Carini, Paul

“ Ca . Pelagibacter ubique” is a key driver of marine biogeochemistry cycles and a model for understanding how minimal genomes evolved in free-living anucleate organisms. This study explores the unusual sulfur acquisition strategy that has evolved in these cells, which lack assimilatory sulfate reduction and instead rely on reduced sulfur compounds found in oxic marine environments to meet their cellular quotas. Our findings demonstrate that the sulfur acquisition systems are constitutively expressed but the enzymatic steps leading to the essential sulfur-containing amino acid methionine are regulated by a unique array of riboswitches and genes, many of which are encoded in a rapidly evolving genome region. These findings support mounting evidence that streamlined cells have evolved regulatory mechanisms that minimize transcriptional switching and, unexpectedly, localize essential sulfur acquisition genes in a genome region normally associated with adaption to environmental variation.

ABSTRACT Nitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium “ Candidatus Pelagibacter ubique,” a cultured member of the order Pelagibacterales (SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266 Alphaproteobacteria genomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the order Pelagibacterales , including “ Ca . Pelagibacter ubique.” Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in “ Ca. Pelagibacter ubique” strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrient-deprived “ Ca. Pelagibacter ubique” cells. Overall, these data reveal a streamlined, P II -independent response to nitrogen stress in “ Ca. Pelagibacter ubique,” and likely other Pelagibacterales , and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds. IMPORTANCE Pelagibacterales are extraordinarily abundant and play a pivotal role in marine geochemical cycles, as one of the major recyclers of labile dissolved organic matter. They are also models for understanding how streamlining selection can reshape chemoheterotroph metabolism. Streamlining and its broad importance to environmental microbiology are emerging slowly from studies that reveal the complete genomes of uncultured organisms. Here, we report another remarkable example of streamlined metabolism in Pelagibacterales , this time in systems that control nitrogen assimilation. Pelagibacterales are major contributors to metatranscriptomes and metaproteomes from ocean systems, where patterns of gene expression are used to gain insight into ocean conditions and geochemical cycles. The data presented here supply background that is essential to interpreting data from field studies.

ABSTRACT SAR11 is an ancient and diverse clade of heterotrophic bacteria that are abundant throughout the world’s oceans, where they play a major role in the ocean carbon cycle. Correlations between the phylogenetic branching order and spatiotemporal patterns in cell distributions from planktonic ocean environments indicate that SAR11 has evolved into perhaps a dozen or more specialized ecotypes that span evolutionary distances equivalent to a bacterial order. We isolated and sequenced genomes from diverse SAR11 cultures that represent three major lineages and encompass the full breadth of the clade. The new data expand observations about genome evolution and gene content that previously had been restricted to the SAR11 Ia subclade, providing a much broader perspective on the clade’s origins, evolution, and ecology. We found small genomes throughout the clade and a very high proportion of core genome genes (48 to 56%), indicating that small genome size is probably an ancestral characteristic. In their level of core genome conservation, the members of SAR11 are outliers, the most conserved free-living bacteria known. Shared features of the clade include low GC content, high gene synteny, a large hypervariable region bounded by rRNA genes, and low numbers of paralogs. Variation among the genomes included genes for phosphorus metabolism, glycolysis, and C1 metabolism, suggesting that adaptive specialization in nutrient resource utilization is important to niche partitioning and ecotype divergence within the clade. These data provide support for the conclusion that streamlining selection for efficient cell replication in the planktonic habitat has occurred throughout the evolution and diversification of this clade. IMPORTANCE The SAR11 clade is the most abundant group of marine microorganisms worldwide, making them key players in the global carbon cycle. Growing knowledge about their biochemistry and metabolism is leading to a more mechanistic understanding of organic carbon oxidation and sequestration in the oceans. The discovery of small genomes in SAR11 provided crucial support for the theory that streamlining selection can drive genome reduction in low-nutrient environments. Study of isolates in culture revealed atypical organic nutrient requirements that can be attributed to genome reduction, such as conditional auxotrophy for glycine and its precursors, a requirement for reduced sulfur compounds, and evidence for widespread cycling of C1 compounds in marine environments. However, understanding the genetic variation and distribution of such pathways and characteristics like streamlining throughout the group has required the isolation and genome sequencing of diverse SAR11 representatives, an analysis of which we provide here.