Prophage enhances the ability of deep-sea bacterium Shewanella psychrophila WP2 to utilize D-amino acid.

Prophages are prevalent in the marine bacterial genomes and reshape the physiology and metabolism of their hosts. However, whether and how prophages influence the microbial degradation of D-amino acids (D-AAs), which is one of the widely distributed recalcitrant dissolved organic matters (RDOMs) in the ocean, remain to be explored. In this study, we addressed this issue in a representative marine bacterium, Shewanella psychrophila WP2 (WP2), and its integrated prophage SP1. Notably, compared to the WP2 wild-type strain, the SP1 deletion mutant of WP2 (WP2ΔSP1) exhibited a significantly lower D-glutamate (D-Glu) consumption rate and longer lag phase when D-Glu was used as the sole nitrogen source. The subsequent transcriptome analysis identified 1,523 differentially expressed genes involved in diverse cellular processes, especially that multiple genes related to inorganic nitrogen metabolism were highly upregulated. In addition, the dynamic profiles of ammonium, nitrate, and nitrite were distinct between the culture media of WP2 and WP2ΔSP1. Finally, we provide evidence that SP1 conferred a competitive advantage to WP2 when D-Glu was used as the sole nitrogen source and SP1-like phages may be widely distributed in the global ocean. Taken together, these findings offer novel insight into the influences of prophages on host metabolism and RDOM cycling in marine environments.IMPORTANCEThis work represents the first exploration of the impact of prophages on the D-amino acid (D-AA) metabolism of deep-sea bacteria. By using S. psychrophila WP2 and its integrated prophage SP1 as a representative system, we found that SP1 can significantly increase the catabolism rate of WP2 to D-glutamate and produce higher concentrations of ammonium, resulting in faster growth and competitive advantages. Our findings not only deepen our understanding of the interaction between deep-sea prophages and hosts but also provide new insights into the ecological role of prophages in refractory dissolved organic matter and the nitrogen cycle in deep oceans.

A systematic analysis of marine lysogens and proviruses.

Viruses are ubiquitous in the oceans, exhibiting high abundance and diversity. Here, we systematically analyze existing genomic sequences of marine prokaryotes to compile a Marine Prokaryotic Genome Dataset (MPGD, consisting of over 12,000 bacterial and archaeal genomes) and a Marine Temperate Viral Genome Dataset (MTVGD). At least 40% of the MPGD genomes contain one or more proviral sequences, indicating that they are lysogens. The MTVGD includes over 12,900 viral contigs or putative proviruses, clustered into 10,897 viral genera. We show that lysogens and proviruses are abundant in marine ecosystems, particularly in the deep sea, and marine lysogens differ from non-lysogens in multiple genomic features and growth properties. We reveal several virus-host interaction networks of potential ecological relevance, and identify proviruses that appear to be able to infect (or to be transferred between) different bacterial classes and phyla. Auxiliary metabolic genes in the MTVGD are enriched in functions related to carbohydrate metabolism. Finally, we experimentally demonstrate the impact of a prophage on the transcriptome of a representative marine Shewanella bacterium. Our work contributes to a better understanding of the ecology of marine prokaryotes and their viruses.

Unexplored diversity and ecological functions of transposable phages.

Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable phages (TBPs) are exceptional in terms of their unique lifestyle, especially their replicative transposition. Although several TBPs have been isolated and the life cycle of the representative phage Mu has been extensively studied, the diversity distribution and ecological functions of TBPs on the global scale remain unknown. Here, by mining TBPs from enormous microbial genomes and viromes, we established a TBP genome dataset (TBPGD), that expands the number of accessible TBP genomes 384-fold. TBPs are prevalent in diverse biomes and show great genetic diversity. Based on taxonomic evaluations, we propose the categorization of TBPs into four viral groups, including 11 candidate subfamilies. TBPs infect multiple bacterial phyla, and seem to infect a wider range of hosts than non-TBPs. Diverse auxiliary metabolic genes (AMGs) are identified in the TBP genomes, and genes related to glycoside hydrolases and pyrimidine deoxyribonucleotide biosynthesis are highly enriched. Finally, the influences of TBPs on their hosts are experimentally examined by using the marine bacterium Shewanella psychrophila WP2 and its infecting transposable phage SP2. Collectively, our findings greatly expand the genetic diversity of TBPs, and comprehensively reveal their potential influences in various ecosystems.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes.

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Phylogenomic analysis reveals a two-stage process of the evolutionary transition of Shewanella from the upper ocean to the hadal zone.

Shewanella strains are characterized by versatile metabolic capabilities, resulting in their wide distribution in the ocean at different depths. Considering that particle sedimentation is an important dynamic process in the ocean, we hypothesized that hadal Shewanella species evolved from the upper ocean. In this study, we isolated three novel Shewanella strains from deep-sea sediments in the Southwest Indian Ocean. Genome sequencing indicated that strains YLB-06 and YLB-08 represent two novel species in the genus Shewanella. Through phylogenomic analysis, we showed that speciation and genomic changes in marine Shewanella strains are related to water depth. We further confirmed the aforementioned hypothesis and revealed a two-stage process of the evolutionary transition of Shewanella from the upper ocean to the hadal zone by comparative genomics and gene gain/loss analysis. Finally, the transcriptomic analysis demonstrated that recently obtained genes are strictly repressed and may thus play a minor role in the response to environmental changes.

Multiple Mechanisms Are Involved in Repression of Filamentous Phage SW1 Transcription by the DNA-Binding Protein FpsR.

SW1 is the first filamentous phage isolated from a deep-sea environment. Nevertheless, the mechanism by which the SW1 genetic switch is controlled is largely unknown. In this study, the function of the phage-encoded FpsR protein was characterized by molecular biological and biochemical analyses. The deletion of fpsR increased the copy number of SW1 ssDNA and mRNA, indicating that FpsR functions as a repressor. In addition, transcription from the fpsR promoter was shown to be increased in an fpsR deletion mutant, suggesting self-repression by FpsR. Purified FpsR bound to four adjacent operator sites (O1-O4) embedded within the fpsA promoter and the fpsA-fpsR intergenic region. A surface plasmon resonance experiment showed that FpsR can bind to the O1-O4 operators separately and with different binding affinity, and the dissociation constants of FpsR with O2 and O3 were found to be lower at 4 °C than at 20 °C. A gel permeation chromatography assay revealed that FpsR oligomerized to form tetramers. Point mutation analysis indicated that the C-terminal domain influenced the binding affinity and regulatory function of FpsR. Collectively, these data support a model in which FpsR actively regulates phage production by interacting with the corresponding operators, thus playing a crucial role in the SW1 genetic switch.

Validation of reference genes for reverse transcription real-time quantitative PCR analysis in the deep-sea bacterium Shewanella psychrophila WP2.

Reference genes are critical to obtain reliable results of reverse transcription real-time quantitative PCR (RT-qPCR), which is widely used for relative quantification of gene expression. In this study, we evaluated the validity of seven candidate reference genes for normalization in RT-qPCR analysis in the deep-sea bacterium Shewanella psychrophila WP2 under different environmental conditions. Among the set of genes investigated, gyrA, 16S rRNA and rho were identified as the most suitable reference genes for WP2 at different temperatures, hydrostatic pressures and salinities, respectively. Notably, the rho gene is conserved in Shewanella genus and other deep-sea bacteria, thus, could be used as a versatile reference gene for RT-qPCR analysis of these microorganisms under extreme environmental conditions.