Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments.

Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons (intI1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK, mexB, mexF, and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, ß-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics.IMPORTANCEThe United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events.

Corrigendum: New perspectives on an old grouping: the genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

[This corrects the article DOI: 10.3389/fmicb.2022.1011102.].

Microbial communities in sandy beaches from the three domains of life differ by microhabitat and intertidal location.

The microbial communities of sandy beaches are poorly described despite the biogeochemical importance and ubiquity of these ecosystems. Using metabarcoding of the 16S and 18S rRNA genes, we investigated the diversity, microhabitats (with or between sand grains) and intertidal distributions of microorganisms (including meiofauna) from pristine sandy beaches in British Columbia, Canada, and hypothesized that abiotic variations due to microhabitat or intertidal gradients influence the distribution of microorganisms on local scales. Bacterial, archaeal and protistan communities of the sand were clearly distinct from interstitial communities, and from planktonic communities of the overlying seawater, which correlated with differences in function and lifestyle (e.g., sulphur reduction and gliding motility). In contrast, meiofaunal communities could not be distinguished by sample type, suggesting that they are more frequently mobilized between these microhabitats. Across intertidal zones, high intertidal, mid intertidal and low intertidal/swash communities were distinct and correlated with moisture, organic carbon and phosphate content, implying that the distribution of microorganisms is influenced by intertidal abiotic gradients. However, few taxa at the genus or species level individually contributed to this zonation pattern; rather, a unique combination of multiple microbial taxa was probably responsible. Although significant differences in microbial community composition on sandy beaches can be attributed to microhabitat and intertidal gradients, further investigations are needed to assess community assembly processes, the consistency of these distributions, and the functions of the majority of the microorganisms observed in the sand and their effects on the biogeochemistry and ecology of sandy beaches.

Responses of the Soil Bacterial Community, Resistome, and Mobilome to a Decade of Annual Exposure to Macrolide Antibiotics.

Biosolids that are applied to agricultural soil as an organic fertilizer are frequently contaminated with pharmaceutical residues that have persisted during wastewater treatment and partitioned into the organic phase. Macrolide antibiotics, which serve as a critically important human medicine, have been detected within biosolids. To determine the impacts of macrolide antibiotics on soil bacteria, every year for a decade, a series of replicated field plots received an application of a mixture of erythromycin, clarithromycin, and azithromycin at a realistic (0.1 mg kg soil-1) or an unrealistically high (10 mg kg soil-1) dose or were left untreated. The effects of repeated antibiotic exposure on the soil bacterial community, resistome, mobilome, and integron gene cassette content were evaluated by 16S rRNA and integron gene cassette amplicon sequencing, as well as whole-metagenome sequencing. At the unrealistically high dose, the overall diversity of the resistome and mobilome was altered, as 21 clinically important antibiotic resistance genes predicted to encode resistance to 10 different antibiotic drug classes were increased and 20 mobile genetic element variants (tnpA, intI1, tnpAN, and IS91) were increased. In contrast, at the realistic dose, no effect was observed on the overall diversity of the soil bacterial community, resistome, mobilome, or integron gene cassette-carrying genes. Overall, these results suggest that macrolide antibiotics entrained into soil at concentrations anticipated with biosolid applications would not result in major changes to these endpoints. IMPORTANCE Biosolids, produced from the treatment of sewage sludge, are rich in plant nutrients and are a valuable alternative to inorganic fertilizer when applied to agricultural soil. However, the use of biosolids in agriculture, which are frequently contaminated with pharmaceuticals, such as macrolide antibiotics, may pose a risk to human health by selecting for antibiotic resistance genes that could be transferred to plant-based food destined for human consumption. The consequences of long-term, repeated macrolide antibiotic exposure on the diversity of the soil bacterial community, resistome, and mobilome were evaluated. At unrealistically high concentrations, macrolide antibiotics alter the overall diversity of the resistome and mobilome, enriching for antibiotic resistance genes and mobile genetic elements of concern to human health. However, at realistic antibiotic concentrations, no effect on these endpoints was observed, suggesting that current biosolids land management practices are unlikely to pose a risk to human health due to macrolide antibiotic contamination alone.

New perspectives on an old grouping: The genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

Oxalobacter formigenes is a unique bacterium with the ability to metabolize oxalate as a primary carbon source. Most kidney stones in humans are composed of calcium and oxalate. Therefore, supplementation with an oxalate-degrading bacterium may reduce stone burden in patients suffering from recurrent calcium oxalate-based urolithiasis. Strains of O. formigenes are divided into two groups: group I and group II. However, the differences between strains from each group remain unclear and elucidating these distinctions will provide a better understanding of their physiology and potential clinical applications. Here, genomes from multiple O. formigenes strains underwent whole genome sequencing followed by phylogenetic and functional analyses. Genetic differences suggest that the O. formigenes taxon should be divided into an additional three species: Oxalobacter aliiformigenes sp. nov, Oxalobacter paeniformigenes sp. nov, and Oxalobacter paraformigenes sp. nov. Despite the similarities in the oxalyl-CoA gene (oxc), which is essential for oxalate degradation, these strains have multiple unique genetic features that may be potential exploited for clinical use. Further investigation into the growth of these strains in a simulated fecal environment revealed that O. aliiformigenes strains are capable of thriving within the human gut microbiota. O. aliiformigenes may be a better therapeutic candidate than current group I strains (retaining the name O. formigenes), which have been previously tested and shown to be ineffective as an oral supplement to mitigate stone disease. By performing genomic analyses and identifying these novel characteristics, Oxalobacter strains better suited to mitigation of calcium oxalate-based urolithiasis may be identified in the future.

EukRef-excavates: seven curated SSU ribosomal RNA gene databases.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

A Leak in the Academic Pipeline: Identity and Health Among Postdoctoral Women.

Several challenges (e.g., sexism, parental leave, the glass ceiling, etc.) disproportionately affect women in academia (and beyond), and thus perpetuate the leaky pipeline metaphor for women who opt-out of an academic career. Although this pattern can be seen at all levels of the academic hierarchy, a critical time for women facing such challenges is during the postdoctoral stage, when personal life transitions and professional ambitions collide. Using a social identity approach, we explore factors affecting the mental health of postdoctoral women, including identity development (e.g., as a mother, a scientist) and lack of control (uncertainty about one's future personal and professional prospects), which likely contribute to the leak from academia. In this mixed-method research, Study 1 comprised interviews with postdoctoral women in North America (n = 13) and Europe (n = 8) across a range disciplines (e.g., psychology, physics, political science). Common themes included the negative impact of career uncertainty, gender-based challenges (especially sexism and maternity leave), and work-life balance on mental and physical health. However, interviewees also described attempts to overcome gender inequality and institutional barriers by drawing on support networks. Study 2 comprised an online survey of postdoctoral women (N = 146) from a range of countries and academic disciplines to assess the relationships between social identification (e.g., disciplinary, gender, social group), perceived control (i.e., over work and life), and mental health (i.e., depression, anxiety, stress, and life satisfaction). Postdoctoral women showed mild levels of stress and depression, and were only slightly satisfied with life. They also showed only moderate levels of perceived control over one's life and work. However, hierarchical regression analyses revealed that strongly identifying with one's discipline was most consistently positively associated with both perceived control and mental health. Collectively, these findings implicate the postdoctoral stage as being stressful and tenuous for women regardless of academic background or nationality. They also highlight the importance of disciplinary identity as a potentially protective factor for mental health that, in turn, may diminish the rate at which postdoctoral women leak from the academic pipeline.

A systematic, deep sequencing-based methodology for identification of mixed-genotype hepatitis C virus infections.

Hepatitis C virus (HCV) mixed genotype infections can affect treatment outcomes and may have implications for vaccine design and disease progression. Previous studies demonstrate 0-39% of high-risk, HCV-infected individuals harbor mixed genotypes however standardized, sensitive methods of detection are lacking. This study compared PCR amplicon, random primer (RP), and probe enrichment (PE)-based deep sequencing methods coupled with a custom sequence analysis pipeline to detect multiple HCV genotypes. Mixed infection cutoff values, based on HCV read depth and coverage, were identified using receiver operating characteristic curve analysis. The methodology was validated using artificially mixed genotype samples and then applied to two clinical trials of HCV treatment in high-risk individuals (ACTIVATE, 114 samples from 90 individuals; DARE-C II, 26 samples from 18 individuals) and a cohort of HIV/HCV co-infected individuals (Canadian Coinfection Cohort (CCC), 3 samples from 2 individuals with suspected mixed genotype infections). Amplification bias of genotype (G)1b, G2, G3 and G5 was observed in artificially mixed samples using the PCR method while no genotype bias was observed using RP and PE. RP and PE sequencing of 140 ACTIVATE and DARE-C II samples identified the following primary genotypes: 15% (n = 21) G1a, 76% (n = 106) G3, and 9% (n = 13) G2. Sequencing of ACTIVATE and DARE-C II demonstrated, on average, 2% and 1% of HCV reads mapping to a second genotype using RP and PE, respectively, however none passed the mixed infection cutoff criteria and phylogenetics confirmed no mixed infections. From CCC, one mixed infection was confirmed while the other was determined to be a recombinant genotype. This study underlines the risk for false identification of mixed HCV infections and stresses the need for standardized methods to improve prevalence estimates and to understand the impact of mixed infections for management and elimination of HCV.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches.

UNLABELLED: By combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution of Bacteroidales ectosymbionts of the protist Barbulanympha from the hindguts of the wood-eating cockroach Cryptocercus punctulatus In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of the Barbulanympha ectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis of C. punctulatus hindgut symbionts exposed in vivo to (15)N2 supports the hypothesis that Barbulanympha ectosymbionts are capable of nitrogen fixation. This genomic and in vivo functional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions. IMPORTANCE: The ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protist Barbulanympha from the hindgut of the wood-eating cockroach Cryptocercus punctulatus In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

Moramonas marocensis gen. nov., sp. nov.: a jakobid flagellate isolated from desert soil with a bacteria-like, but bloated mitochondrial genome.

A new jakobid genus has been isolated from Moroccan desert soil. The cyst-forming protist Moramonas marocensis gen. nov., sp. nov. has two anteriorly inserted flagella of which one points to the posterior cell pole accompanying the ventral feeding groove and is equipped with a dorsal vane-a feature typical for the Jakobida. It further shows a flagellar root system consisting of singlet microtubular root, left root (R1), right root (R2) and typical fibres associated with R1 and R2. The affiliation of M. marocensis to the Jakobida was confirmed by molecular phylogenetic analyses of the SSU rRNA gene, five nuclear genes and 66 mitochondrial protein-coding genes. The mitochondrial genome has the high number of genes typical for jakobids, and bacterial features, such as the four-subunit RNA polymerase and Shine-Dalgarno sequences upstream of the coding regions of several genes. The M. marocensis mitochondrial genome encodes a similar number of genes as other jakobids, but is unique in its very large genome size (greater than 264 kbp), which is three to four times higher than that of any other jakobid species investigated yet. This increase seems to be due to a massive expansion in non-coding DNA, creating a bloated genome like those of plant mitochondria.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

The role of host phylogeny varies in shaping microbial diversity in the hindguts of lower termites.

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

Trichonympha burlesquei n. sp. from Reticulitermes virginicus and evidence against a cosmopolitan distribution of Trichonympha agilis in many termite hosts.

Historically, symbiotic protists in termite hindguts have been considered to be the same species if they are morphologically similar, even if they are found in different host species. For example, the first-described hindgut and hypermastigote parabasalian, Trichonympha agilis (Leidy, 1877) has since been documented in six species of Reticulitermes, in addition to the original discovery in Reticulitermes flavipes. Here we revisit one of these, Reticulitermes virginicus, using molecular phylogenetic analysis from single-cell isolates and show that the Trichonympha in R. virginicus is distinct from isolates in the type host and describe this novel species as Trichonympha burlesquei n. sp. We also show the molecular diversity of Trichonympha from the type host R. flavipes is greater than supposed, itself probably representing more than one species. All of this is consistent with recent data suggesting a major underestimate of termite symbiont diversity.

Termite hindguts and the ecology of microbial communities in the sequencing age.

Advances in high-throughput nucleic acid sequencing have improved our understanding of microbial communities in a number of ways. Deeper sequence coverage provides the means to assess diversity at the resolution necessary to recover ecological and biogeographic patterns, and at the same time single-cell genomics provides detailed information about the interactions between members of a microbial community. Given the vastness and complexity of microbial ecosystems, such analyses remain challenging for most environments, so greater insight can also be drawn from analysing less dynamic ecosystems. Here, we outline the advantages of one such environment, the wood-digesting hindgut communities of termites and cockroaches, and how it is a model to examine and compare both protist and bacterial communities. Beyond the analysis of diversity, our understanding of protist community ecology will depend on using statistically sound sampling regimes at biologically relevant scales, transitioning from discovery-based to experimental ecology, incorporating single-cell microbiology and other data sources, and continued development of analytical tools.

Single-Cell DNA barcoding using sequences from the small subunit rRNA and internal transcribed spacer region identifies new species of Trichonympha and Trichomitopsis from the hindgut of the termite Zootermopsis angusticollis.

To aid in their digestion of wood, lower termites are known to harbour a diverse community of prokaryotes as well as parabasalid and oxymonad protist symbionts. One of the best-studied lower termite gut communities is that of Zootermopsis angusticollis which has been known for almost 100 years to possess 3 species of Trichonympha (T. campanula, T. collaris, and T. sphaerica), 1 species of Trichomitopsis (T. termopsidis), as well as smaller flagellates. We have re-assessed this community by sequencing the small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) region from a large number of single Trichonympha and Trichomitopsis cells for which morphology was also documented. Based on phylogenetic clustering and sequence divergence, we identify 3 new species: Trichonympha postcylindrica, Trichomitopsis minor, and Trichomitopsis parvus spp. nov. Once identified by sequencing, the morphology of the isolated cells for all 3 new species was re-examined and found to be distinct from the previously described species: Trichonympha postcylindrica can be morphologically distinguished from the other Trichonympha species by an extension on its posterior end, whereas Trichomitopsis minor and T. parvus are smaller than T. termopsidis but similar in size to each other and cannot be distinguished based on morphology using light microscopy. Given that Z. angusticollis has one of the best characterized hindgut communities, the near doubling of the number of the largest and most easily identifiable symbiont species suggests that the diversity of hindgut symbionts is substantially underestimated in other termites as well. Accurate descriptions of the diversity of these microbial communities are essential for understanding hindgut ecology and disentangling the interactions among the symbionts, and molecular barcoding should be a priority for these systems.

Selection in coastal Synechococcus (cyanobacteria) populations evaluated from environmental metagenomes.

Environmental metagenomics provides snippets of genomic sequences from all organisms in an environmental sample and are an unprecedented resource of information for investigating microbial population genetics. Current analytical methods, however, are poorly equipped to handle metagenomic data, particularly of short, unlinked sequences. A custom analytical pipeline was developed to calculate dN/dS ratios, a common metric to evaluate the role of selection in the evolution of a gene, from environmental metagenomes sequenced using 454 technology of flow-sorted populations of marine Synechococcus, the dominant cyanobacteria in coastal environments. The large majority of genes (98%) have evolved under purifying selection (dN/dS<1). The metagenome sequence coverage of the reference genomes was not uniform and genes that were highly represented in the environment (i.e. high read coverage) tended to be more evolutionarily conserved. Of the genes that may have evolved under positive selection (dN/dS>1), 77 out of 83 (93%) were hypothetical. Notable among annotated genes, ribosomal protein L35 appears to be under positive selection in one Synechococcus population. Other annotated genes, in particular a possible porin, a large-conductance mechanosensitive channel, an ATP binding component of an ABC transporter, and a homologue of a pilus retraction protein had regions of the gene with elevated dN/dS. With the increasing use of next-generation sequencing in metagenomic investigations of microbial diversity and ecology, analytical methods need to accommodate the peculiarities of these data streams. By developing a means to analyze population diversity data from these environmental metagenomes, we have provided the first insight into the role of selection in the evolution of Synechococcus, a globally significant primary producer.

Whole-genome microarray analyses of Synechococcus-Vibrio interactions.

Microbes live in diverse communities yet their physiologies are typically studied in axenic culture. To begin to address this dichotomy, whole-genome microarray analyses were used and revealed that several major metabolic pathways were affected in Synechococcus sp. WH8102, a model phototroph, when grown with Vibrio parahaemolyticus, a model heterotroph. In co-cultures with V. parahaemolyticus, although phosphate was not depleted, Synechococcus sp. WH8102 may have experienced phosphate stress since the expression of phosphate acquisition genes increased and alkaline phosphatase activity was higher than in monocultures. Expression of cell wall synthesis genes and the components of a zinc transporter were also upregulated. In contrast, a ferric uptake regulation (Fur) family gene was downregulated as were genes that encode proteins rich in iron or involved in detoxifying oxygen radicals. Nitrogen use may also have been affected in co-cultures as the gene expression changes share similarities with ammonia-grown Synechococcus. This study demonstrates the multiple impacts that interspecific microbial interactions can have on the physiology of a major primary producer and the importance of investigating microbial physiology from a community perspective.

Temporal variation of Synechococcus clades at a coastal Pacific Ocean monitoring site.

Marine cyanobacteria from the genus Synechococcus are found throughout the world's oceans and are important contributors to global primary productivity and carbon cycling. Cultured isolates and environmental DNA clone libraries of Synechococcus have demonstrated the diversity of these microbes. However, the natural distribution of this diversity through space and time and the ecological significance of their distribution are still poorly understood. To understand the seasonal dynamics of Synechococcus diversity, we have developed a quantitative PCR strategy using the gene encoding as a subunit of DNA-dependent RNA polymerase (rpoC1) and applied it to a 3-year time series of surface samples from the Scripps Institution of Oceanography pier (La Jolla, CA, USA), a coastal site in the northeastern Pacific Ocean. Synechococcus from clades I and IV were dominant throughout the time series and correlated with total Synechococcus abundance. The relative abundance of these two dominant clades showed evidence of a seasonal cycle. Synechococcus from clade IV were typically more abundant, but those from clade I dominated during periods just before the annual spring bloom of Synechococcus. Synechococcus from clades II and III were absent during spring and early summer, but appeared at low abundances in late summer and winter possibly due to changes in circulation in the Southern California Bight. As the first long-term time series describing Synechococcus population diversity, these temporal dynamics were used to interpret the genetic/genomic diversity observed in the environment and the potential factors regulating their distribution.