Debbie Lindell

Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation

The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph. It numerically dominates the phytoplankton in the tropical and subtropical oceans, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage and that have different minimum, maximum and optimal light intensities for growth. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in the oceans.

Photosynthesis genes in marine viruses yield proteins during host infection

Cyanobacteria, and the viruses (phages) that infect them, are significant contributors to the oceanic ‘gene pool’. This pool is dynamic, and the transfer of genetic material between hosts and their phages probably influences the genetic and functional diversity of both. For example, photosynthesis genes of cyanobacterial origin have been found in phages that infect Prochlorococcus and Synechococcus, the numerically dominant phototrophs in ocean ecosystems. These genes include psbA, which encodes the photosystem II core reaction centre protein D1, and high-light-inducible (hli) genes. Here we show that phage psbA and hli genes are expressed during infection of Prochlorococcus and are co-transcribed with essential phage capsid genes, and that the amount of phage D1 protein increases steadily over the infective period. We also show that the expression of host photosynthesis genes declines over the course of infection and that replication of the phage genome is a function of photosynthesis. We thus propose that the phage genes are functional in photosynthesis and that they may be increasing phage fitness by supplementing the host production of these proteins.

Genomic island variability facilitates Prochlorococcus-virus coexistence

Prochlorococcus cyanobacteria are extremely abundant in the oceans, as are the viruses that infect them. How hosts and viruses coexist in nature remains unclear, although the presence of both susceptible and resistant cells may allow this coexistence. Combined whole-genome sequencing and PCR screening technology now enables us to investigate the effect of resistance on genome evolution and the genomic mechanisms behind the long-term coexistence of Prochlorococcus and their viruses. Here we present a genome analysis of 77 substrains selected for resistance to ten viruses, revealing mutations primarily in non-conserved, horizontally transferred genes that localize to a single hypervariable genomic island. Mutations affected viral attachment to the cell surface and imposed a fitness cost to the host, manifested by significantly lower growth rates or a previously unknown mechanism of more rapid infection by other viruses. The mutant genes are generally uncommon in nature yet some carry polymorphisms matching those found experimentally. These data are empirical evidence indicating that viral-attachment genes are preferentially located in genomic islands and that viruses are a selective pressure enhancing the diversity of both island genes and island gene content. This diversity emerges as a genomic mechanism that reduces the effective host population size for infection by a given virus, thus facilitating long-term coexistence between viruses and their hosts in nature.

Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, prochlorococcus

The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night— metabolic processes that share some of the same enzymes — appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a ‘deficient’ circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.

Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability

Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium‐replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.

Ecological aspects of ntcA gene expression and its use as an indicator of the nitrogen status of marine Synechococcus spp.

ABSTRACT Nitrogen nutrition in cyanobacteria is regulated by NtcA, a transcriptional activator that is subject to negative control by ammonium. Using Synechococcus sp. strain WH7803 as a model organism, we show that ntcAexpression was induced when cells were exposed to nitrogen stress but not when they were subjected to phosphorus or iron deprivation. Transcript levels accumulated in cells grown on a variety of inorganic and organic nitrogen sources, with the sole exception of ammonium. ntcA transcription was induced when ammonium levels dropped below 1 μM and reached maximal levels within 2 h. Furthermore, the addition of more than 1 μM ammonium led to a rapid decline in ntcA mRNA. The negative effect of ammonium was prevented by the addition ofl-methionine-d,l-sulfoximine (MSX) and azaserine, inhibitors of ammonium assimilation. Thus, basalntcA transcript levels are indicative of ammonium utilization. Conversely, the highest ntcA transcript levels were found in cells lacking a nitrogen source capable of supporting growth. Therefore, maximal ntcA expression would indicate nitrogen deprivation. This state of nitrogen deprivation was induced by a 1-h incubation with MSX. The rapid response of ntcA gene expression to the addition of ammonium and MSX was used to design a protocol for assessing relative ntcA transcript levels in field populations of cyanobacteria, from which their nitrogen status can be inferred. ntcA was basally expressed in Synechococcus at a nutrient-enriched site at the northern tip of the Gulf of Aqaba, Red Sea. Therefore, these cyanobacteria were not nitrogen stressed, and their nitrogen requirements were met by regenerated nitrogen in the form of ammonium.

Antisense RNA protects mRNA from RNase E degradation by RNA–RNA duplex formation during phage infection

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg2+, pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA–mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

Short RNA half-lives in the slow-growing marine cyanobacterium Prochlorococcus

BackgroundRNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans.ResultsUsing a combination of microarrays, quantitative RT-PCR and a new fitting method for determining RNA decay rates, we found a median half-life of 2.4 minutes and a median decay rate of 2.6 minutes for expressed genes - twofold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (approximately 18 minutes) were for genes with high transcript levels. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb.ConclusionsWe hypothesize that the fast turnover relative to the slow generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will help us interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms.

A novel lineage of myoviruses infecting cyanobacteria is widespread in the oceans

Viruses infecting bacteria (phages) are thought to greatly impact microbial population dynamics as well as the genome diversity and evolution of their hosts. Here we report on the discovery of a novel lineage of tailed dsDNA phages belonging to the family Myoviridae and describe its first representative, S-TIM5, that infects the ubiquitous marine cyanobacterium, Synechococcus. The genome of this phage encodes an entirely unique set of structural proteins not found in any currently known phage, indicating that it uses lineage-specific genes for virion morphogenesis and represents a previously unknown lineage of myoviruses. Furthermore, among its distinctive collection of replication and DNA metabolism genes, it carries a mitochondrial-like DNA polymerase gene, providing strong evidence for the bacteriophage origin of the mitochondrial DNA polymerase. S-TIM5 also encodes an array of bacterial-like metabolism genes commonly found in phages infecting cyanobacteria including photosynthesis, carbon metabolism and phosphorus acquisition genes. This suggests a common gene pool and gene swapping of cyanophage-specific genes among different phage lineages despite distinct sets of structural and replication genes. All cytosines following purine nucleotides are methylated in the S-TIM5 genome, constituting a unique methylation pattern that likely protects the genome from nuclease degradation. This phage is abundant in the Red Sea and S-TIM5 gene homologs are widespread in the oceans. This unusual phage type is thus likely to be an important player in the oceans, impacting the population dynamics and evolution of their primary producing cyanobacterial hosts.

Virus-host swinging party in the oceans: Incorporating biological complexity into paradigms of antagonistic coexistence

Bacteria and their viruses (phages) are antagonists, yet have coexisted in nature for billions of years. Models proposed to explain the paradox of antagonistic coexistence generally reach two types of solutions: Arms race-like dynamics that lead to hosts and viruses with increasing resistance and infection ranges; and population fluctuations between diverse host and viral types due to a metabolic cost of resistance. Recently, we found that populations of the marine cyanobacterium, Prochlorococcus, consist of cells with extreme hypervariability in gene sequence and gene content in a viral susceptibility region of the genome. Furthermore, we found a novel cost of resistance where resistance to one set of viruses is accompanied by changes in infection dynamics by other viruses. In this combined mini-review and commentary paper we discuss these findings in the context of existing ecological, evolutionary and genetic models of host-virus coexistence. We suggest that this coexistence is governed mainly by fluctuations between microbial subpopulations with differing viral susceptibility regions and that these fluctuations are driven by both metabolic and enhanced infection costs of resistance. Furthermore, we suggest that enhanced infection leads to passive host-switching by viruses, preventing the development of hosts with universal resistance. These findings highlight the vital importance of community complexity for host-virus coexistence.