New insights on Pseudoalteromonas haloplanktis TAC125 genome organization and benchmarks of genome assembly applications using next and third generation sequencing technologies

Abstract

Pseudoalteromonas haloplanktis TAC125 is among the most commonly studied bacteria adapted to cold environments. Aside from its ecological relevance, P. haloplanktis has a potential use for biotechnological applications. Due to its importance, we decided to take advantage of next generation sequencing (Illumina) and third generation sequencing (PacBio and Oxford Nanopore) technologies to resequence its genome. The availability of a reference genome, obtained using whole genome shotgun sequencing, allowed us to study and compare the results obtained by the different technologies and draw useful conclusions for future de novo genome assembly projects. We found that assembly polishing using Illumina reads is needed to achieve a consensus accuracy over 99.9% when using Oxford Nanopore sequencing, but not in PacBio sequencing. However, the dependency of consensus accuracy on coverage is lower in Oxford Nanopore than in PacBio, suggesting that a cost-effective solution might be the use of low coverage Oxford Nanopore sequencing together with Illumina reads. Despite the differences in consensus accuracy, all sequencing technologies revealed the presence of a large plasmid, pMEGA, which was undiscovered until now. Among the most interesting features of pMEGA is the presence of a putative error-prone polymerase regulated through the SOS response. Aside from the characterization of the newly discovered plasmid, we confirmed the sequence of the small plasmid pMtBL and uncovered the presence of a potential partitioning system. Crucially, this study shows that the combination of next and third generation sequencing technologies give us an unprecedented opportunity to characterize our bacterial model organisms at a very detailed level.

Complete Genome Insights into Lactococcus petauri CF11 Isolated from a Healthy Human Gut Using Second- and Third-Generation Sequencing

“…Sequencing technology can truly reflect the genetic information of genomic DNA, providing important functional predictions. The Pacific Bio Sciences (Pac Bio) sequencing platform, which is a single molecular sequencing technology (Iso-Seq), offers significant improvements over current sequencing technologies because of its high throughput nature, fast speed, and longer reads (Qi et al, 2019). However, the error rate of Pac Bio is higher than in secondgeneration sequencing (SGS) technologies, leading to a reduced accuracy of assembly (Koren et al, 2012).…”

Diauxie and co-utilization of carbon sources can coexist during bacterial growth in nutritionally complex environments

“…Their metabolism is pivotal for the maintenance and the correct balance of oceanic biogeochemical cycles as they are central to the so-called microbial loop, i.e., the trophic pathway of the marine food web responsible for the microbial assimilation of dissolved organic matter, by transforming phytoplankton-derived organic matter and fuelling the entire ocean biogeochemical nutrient cycle. In this regard, the metabolism of Pseudoalteromonas haloplanktis TAC125 (PhTAC125), a heterotrophic marine bacterium isolated from Antarctica, has recently gained a certain attention due to its potential biotechnological exploitation 22 , its capability to synthesize anti-biofilm compounds 23 , the necessity to set up efficient culture conditions 24,25 and the metabolic reprogramming during growth in complex environments 26 . In particular, the analysis of its growth phenotype in an amino acid rich medium has shown the presence of metabolic switches among different groups of amino acids 25 , although nothing could be said about the molecular mechanisms underlying such phenotype and the possible regulation involved.…”

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

“…In this ~24 kb region, lacZ clusters with other genes involved in carbohydrates and amino acids metabolism ( Figure 1 , blue arrows) and, intriguingly, with three predicted CDSs involved in cut and paste mechanisms (black arrows) and a putative RepB protein involved in plasmid replication (red arrow) [ 25 ]. In particular, the RepB is highly conserved (65–95% nucleotide identity) in plasmids harbored by three marine Pseudoalteromonadales , P. haloplanktis TAC125 (MN400773.1), P. nigrifaciens KMM 661 (CP011038.1), P. arctica A 37-1-2 (CP011027.1), whose reciprocal similarities have been recently examined [ 26 ]. Considering that the whole analyzed contig is almost totally conserved in P. nigrifaciens plasmid (88% coverage with 99% identity), it is very likely that the DNA containing the lacZ gene is the result of horizontal gene transfer also in Ph TAE79.…”

“…To approach this problem, we applied a strain engineering strategy based on the combination of the psychrophilic regulatory elements derived from Ph TAE79 with the mesophilic lactose permease LacY from E. coli . Despite the use of plasmids with a low copy number [ 26 ] and two different constitutive promoters with medium and high strength, the accumulation of the permease and the alteration of membrane properties probably caused serious toxic phenomena in Ph TAC125 [ 43 ]. This led to the failure of the first attempt of heterologous expression of LacY.…”