Sébastien Rodrigue

Patterns and Implications of Gene Gain and Loss in the Evolution of Prochlorococcus

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the worlds oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.

Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus.

Cyanobacterial Diversity What does it mean to be a global species? The marine cyanobacterium Prochlorococcus is ubiquitous and, arguably, the most abundant and productive of all living organisms. Although to our eyes the seas look uniform, to a bacterium the oceans bulk is a plethora of microhabitats, and by large-scale single-cell genomic analysis of uncultured cells, Kashtan et al. (p. 416; see the Perspective by Bowler and Scanlan) reveal that Prochlorococcus has diversified to match. This “species” constitutes a mass of subpopulations—each with million-year ancestry—that vary seasonally in abundance. The subpopulations in turn have clades nested within that show covariation between sets of core alleles and variable gene content, indicating flexibility of responses to rapid environmental changes. Large sets of coexisting populations could be a general feature of other free-living bacterial species living in highly mixed habitats. Covariation between the core alleles and flexible gene content of a marine cyanobacterium underpins vast diversity. [Also see Perspective by Bowler and Scanlan] Extensive genomic diversity within coexisting members of a microbial species has been revealed through selected cultured isolates and metagenomic assemblies. Yet, the cell-by-cell genomic composition of wild uncultured populations of co-occurring cells is largely unknown. In this work, we applied large-scale single-cell genomics to study populations of the globally abundant marine cyanobacterium Prochlorococcus. We show that they are composed of hundreds of subpopulations with distinct “genomic backbones,” each backbone consisting of a different set of core gene alleles linked to a small distinctive set of flexible genes. These subpopulations are estimated to have diverged at least a few million years ago, suggesting ancient, stable niche partitioning. Such a large set of coexisting subpopulations may be a general feature of free-living bacterial species with huge populations in highly mixed habitats.

Whole Genome Amplification and De novo Assembly of Single Bacterial Cells

Background Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells. Methodology/Principal Findings We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. Conclusions/Significance The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

Unlocking Short Read Sequencing for Metagenomics

Background Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved. Methodology/Principal Findings We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read. Conclusions/Significance This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.

Polyphosphate kinase is involved in stress-induced mprAB-sigE-rel signalling in mycobacteria.

Polyphosphate kinase 1 (PPK1) helps bacteria to survive under stress. The ppk1 gene of Mycobacterium tuberculosis was overexpressed in Escherichia coli and characterized. Residues R230 and F176, predicted to be present in the head domain of PPK1, were identified as residues critical for polyphosphate (polyP)‐synthesizing ability and dimerization of PPK1. A ppk1 knockout mutant of Mycobacterium smegmatis was compromised in its ability to survive under long‐term hypoxia. The transcription of the rel gene and the synthesis of the stringent response regulator ppGpp were impaired in the mutant and restored after complementation with ppk1 of M.u2003tuberculosis, providing evidence that PPK1 is required for the stringent response. We present evidence that PPK1 is likely required for mprAB‐sigE‐rel signalling. σE regulates the transcription of rel, and we hypothesize that under conditions of stress polyP acts as a preferred donor for MprB‐mediated phosphorylation of MprA facilitating transcription of the sigE gene thereby leading finally to the enhancement of the transcription of rel in M.u2003smegmatis and M.u2003tuberculosis. Downregulation of ppk1 led to impaired survival of M.u2003tuberculosis in macrophages. PolyP plays a central role in the stress response of mycobacteria.

Transcriptome and Proteome Dynamics of a Light-Dark Synchronized Bacterial Cell Cycle

Background Growth of the oceans most abundant primary producer, the cyanobacterium Prochlorococcus, is tightly synchronized to the natural 24-hour light-dark cycle. We sought to quantify the relationship between transcriptome and proteome dynamics that underlie this obligate photoautotrophs highly choreographed response to the daily oscillation in energy supply. Methodology/Principal Findings Using RNA-sequencing transcriptomics and mass spectrometry-based quantitative proteomics, we measured timecourses of paired mRNA-protein abundances for 312 genes every 2 hours over a light-dark cycle. These temporal expression patterns reveal strong oscillations in transcript abundance that are broadly damped at the protein level, with mRNA levels varying on average 2.3 times more than the corresponding protein. The single strongest observed protein-level oscillation is in a ribonucleotide reductase, which may reflect a defense strategy against phage infection. The peak in abundance of most proteins also lags that of their transcript by 2–8 hours, and the two are completely antiphase for some genes. While abundant antisense RNA was detected, it apparently does not account for the observed divergences between expression levels. The redirection of flux through central carbon metabolism from daytime carbon fixation to nighttime respiration is associated with quite small changes in relative enzyme abundances. Conclusions/Significance Our results indicate that expression responses to periodic stimuli that are common in natural ecosystems (such as the diel cycle) can diverge significantly between the mRNA and protein levels. Protein expression patterns that are distinct from those of cognate mRNA have implications for the interpretation of transcriptome and metatranscriptome data in terms of cellular metabolism and its biogeochemical impact.

Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial σ factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for σF, σC, and σK, and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of σ factor genes. Our results revealed that many σ factors are expressed from autoregulated promoters.

Novel Mycobacterium tuberculosis anti-σ factor antagonists control σf activity by distinct mechanisms

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 σ factors, as well as several putative anti‐, and anti‐anti‐ σ factors. Here we show that a σ factor that has been previously shown to be involved in virulence and persistence processes, σF, can be specifically inhibited by the anti‐σ factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti‐anti‐σ factors. The first anti‐anti‐σ factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non‐functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti‐anti‐σ factor. These results suggest that σF activity might be post‐translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacterias ability to adapt to diverse host environments during disease progression, latency and reactivation.

Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth.

There is growing evidence that strains of Mycobacterium tuberculosis differ in pathogenicity and transmissibility, but little is understood about the contributory factors. We have previously shown that increased expression of the principal sigma factor, SigA, mediates the capacity of M. tuberculosis strain 210 to grow more rapidly in human monocytes, compared with other strains. Strain 210 is part of the widespread W-Beijing family of M. tuberculosis strains and includes clinical isolate TB294. To identify genes that respond to changes in SigA levels and that might enhance intracellular growth, we examined RNA and protein expression patterns in TB294-pSigA, a recombinant strain of TB294 that overexpresses sigA from a multicopy plasmid. Lysates from broth-grown cultures of TB294-pSigA contained high levels of Eis, a protein known to modulate host-pathogen interactions. DNA microarray analysis indicated that the eis gene, Rv2416c, was expressed at levels in TB294-pSigA 40-fold higher than in the vector control strain TB294-pCV, during growth in the human monocyte cell line MonoMac6. Other genes with elevated expression in TB294-pSigA showed much smaller changes from TB294-pCV, and the majority of genes with expression differences between the two strains had reduced expression in TB294-pSigA, including an unexpected number of genes associated with the DNA-damage response. Real-time PCR analyses confirmed that eis was expressed at very high levels in TB294-pSigA in monocytes as well as in broth culture, and further revealed that, like sigA, eis was also more highly expressed in wild-type TB294 than in the laboratory strain H37Rv, during growth in monocytes. These findings suggested an association between increased SigA levels and eis activation, and results of chromatin immunoprecipitation confirmed that SigA binds the eis promoter in live TB294 cells. Deletion of eis reduced growth of TB294 in monocytes, and complementation of eis reversed this effect. We conclude that SigA regulates eis, that there is a direct correlation between upregulation of SigA and high expression levels of eis, and that eis contributes to the enhanced capacity of a clinical isolate of M. tuberculosis strain 210 to grow in monocytes.

Detection of prokaryotic promoters from the genomic distribution of hexanucleotide pairs

BackgroundIn bacteria, sigma factors and other transcriptional regulatory proteins recognize DNA patterns upstream of their target genes and interact with RNA polymerase to control transcription. As a consequence of evolution, DNA sequences recognized by transcription factors are thought to be enriched in intergenic regions (IRs) and depleted from coding regions of prokaryotic genomes.ResultsIn this work, we report that genomic distribution of transcription factors binding sites is biased towards IRs, and that this bias is conserved amongst bacterial species. We further take advantage of this observation to develop an algorithm that can efficiently identify promoter boxes by a distribution-dependent approach rather than a direct sequence comparison approach. This strategy, which can easily be combined with other methodologies, allowed the identification of promoter sequences in ten species and can be used with any annotated bacterial genome, with results that rival with current methodologies. Experimental validations of predicted promoters also support our approach.ConclusionConsidering that complete genomic sequences of over 1000 bacteria will soon be available and that little transcriptional information is available for most of them, our algorithm constitutes a promising tool for the prediction of promoter sequences. Importantly, our methodology could also be adapted to identify DNA sequences recognized by other regulatory proteins.