Biology subjects

Du, S.

Publications and source records attributed to Du, S..

3 recordsLinked to original sources

bioRxiv2018

Pelagiphages in the Podoviridiae family integrate into host genomes

The Pelagibacterales order (SAR11) in Alphaproteobacteria dominates marine surface bacterioplankton communities, where it plays a key role in carbon and nutrient cycling. SAR11 phages, known as pelagiphages, are among the most abundant phages in the ocean. Four pelagiphages that infect Pelagibacter HTCC1062 have been reported. Here we report 11 new pelagiphages in the Podoviridae family. Comparative genomic analysis revealed that they are all closely related to previously reported pelagiphages HTVC011P and HTVC019P, in the HTVC019Pvirus genus. HTVC019Pvirus pelagiphages share a core genome of 15 genes, with a pan-genome of 234 genes. Phylogenomic analysis clustered these pelagiphages into three subgroups. Integrases were identified in all but one pelagiphage genomes. Evidence of site-specific integration was obtained by high-throughput sequencing and sequencing PCR amplicons containing predicted integration sites, demonstrating the capacity of these pelagiphages to propagate by both lytic and lysogenic infection. HTVC019P, HTVC021P, HTVC022P, HTVC201P and HTVC121P integrate into tRNA-Cys genes. HTVC011P, HTVC025P, HTVC105P, HTVC109P, HTVC119P and HTVC200P target tRNA-Leu genes, while HTVC120P integrates into the tRNA-Arg. Evidence of pelagiphage integration was also retrieved from Global Ocean Survey (GOS) database, suggesting the occurrence of pelagiphage integration in situ. The capacity of HTVC019Pvirus pelagiphages to integrate into host genomes suggests they could impact SAR11 populations by a variety of mechanisms, including mortality, genetic transduction, and prophage-induced viral immunity. HTVC019Pvirus pelagiphages are a rare example of a lysogenic phage that can be implicated in ecological processes on broad scales, and thus have potential to become a useful model for investigating strategies of host infection and phage-dependent horizontal gene transfer.\n\nIMPORTANCEPelagiphages are ecologically important because of their extraordinarily high census numbers, which makes them potentially significant agents in the viral shunt, a concept that links viral predation to the recycling of dissolved organic matter released from lysing plankton cells. Lysogenic Pelagiphages, such as the HTVC019Pvirus pelagiphages we investigate here, are also important because of their potential to contribute to the hypothesized processes such as the \"Piggy-Back-the-Winner\" and \"King-of-the-Mountain\". The former explains nonlinearities in virus to host ratios by postulating increased lysogenization of successful host cells, while the latter postulates host-density dependent propagation of defensive alleles. Here we report multiple Pelagiphage isolates, and provided detailed evidence of their integration into SAR11 genomes. The development of this ecologically significant experimental system for studying phage-dependent processes is progress towards the validation of broad hypotheses about phage ecology with specific examples based on knowledge of mechanisms.

microbiology↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗

bioRxiv2018

4-hydroxyphenylpyruvate dioxygenase thermolability is responsible for temperature-dependent melanogenesis in Aeromonas salmonicida subsP salmonicida

Aeromonas salmonicida subsp. salmonicida (A.s.s) is a major pathogen affecting fisheries worldwide. It is a well-known member of the pigmented Aeromonas species, which produces melanin at [≤] 22 {degrees}C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30-35 {degrees}C while bacterial growth is not affected. The mechanism and biological significance of this temperature-dependent melanogenesis are not clear. Heterologous expression of an A.s.s. 4-hydroxyphenylpyruvate dioxygenase (HppD), the most crucial enzyme in the HGA-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In the current study, we demonstrated that the extreme thermolability of HppD is responsible for the temperature-dependent melanization of A.s.s. Substitutions in three residues, Ser18, Pro103, or Leu119 of HppD from A.s.s increases the thermolability of this enzyme and results in temperature-independent melanogenesis. Moreover, replacing the corresponding residues of HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A.s.s HppD leads to thermosensitive melanogenesis. Structural analysis suggested that mutations at these sites, especially at position P103, can strengthen the secondary structure of HppD and greatly improve its thermal stability. In addition, we found that HppD sequences of all A.s.s isolates are identical and that two of the three residues are completely conserved within A.s.s isolates, which clearly distinguishes these from other Aeromonas strains. We suggest that this property represents an adaptive strategy to the psychrophilic lifestyle of A.s.s.\n\nImportanceAeromonas salmonicida subsp. salmonicida (A.s.s) is the causative agent of furunculosis, a bacterial septicemia of cold water fish of the Salmonidae family. As it has a well-defined host range, A.s.s has become an ideal model to investigate the co-evolution of host and pathogen. For many pathogens, melanin production is associated with virulence. Although other species of Aeromonas can produce melanin, A.s.s is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here we demonstrate that thermosensitive melanogenesis in A.s.s strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). The strictly conserved hppD sequences among A.s.s and the exclusive thermosensitive pigmentation of these strains might provide insight into the role of melanin in the adaptation to a particular host, and offer a novel molecular marker to readily differentiate A.s.s strains from other A. salmonicida subspecies and Aeromonas species.

molecular biology↗