME Journal

Marine Group A (MGA) is a candidate phylum of Bacteria that is ubiquitous and abundant in the ocean. Despite being prevalent, the structural and functional properties of MGA populations remain poorly constrained. Here, we quantified MGA diversity and population structure in relation to nutrients and O2 concentrations in the oxygen minimum zone (OMZ) of the Northeast subarctic Pacific Ocean using a combination of catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and 16S small subunit ribosomal RNA (16S rRNA) gene sequencing (clone libraries and 454-pyrotags). Estimates of MGA abundance as a proportion of total bacteria were similar across all three methods although estimates based on CARD-FISH were consistently lower in the OMZ (5.6%±1.9%) than estimates based on 16S rRNA gene clone libraries (11.0%±3.9%) or pyrotags (9.9%±1.8%). Five previously defined MGA subgroups were recovered in 16S rRNA gene clone libraries and five novel subgroups were defined (HF770D10, P262000D03, P41300E03, P262000N21 and A714018). Rarefaction analysis of pyrotag data indicated that the ultimate richness of MGA was very nearly sampled. Spearman’s rank analysis of MGA abundances by CARD-FISH and O2 concentrations resulted in significant correlation. Analyzed in more detail by 16S rRNA pyrotag sequencing, MGA operational taxonomic units affiliated with subgroups Arctic95A-2 and A714018 comprised 0.3–2.4% of total bacterial sequences and displayed strong correlations with decreasing O2 concentration. This study is the first comprehensive description of MGA diversity using complementary techniques. These results provide a phylogenetic framework for interpreting future studies on ecotype selection among MGA subgroups, and suggest a potentially important role for MGA in the ecology and biogeochemistry of OMZs.

39. Anderson, R.E., M.T. Beltrán, S.J. Hallam, J.A. Baross, 2012. Microbial community structure across fluid gradients in the Juan de Fuca Ridge hydrothermal system, FEMS Microbial Ecology 83(2):324-339

Physical and chemical gradients are dominant factors in shaping hydrothermal vent microbial ecology, where archaeal and bacterial habitats encompass a range between hot, reduced hydrothermal fluid and cold, oxidized seawater. To determine the impact of these fluid gradients on microbial communities inhabiting these systems, we surveyed bacterial and archaeal community structure among and between hydrothermal plumes, diffuse flow fluids, and background seawater in several hydrothermal vent sites on the Juan de Fuca Ridge using 16S rRNA gene diversity screening (clone libraries and terminal restriction length polymorphisms) and quantitative polymerase chain reaction methods. Community structure was similar between hydrothermal plumes and background seawater, where a number of taxa usually associated with low-oxygen zones were observed, whereas high-temperature diffuse fluids exhibited a distinct phylogenetic profile. SUP05 and Arctic96BD-19 sulfur-oxidizing bacteria were prevalent in all three mixing regimes where they exhibited overlapping but not identical abundance patterns. Taken together, these results indicate conserved patterns of redox-driven niche partitioning between hydrothermal mixing regimes and microbial communities associated with sinking particles and oxygen-deficient waters. Moreover, the prevalence of SUP05 and Arctic96BD-19 in plume and diffuse flow fluids indicates a more cosmopolitan role for these groups in the ecology and biogeochemistry of the dark ocean.

38. Chen, H., S. Zhu, I. Casabon, S.J. Hallam, F.H. Crocker, W.W. Mohn, K.J. Indest, L.D. Eltis, 2012. Genomic and transcriptomic studies of an RDX-degrading actinobacterium, Applied and Environmental Microbiology 78(21):7798-7800

Whole genome sequencing, transcriptomic analyses and metabolic reconstruction were used to investigate Gordonia sp. KTR9's ability to catabolize a range of compounds including explosives and steroids. Aspects of this mycolic acid-containing actinobacterium's catabolic potential were experimentally verified and compared with those of rhodococci and mycobacteria.


37. Hartmann, M., C.G. Howes, D. VanInsberghe, H. Yu, D. Bachar, R. Christen, R.H. Nilsson, S.J. Hallam, W.W. Mohn, 2012. Significant and persistent impact of timber harvesting on soil microbial communities in Northern coniferous forests, ISME Journal 6(12):2199-2218

Forest ecosystems have integral roles in climate stability, biodiversity and economic development. Soil stewardship is essential for sustainable forest management. Organic matter (OM) removal and soil compaction are key disturbances associated with forest harvesting, but their impacts on forest ecosystems are not well understood. Because microbiological processes regulate soil ecology and biogeochemistry, microbial community structure might serve as indicator of forest ecosystem status, revealing changes in nutrient and energy flow patterns before they have irreversible effects on long-term soil productivity. We applied massively parallel pyrosequencing of over 4.6 million ribosomal marker sequences to assess the impact of OM removal and soil compaction on bacterial and fungal communities in a field experiment replicated at six forest sites in British Columbia, Canada. More than a decade after harvesting, diversity and structure of soil bacterial and fungal communities remained significantly altered by harvesting disturbances, with individual taxonomic groups responding differentially to varied levels of the disturbances. Plant symbionts, like ectomycorrhizal fungi, and saprobic taxa, such as ascomycetes and actinomycetes, were among the most sensitive to harvesting disturbances. Given their significant ecological roles in forest development, the fate of these taxa might be critical for sustainability of forest ecosystems. Although abundant bacterial populations were ubiquitous, abundant fungal populations often revealed a patchy distribution, consistent with their higher sensitivity to the examined soil disturbances. These results establish a comprehensive inventory of bacterial and fungal community composition in northern coniferous forests and demonstrate the long-term response of their structure to key disturbances associated with forest harvesting.

36. Bourbonnais, A., S.K. Juniper, D.A. Butterfield, A.H. Devol, M.M.M. Kuypers, G. Lavik, S.J. Hallam, C.B. Wenk, B.X. Chang, S.A. Murdock, M.F. Lehmann, 2012. Activity and abundance of denitrifying bacteria in the subsurface biosphere of diffuse hydrothermal vents of the Juan de Fuca Ridge, Biogeosciences Discuss 9(4):4177-4223

Little is known about nitrogen (N) transformations in general, and the elimination of N in particular, at diffuse vents where anoxic hydrothermal fluids have mixed with oxygenated crustal seawater prior to discharge. Oceanic N sinks that remove bio-available N ultimately affect chemosynthetic primary productivity in these ecosystems. Using 15N paired isotope techniques, we determined potential rates of fixed N-loss pathways (denitrification, anammox) and dissimilative nitrate reduction to ammonium (DNRA) in sulfidic hydrothermal vent fluids discharging from the subsurface at several sites at Axial Volcano and the Endeavour Segment on the Juan de Fuca Ridge. We also measured physico-chemical parameters (i.e. temperature, pH, nutrients, H2S and N2O concentrations) as well as the biodiversity and abundance of chemolithotrophic nitratereducing, sulfur-oxidizing -proteobacteria (SUP05 cluster) using sequence analysis of amplified small subunit ribosomal RNA (16S rRNA) genes in combination with taxonspecific quantitative polymerase chain reaction (qPCR) assays. Denitrification was the dominant N-loss pathway in the subsurface biosphere of the Juan de Fuca Ridge, with rates of up to 1000 nmolNl−1 day−1. In comparison, anammox rates were always < 5 nmolNl−1 day−1 and below the detection limit at most of the sites. DNRA rates were up to 152 nmolNl−1 day−1. These results suggest that bacterial denitrification out-competes anammox in sulfidic hydrothermal vent waters. Taxon-specific qPCR revealed that -proteobacteria of the SUP05 cluster sometimes dominated the microbial community (SUP05/total bacteria up to 38 %). Significant correlation existed between fixed N-loss (i.e., denitrification, anammox) rates and in-situ nitrate and dissolved inorganic nitrogen (DIN) deficits in the fluids, indicating that DIN availability may ultimately regulate N-loss in the subsurface. Based on our rate measurements, and on published data on hydrothermal fluid fluxes and residence times, we estimated that, on average, 10 TgNyr−1 could globally be removed in the subsurface biosphere of hydrothermal vents systems, and could thus represent a small, but significant, fraction of the total marine N loss (240–400 TgNyr−1).


35. Orsi, W., Y.C. Song, S.J. Hallam, and V. Edgcomb, 2012. Effect of oxygen minimum zone formation on communities of marine protists, ISME Journal 6(8):1586-1601

Changes in ocean temperature and circulation patterns compounded by human activities are leading to oxygen minimum zone (OMZ) expansion with concomitant alteration in nutrient and climate active trace gas cycling. Here, we report the response of microbial eukaryote populations to seasonal changes in water column oxygen-deficiency using Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island British Columbia, as a model ecosystem. We combine small subunit ribosomal RNA gene sequencing approaches with multivariate statistical methods to reveal shifts in operational taxonomic units during successive stages of seasonal stratification and renewal. A meta-analysis is used to identify common and unique patterns of community composition between Saanich Inlet and the anoxic/sulfidic Cariaco Basin (Venezuela) and Framvaren Fjord (Norway) to show shared and unique responses of microbial eukaryotes to oxygen and sulfide in these three environments. Our analyses also reveal temporal fluctuations in rare populations of microbial eukaryotes, particularly anaerobic ciliates, that may be of significant importance to the biogeochemical cycling of methane in OMZs.

34. Wright, J.J., K.M. Konwar, S.J. Hallam, 2012. Microbial ecology of expanding oxygen minimum zones, Nature Reviews Microbiology 10(6):381-394

Dissolved oxygen concentration is a crucial organizing principle in marine ecosystems. As oxygen levels decline, energy is increasingly diverted away from higher trophic levels into microbial metabolism, leading to loss of fixed nitrogen and to production of greenhouse gases, including nitrous oxide and methane. In this Review, we describe current efforts to explore the fundamental factors that control the ecological and microbial biodiversity in oxygen-starved regions of the ocean, termed oxygen minimum zones. We also discuss how recent advances in microbial ecology have provided information about the potential interactions in distributed co-occurrence and metabolic networks in oxygen minimum zones, and we provide new insights into coupled biogeochemical processes in the ocean.

33. Leung, K., H. Zahn, T. Leaver, K.M. Konwar, N.W. Hanson, A.P. Pagé, C.C. Lo, P.S. Chain, S.J. Hallam, C.L. Hansen, 2012. A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities, Proceedings of the National Academy of Sciences of the United States of America 109(20):7665-7670

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by “flow-controlled wetting,” a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

32. Sauter, L.M., E. Latypova, N.E. Smalley, M.E. Lidstrom, S.J. Hallam, M.G. Kalyuzhnaya, 2012. Methanotrophic communities of Saanich Inlet: A microcosm perspective, Systematic and Applied Microbiology 35(3):198-203

Saanich Inlet (British Columbia, Canada) is a seasonally anoxic fjord characterized by high rates of both methane production and consumption. In this study, the diversity of microbial populations residing in intermediate waters, characterized by having a high methane content, was assessed using CH4-microcosm experiments coupled with PCR surveys of phylogenetic (16S rRNA gene) and functional gene markers (pmoA and fhcD genes). The experiments revealed that bacteria represented by sequences affiliated with Methylomicrobium within the Methylococcales, Methylophaga and Cycloclasticus within the Thiotrichales, and uncultured Planctomycetes were enriched in response to CH4 addition.

31. Walsh D.A. and S.J. Hallam, 2011. Bacterial community diversity and population dynamics in a seasonally anoxic fjord: Saanich Inlet, Chapter 25:253-267 Handbook of Molecular Microbial Ecology II: Metagenomics and Complementary Approaches, Wiley-Blackwell, Ed. Frans de Bruijn


30. Tuapp, M., K. Mewis, S.J. Hallam, 2011. The art and design of functional metagenomic screens, Current Opinion in Biotechnology 22(3):465-472

This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host–vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.

29. Hallam, S.J., A.P. Pagé, L. Constan, Y.C. Song, A.D. Norbeck, H. Brewer, L. Pasa-Tolic, 2011. Molecular tools for investigatin ANME community structure and function, Methods in Enzymology: Methods in Methane Metabolism, PT A 494:75-90

1. Introduction
2. Exploring ANME Population Structure
2.1. Sample processing and DNA extraction
2.2. Methyl-coenzyme M reductase primer design
2.3. Quantifying mcrA copy number using dye assay chemistry
3. Detecting ANME Proteins in Marine Sediments
3.1. Sample processing and protein extraction
3.2. Protein digestion and HPLC separation
3.3. Tandem mass spectrometry and peptide identification

28. Hartmann, M., C.G. Howes, V. Veldre, S. Schneider, P.A. Vaishampayan, A.C. Yannarell, C. Quince, P. Johansson, K.J. Bjorkroth, K. Abarenkov, S.J. Hallam, W.W. Mohn, R.H. Nilsson, 2011. V-REVCOMP: automated high-throughput detection of reverse complementary 16S rRNA gene sequences in large environmental and taxonomic datasets, FEMS Microbiology Letters, 319(2):140-145

Reverse complementary DNA sequences – sequences that are inadvertently given backwards with all purines and pyrimidines transposed – can affect sequence analysis detrimentally unless taken into account. We present an open-source, high-throughput software tool –v-revcomp ( – to detect and reorient reverse complementary entries of the small-subunit rRNA (16S) gene from sequencing datasets, particularly from environmental sources. The software supports sequence lengths ranging from full length down to the short reads that are characteristic of next-generation sequencing technologies. We evaluated the reliability of v-revcomp by screening all 406 781 16S sequences deposited in release 102 of the curated SILVA database and demonstrated that the tool has a detection accuracy of virtually 100%. We subsequently used v-revcomp to analyse 1 171 646 16S sequences deposited in the International Nucleotide Sequence Databases and found that about 1% of these user-submitted sequences were reverse complementary. In addition, a nontrivial proportion of the entries were otherwise anomalous, including reverse complementary chimeras, sequences associated with wrong taxa, nonribosomal genes, sequences of poor quality or otherwise erroneous sequences without a reasonable match to any other entry in the database. Thus, v-revcomp is highly efficient in detecting and reorienting reverse complementary 16S sequences of almost any length and can be used to detect various sequence anomalies.

27. Zaikova, E., D. A. Walsh, C. P. Stilwell, W.W. Mohn, P. D. Tortell, and S. J. Hallam, 2010. Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet British Columbia, Environmental Microbiology, 12(1):172-91

Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the δ-proteobacteria, SAR406 and γ-proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.

26. Taupp, M., L. Constan L., S. J. Hallam. 2010. The biochemistry of anaerobic methane oxidation, Part 7 in the Handbook of Hydrocarbon and Lipid Microbiology

The anaerobic oxidation of methane (AOM) is a globally significant biogeochemical process that exerts a profound influence on methane flux between oceanic and atmospheric compartments of the biosphere. In marine sediments AOM occurs in a region of sulfate and methane depletion known as the sulfate–methane transition zone (SMTZ) where methane is converted to carbon dioxide and reduced products that are in turn used as electron donors in the conversion of sulfate to hydrogen sulfide and water. From a bioenergetic perspective, AOM represents a major source of maintenance energy within the SMTZ, and despite low estimated free energy yields supports a vigorous microbial metabolism. Lipid biomarker, phylogenetic stain and environmental PCR studies aimed at determining the biological component of AOMconverge on microbial communities dominated by uncultivated anaerobic methane-oxidizing archaea (ANME-1, ANME-2 and ANME-3) and sulfate reducing bacteria (SRB). Specific physical associations between these groups have been observed consistent with syntrophic modes of growth. However, despite extensive mesocosm and labeling studies theprecise modeof electron transfer between ANME and SRB remains unknown. Recent cultivation-independent studies of AOM communities from the Eel River Basin, Hydrate Ridge and the Black Sea have led to preliminary reconstruction of the genes and pathways mediating carbon and energy metabolism within ANME subgroups providing a genomic and proteomic basis for inferring substrate ranges, intermediates and terminal electron acceptors. The following chapter reviews biochemical aspects of AOM with special emphasis on pathway validation, electron flow and enzyme function. We consider how ANME subgroup partitioning and gene expression profiles overlap with prevailing thermodynamic models and speculate on syntrophic growth models as they relate to broader aspects of community metabolism within AOM sediments.

25. Taupp, M. and S. J. Hallam. 2010. The “meta-methanoxgenome”, Part 45 in the Handbook of Hydrocarbon and Lipid Microbiology

Although the vast majority of microbes in nature resist laboratory cultivation, they represent an almost limitless reservoir of genomic diversity and biological innovation. The development of cultivation-independent tools to explore the genomic potential of naturally occurring microbial communities illuminates their hidden enzymatic powers and provides direct insight into the metabolic networks underlying fundamental biogeochemical processes. Microbial mediated anaerobic oxidation of methane (AOM) is a prime exemplar of this paradigm. AOM acts as a biological methane filter beneath the sea, stimulates subsurface metabolism and supports thriving chemosynthetic communities that derive energy from one of its by-products, hydrogen sulfide. Recent environmental genomic surveys of DNA sequences recovered from AOM communities in the Eel River Basin, Hydrate Ridge and Black Sea have identified numerous gene sequences typically associated with canonical methanogenic pathways, including the terminal step, mediated by methyl coenzyme M reductase. Combined nucleotide composition, read density, and linkage analyses have enabled the separation of this sequence information into phylogenetic bins representing the genomic repertoires of anaerobic methane-oxidizing archaeal (ANME) subgroups opening a functional genomic window into the metabolic networks underlying AOM. The following chapter reviews theoretical and pragmatic aspects of environmental genomic library construction and sequence analysis of AOM communities focusing on carbon and energy metabolism. We consider how ANME ecotype variation contributes to adaptation, or niche partitioning at the genotypic level and present a series of experimental recommendations for pathway validation and composite genome assembly.

24. Walsh, D. A., E. Zaikova, C. G. Howes, Y. C. Song., J.J. Wright, S. Tringe, P. D. Tortell, and S. J. Hallam, 2009. Metagenome of a versatile chemolithoautotroph from expanding oceanic dead zones, Science, 326(5952):578-82

Oxygen minimum zones, also known as oceanic “dead zones,” are widespread oceanographic features currently expanding because of global warming. Although inhospitable to metazoan life, they support a cryptic microbiota whose metabolic activities affect nutrient and trace gas cycling within the global ocean. Here, we report metagenomic analyses of a ubiquitous and abundant but uncultivated oxygen minimum zone microbe (SUP05) related to chemoautotrophic gill symbionts of deep-sea clams and mussels. The SUP05 metagenome harbors a versatile repertoire of genes mediating autotrophic carbon assimilation, sulfur oxidation, and nitrate respiration responsive to a wide range of water-column redox states. Our analysis provides a genomic foundation for understanding the ecological and biogeochemical role of pelagic SUP05 in oxygen-deficient oceanic waters and its potential sensitivity to environmental changes.

23. Schmidtova, J., S. J. Hallam, S.A. Baldwin, 2009. Phylogenetic diversity of transition and anoxic zone bacterial communities within a near-shore anoxic basin: Nitinat Lake, Environmental Microbiology, 11(12):3233-51

At two stations surveyed in Nitinat Lake, a 200-m-deep anoxic tidal fjord, sulfide was detected as close as 15 m from the surface. Biological characterization, determined from small subunit ribosomal RNA gene sequencing, of the chemocline and anaerobic zone revealed many sequences related to sulfur-oxidizing bacteria, suggesting that sulfur cycling is a dominant process. γ- and ε-Proteobacteria related to thiotrophic symbionts, as well as Chlorobium sp., dominated the transition zone. These are expected to play a role in dark and phototrophic CO2 fixation, respectively. ε-Proteobacteria phylotype abundance increased with depth, eventually comprising 69–97% of all sequences recovered from the anoxic zone. The vast majority (74%) of these phylotypes were affiliated with a novel Acrobacter sp. group (NITEP5). Quantification of NITEP5 revealed that up to 2.8 × 105 cells ml−1 were present in the anoxic zone. Surprisingly, although sequences related to known sulfate-reducing bacteria were recovered from the transition zone, quantification of the dsr gene and 35SO42− uptake tests suggest that sulfate-reduction within the water column is negligible. Overall, sequence diversity between different vertical zones was high, although the spatial segregation of γ-Proteobacteria, Chlorobi, and ε-Proteobacteria did not appear to vary significantly between seasons.

22. Hartmann, M., S. Lee, S. J. Hallam and W.W. Mohn, 2009. Bacterial, archaeal and eukaryal community structures throughout soil horizons of harvested and naturally disturbed forest stands, Environmental Microbiology 11(12):3045-62

Disturbances caused by timber harvesting have critical long-term effects on the forest soil microbiota and alter fundamental ecosystem services provided by these communities. This study assessed the effects of organic matter removal and soil compaction on microbial community structures in different soil horizons 13 years after timber harvesting at the long-term soil productivity site at Skulow Lake, British Columbia. A harvested stand was compared with an unmanaged forest stand. Ribosomal intergenic spacer profiles of bacteria, archaea and eukarya indicated significantly different community structures in the upper three soil horizons of the two stands, with differences decreasing with depth. Large-scale sequencing of the ribosomal intergenic spacers coupled to small-subunit ribosomal RNA genes allowed taxonomic identification of major microbial phylotypes affected by harvesting or varying among soil horizons. Actinobacteria and Gemmatimonadetes were the predominant phylotypes in the bacterial profiles, with the relative abundance of these groups highest in the unmanaged stand, particularly in the deeper soil horizons. Predominant eukaryal phylotypes were mainly assigned to known mycorrhizal and saprotrophic species of Basidiomycetes and Ascomycetes. Harvesting affected Basidiomycetes to a minor degree but had stronger effects on some Ascomycetes. Archaeal profiles had low diversity with only a few predominant crenarchaeal phylotypes whose abundance appeared to increase with depth. Detection of these effects 13 years after harvesting may indicate a long-term change in processes mediated by the microbial community with important consequences for forest productivity. These effects warrant more comprehensive investigation of the effects of harvesting on the structure of forest soil microbial communities and the functional consequences.

21. Lobban, C. S., S. J. Hallam, P. Mukherjee, and J. W. Petrich. 2007. Photophysics and multifunctionality of hypericin-like pigments in heterotrich ciliates: a phylogenetic perspective. Photochem Photobiol 83:1074-94

In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.

20. Hallam, S. J., K. T. Konstantinidis, N. Putnam, C. Schleper, Y. Watanabe, J. Sugahara, C. Preston, J. de la Torre, P. M. Richardson, and E. F. DeLong. 2006. Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum. Proc Natl Acad Sci U S A 103:18296-301

Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal–sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.

19. Yoshinari, S., T. Itoh, S. J. Hallam, E. F. DeLong, S. Yokobori, A. Yamagishi, T. Oshima, K. Kita, and Y. Watanabe. 2006. Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease. Biochem Biophys Res Commun 346:1024-32

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA–protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.

18. Hallam, S. J., T. J. Mincer, C. Schleper, C. M. Preston, K. Roberts, P. M. Richardson, and E. F. DeLong. 2006. Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine Crenarchaeota. PLoS Biol 4:e95

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.

17. DeLong, E. F., C. M. Preston, T. Mincer, V. Rich, S. J. Hallam, N. U. Frigaard, A. Martinez, M. B. Sullivan, R. Edwards, B. R. Brito, S. W. Chisholm, and D. M. Karl. 2006. Community genomics among stratified microbial assemblages in the ocean's interior. Science 311:496-503

Microbial life predominates in the ocean, yet little is known about its genomic variability, especially along the depth continuum. We report here genomic analyses of planktonic microbial communities in the North Pacific Subtropical Gyre, from the ocean's surface to near–sea floor depths. Sequence variation in microbial community genes reflected vertical zonation of taxonomic groups, functional gene repertoires, and metabolic potential. The distributional patterns of microbial genes suggested depth-variable community trends in carbon and energy metabolism, attachment and motility, gene mobility, and host-viral interactions. Comparative genomic analyses of stratified microbial communities have the potential to provide significant insight into higher-order community organization and dynamics.

16. Cubonova, L., K. Sandman, S. J. Hallam, E. F. Delong, and J. N. Reeve. 2005. Histones in crenarchaea. J Bacteriol 187:5482-5

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.

15. Salerno, J. L., S. A. Macko, S. J. Hallam, M. Bright, Y. J. Won, Z. McKiness, and C. L. Van Dover. 2005. Characterization of symbiont populations in life-history stages of mussels from chemosynthetic environments. Biol Bull 208:145-55

The densities of chemoautotrophic and methanotrophic symbiont morphotypes were determined in life- history stages (post-larvae, juveniles, adults) of two species of mussels (Bathymodiolus azoricus and B. heckerae) from deep-sea chemosynthetic environments (the Lucky Strike hydrothermal vent and the Blake Ridge cold seep) in the Atlantic Ocean. Both symbiont morphotypes were observed in all specimens and in the same relative proportions, regardless of life-history stage. The relative abundance of symbiont morphotypes, determined by transmission electron microscopy, was different in the two species: chemoautotrophs were dominant (13:1-18:1) in B. azoricus from the vent site; methanotrophs were dominant (2:1-3:1) in B. heckerae from the seep site. The ratio of CH4:H2S is proposed as a determinant of the relative abundance of symbiont types: where CH4:H2S is less than 1, as at the Lucky Strike site, chemoautotrophic symbionts dominate; where CH4:H2S is greater than 2, as at the seep site, methanotrophs dominate. Organic carbon and nitrogen isotopic compositions of B. azoricus (delta 13C = -30 per thousand; delta 15N = -9 per thousand) and B. heckerae (delta 13C = -56 per thousand; delta 15N = -2 per thousand) varied little among life-history stages and provided no record of a larval diet of photosynthetically derived organic material in the post-larval and juvenile stages.

14. Hallam, S. J., N. Putnam, C. M. Preston, J. C. Detter, D. Rokhsar, P. M. Richardson, and E. F. DeLong. 2004. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305:1457-62

Microbial methane consumption in anoxic sediments significantly impacts the global environment by reducing the flux of greenhouse gases from ocean to atmosphere. Despite its significance, the biological mechanisms controlling anaerobic methane oxidation are not well characterized. One current model suggests that relatives of methane-producing Archaea developed the capacity to reverse methanogenesis and thereby to consume methane to produce cellular carbon and energy. We report here a test of the “reverse-methanogenesis” hypothesis by genomic analyses of methane-oxidizing Archaea from deep-sea sediments. Our results show that nearly all genes typically associated with methane production are present in one specific group of archaeal methanotrophs. These genome-based observations support previous hypotheses and provide an informed foundation for metabolic modeling of anaerobic methane oxidation.

13. Hallam, S. J., P. R. Girguis, C. M. Preston, P. M. Richardson, and E. F. DeLong. 2003. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea. Appl Environ Microbiol 69:5483-91

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.

12. Girguis, P. R., V. J. Orphan, S. J. Hallam, and E. F. DeLong. 2003. Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor. Appl Environ Microbiol 69:5472-82

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

11. Goffredi, S. K., L. A. Hurtado, S. J. Hallam, and R. C. Vrijenhoek. 2003. Evolutionary relationships of deep-sea vent and cold seep clams (Mollusca : Vesicomyidae) of the "pacifica/lepta" species complex, p. 311-320. Mar Biol, vol. 142

Vesicomyid clams are among the dominant invertebrates of chemosynthesis-based communities found at deep-sea cold seeps and hydrothermal vents, yet there is considerable taxonomic confusion within the family Vesicomyidae. The present study examined phylogenetic relationships among vesicomyid clams belonging to a cryptic-species complex that includes Vesicomya (previously Calyptogena) pacifica (Dall, 1891) and Vesicomya lepta (Dall, 1896). Mitochondrial (mt) COI sequences from clams collected between 1991 and 2001 along the western margin of North America from the San Clemente Basin (32°N latitude) to the Juan de Fuca Ridge (48°N latitude) revealed five discrete evolutionary lineages: V. pacifica (mt-type I), V. lepta (mt-type IV), and three undescribed species defined by mt-types II, III, and V. Separation of the three lineages defined by mt-types I, II, and III was also revealed by an independent nuclear gene (ribosomal ITS-1), and by DNA sequences from vertically transmitted bacterial endosymbionts found in the gills of these clams. Lineages I, II, and III occur in Monterey Bay, California, and the data suggest that bathymetric segregation may have played a role in their isolation and divergence.

10. Won, Y., S. J. Hallam, G. D. O'Mullan, and R. C. Vrijenhoek. 2003. Cytonuclear disequilibrium in a hybrid zone involving deep-sea hydrothermal vent mussels of the genus Bathymodiolus. Mol Ecol 12:3185-90

A hybrid zone involving the deep-sea mussels, Bathymodiolus azoricus and B. puteoserpentis, was recently discovered at Broken Spur hydrothermal vent field (29°10′ N, 43°10′ W) along an intermediate segment of the Mid-Atlantic Ridge axis. Examination of nuclear (allozymes) and cytoplasmic (mitochondrial DNA) gene markers in a new sample from Broken Spur revealed significant cytonuclear disequilibrium caused by an excess of the parental types (coupling phase) and a deficiency of recombinants (repulsion phase). An assignment test of individual multilocus genotypes also revealed an excess of parental genotypes in the admixed population. These results support the hypothesis that the Broken Spur mussel population comprises a nonequilibrium mixture of parental immigrants and hybrid individuals.

9. Won, Y. J., S. J. Hallam, G. D. O'Mullan, I. L. Pan, K. R. Buck, and R. C. Vrijenhoek. 2003. Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus Bathymodiolus. Appl Environ Microbiol 69:6785-92

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.

8. Hallam, S. J., A. Goncharov, J. McEwen, R. Baran, and Y. Jin. 2002. SYD-1, a presynaptic protein with PDZ, C2 and rhoGAP-like domains, specifies axon identity in C. elegans. Nat Neurosci 5:1137-46

Axons are defined by the presence of presynaptic specializations at specific locations. We show here that loss-of-function mutations in the C. elegans gene syd-1 cause presynaptic specializations to form in the dendritic processes of GABA-expressing motor neurons during initial differentiation. At a later developmental stage, however, syd-1 is not required for the polarity respecification of a subset of these neurons. The SYD-1 protein contains PDZ, C2 and rho−GTPase activating protein (GAP)-like domains, and is localized to presynaptic terminals in mature neurons. A truncated SYD-1 that lacks the rhoGAP domain interferes with neurite outgrowth and guidance. Our data indicate that syd-1 may be involved in specifying axon identity during initial polarity acquisition.

7. Hallam, S. J., E. Singer, D. Waring, and Y. Jin. 2000. The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification. Development 127:4239-52

The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. Here we report the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family.

6. Hallam, S. J., and Y. Jin. 1998. lin-14 regulates the timing of synaptic remodelling in Caenorhabditis elegans. Nature 395:78-82

In the nematode Caenorhabditis elegans six GABAergic motor neurons, known as DDs1,2, remodel their patterns of synaptic connectivity during larval development3. DD remodelling involves a complete reversal of the direction of information flow within nerve processes without marked changes in process morphology. We used a marker localized in vivo to DD presynaptic zones to analyse how the timing of DD remodelling is controlled. In wild-type animals, DDs remodel their synaptic outputs within a 3–5-hour period at the end of the first larval stage.We show that the heterochronic gene lin-14, which controls the timing of stagespecific cell lineages4,5, regulates the timing of DD synaptic output remodelling. In lin-14 loss-of-function mutants, DDs remodel precociously. The degree of precocious remodelling is correlated with the level of lin-14 activity. Expression of lin-14(+) in the DDs of lin-14-null mutants rescues the precocious remodelling, indicating that lin-14 can act cell-autonomously. Consistent with this hypothesis, LIN-14 protein levels decrease in the DDs before remodelling. Our observations reveal a role of heterochronic genes in non-dividing cells, and provide an example of cellautonomous respecification of neuronal connectivity.

5. Zhou, D. C., S. J. Hallam, S. J. Lee, R. S. Klein, P. H. Wiernik, M. S. Tallman, and R. E. Gallagher. 1998. Constitutive expression of cellular retinoic acid binding protein II and lack of correlation with sensitivity to all-trans retinoic acid in acute promyelocytic leukemia cells

The up-regulation of cellular retinoic acid binding protein-II (CRABP-II) has been invoked as an important mechanism of clinically acquired resistance to all-trans retinoic acid (RA) therapy in acute promyelocytic leukemia (APL). To test this hypothesis, we used quantitative reverse transcription-PCR and fast performance liquid chromatography procedures to examine the levels of CRABP-II mRNA and RA binding activity in APL patient samples. We found that CRABP-II mRNA in APL cells from pretreatment patients (n = 36) was constitutively expressed at relatively high levels (median, 0.92; range, 0.16–4.13) relative to the level in CRABP-II protein-expressing NB4 cells (arbitrarily set at 1.0 unit). Consistent with this finding, the RA binding activity of CRABP in APL cells from three pretreatment cases (range, 27.2–53.2 fmol/mg protein) was similar to that of NB4 cells (22.6 ± 5.4 fmol/mg protein). Furthermore, in the pretreatment samples, there was no association between CRABP-II mRNA expression level and APL cellular sensitivity to RA-induced differentiation in vitro. After 45 days of remission induction therapy on Eastern Cooperative Oncology Group protocol E2491, CRABP-II mRNA was modestly increased from day 0 values in patients treated with either RA (median increase, 0.41) or chemotherapy (median increase, 0.56), and there was no significant difference between the two treatment groups (P = 0.91). In patients studied after relapse from RA therapy (n = 7), there was a significant decline in APL cell sensitivity to RA-induced differentiation in vitro compared with patients after relapse from chemotherapy (n = 5; P = 0.015-0.055 at three RA concentrations tested), but in the RA relapse cases, there was no change from pretreatment levels of CRABP-II mRNA (median, 0.98) or, in three relapse cases studied, of RA protein binding activity (range, 22.1–70.7 fmol/mg protein). Taken together, our data strongly imply that variations in CRABP-II expression and RA binding activity are not causally related to the development of clinically acquired APL cellular RA resistance, but rather, they suggest that constitutive expression of CRABP-II could have a facilitative role in the response of APL cells to RA.

4. Wadler, S., E. L. Schwartz, H. Haynes, R. Rameau, A. Quish, J. Mandeli, R. Gallagher, S. J. Hallam, A. Fields, G. Goldberg, F. McGill, S. Jennings, R. C. Wallach, and C. D. Runowicz. 1997. All-trans retinoic acid and interferon-alpha-2a in patients with metastatic or recurrent carcinoma of the uterine cervix: clinical and pharmacokinetic studies. New York Gynecologic Oncology Group. Cancer 79:1574-80

Carcinoma of the cervix that is beyond the scope of definitive radiation therapy or surgery has a poor prognosis. Macroscopic involvement of paraaortic lymph nodes, pelvic recurrence after primary radiation therapy, and visceral involvement are associated with a 1-year survival of <15%. In addition, patients often have severe symptoms related to a sacral plexopathy, ureteral obstruction and renal failure, and thromboembolic events. Systemic therapy with cytotoxic agents has only a limited role in controlling these symptoms and preventing disease progression. Thus, other therapeutic approaches remain to be investigated.

3. Schwartz, E. L., S. J. Hallam, R. E. Gallagher, and P. H. Wiernik. 1995. Inhibition of all-trans-retinoic acid metabolism by fluconazole in vitro and in patients with acute promyelocytic leukemia. Biochem Pharmacol 50:923-8

All-trans-retinoic acid induces acute promyelocytic leukemia cell differentiation in vitro, and it produces greater than 90% complete remissions in patients with acute promyelocytic leukemia. Despite the high response rate, the majority of patients relapse with continued trans-retinoic acid therapy, and disease progression has been observed to be accompanied by an increase in the metabolism of trans-retinoic acid in the patients. In this study, the pharmacokinetic disposition of trans-retinoic acid was determined by HPLC in patients with acute promyelocytic leukemia before and after concurrent therapy with the triazole antimycotic agent fluconazole. Treatment with trans-retinoic acid for 1 week reduced the area under the plasma trans-retinoic acid concentration vs time curve in one patient by 67%, from 277 to 91 ng/mL/hr. Trans-retinoic acid pharmacokinetics were repeated after the second dose of fluconazole, administered 1 hour prior to the retinoid, and the AUC was found to be 401 ng/mL/hr, a greater than 4-fold increase from the pre-fluconazole level. A similar, though more modest, effect of fluconazole was seen in a second acute promyelocytic leukemia patient. The effect of fluconazole on trans-retinoic acid metabolism was examined in vitro using isolated human hepatic microsomes. Fluconazole inhibited the NADPH-dependent cytochrome P450-mediated catabolism of trans-retinoic acid in a concentration-dependent manner. Although fluconazole was approximately one-half as potent an inhibitor when compared with ketoconazole, a related antifungal drug, 60–90% inhibition was observed at the concentrations of fluconazole measured in the acute promyelocytic leukemia patients. Neither fluconazole nor ketoconazole inhibited lipid hydroperoxide-mediated metabolism of trans-retinoic acid. Since fluconazole is a well-tolerated agent frequently administered to leukemia patients, its use in combination with trans-retinoic acid merits further consideration.

2. Sagayadan, G. E., P. H. Wiernik, N. Sun, G. Ahearn, D. Thompson, S. J. Hallam, X. P. Hu, J. P. Dutcher, and R. E. Gallagher. 1994. Effect of retinoic acid and interferon alpha on granulocyte-macrophage colony forming cells in chronic myeloid leukemia: increased inhibition by all-trans- and 13-cis-retinoic acids in advanced stage disease. Leuk Res 18:741-8

Granulocyte-macrophage colony forming units (CFU-GM) from patients with advanced stage chronic myelogenous leukemia (CML), i.e. in blastic crisis (BC) or accelerated phase (AP), were inhibited by all-trans-retinoic acid (tRA) approximately 1000-fold more potently than those from chronic phase (CP) CML patients (median ic50 = 10−9 M tRA for six CML-AP/BC cases vs > 10−6 M tRA for seven CML-CP cases). A similar activity pattern was observed for the stereoisomer 13-cis-RA (cRA). There was no apparent correlation of CFU-GM retinoid sensitivity with cloning efficiency or other colony characteristics. Interferon alpha-2a (INFα) alone strongly inhibited CFU-GM growth in all four CML-AP/BC cases (ic50 250 IU/ml) and three out of seven CML-CP cases (ic50 500 IU/ml), but there was little or no interactive effect between various concentrations of tRA and INFα (50 IU/ml) on CFU-GM from either CML-AP/BC or CML-CP cases. These results suggest that CML-AP/BC CFU-GM have some intrinsic molecular alteration(s) which markedly enhances their responsiveness to tRA and cRA, which may be clinically exploitable.

1. Wadler, S., X. Mao, R. Bajaj, S. J. Hallam, and E. L. Schwartz. 1993. N-(phosphonacetyl)-L-aspartate synergistically enhances the cytotoxicity of 5-fluorouracil/interferon-alpha-2a against human colon cancer cell lines. Mol Pharmacol 44:1070-6

Recombinant interferon-alpha (IFN) enhances the cytotoxic effects of the fluorinated pyrimidine, 5-fluorouracil (5FU), against two human colon cancer cell lines. The aspartate transcarbamylase (ATCase) inhibitor, N-(phosphonacetyl)-L-aspartate (PALA), was studied in combination with 5FU/IFN to determine whether further anti-pyrimidine effects would result in greater cytotoxicity. By median effects analysis PALA synergistically augmented the cytotoxic effects of 5FU/IFN against both human colon cancer cell lines. This occurred in the absence of any effects of 5FU/IFN on ATCase and without further potentiation of the PALA-mediated inhibition of ATCase. To explore the mechanism by which this interaction occurred, detailed studies of pools of dNTPs were performed. Both 5FU/IFN and PALA/5FU/IFN treatments resulted in early (2-8 hr) depletion of pools of dTTP, but no effects on pools of dCTP. PALA had no effect on dTTP pools either alone or in the combination. In contrast, both PALA and PALA/5FU/IFN treatments resulted in later (12-24 hr) depletion of pools of dCTP. 5FU/IFN treatment had no effect on these pools. When pools of dCTP and dTTP were repleted by treatment with cytidine or thymidine, 20 microM, however, there was only partial reversal of cytotoxicity induced by 5FU/IFN + PALA, suggesting that the synergy observed did not result solely from a sequential anti-pyrimidine effect. The incorporation of 5FU into RNA was also studied; PALA enhanced the incorporation of [6-3H]5FU into RNA by 83-150%, but not into DNA, suggesting an alternative mechanism of drug interaction.