Scaling down the microbial loop: data-driven modelling of growth interactions in a diatom-bacterium co-culture

An intricate set of interactions characterizes marine ecosystems. One of the most important is represented by the so-called microbial loop, which includes the exchange of dissolved organic matter (DOM) from phototrophic organisms to heterotrophic bacteria. Here, it can be used as the major carbon and energy source. Arguably, this interaction is one of the foundations of the entire ocean food-web. Carbon fixed by phytoplankton can be redirected to bacterial cells in two main ways; either i) bacteria feed on dead (eventually lysed) phytoplankton cells or ii) DOM is actively released by phytoplankton cells (a widespread process that may result in up to 50% of the fixed carbon leaving the cell). In this work, we have set up a co-culture of the model diatom Phaeodactylum tricornutum and the model chemoheterotrophic bacterium Pseudoalteromonas haloplanktis TAC125 and used this system to study the interactions between these two representatives of the microbial loop. We show that the bacterium can indeed thrive on diatom-derived carbon and that this growth can be sustained by both diatom dead cells and diatom-released compounds. These observations were formalized in a network of putative interactions between P. tricornutum and P. haloplanktis and implemented in a mathematical model that reproduces the observed co-culture dynamics, suggesting that our hypotheses on the interactions occurring in this two-player system can accurately explain the experimental data.

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

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

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

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.

Activation Studies of the γ-Carbonic Anhydrases from the Antarctic Marine Bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea with Amino Acids and Amines

The γ-carbonic anhydrases (CAs, EC 4.2.1.1) present in the Antarctic marine bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea, herein referred to as PhaCA and CpsCA, respectively, were investigated for their activation with a panel of 24 amino acids and amines. Both bacteria are considered Antarctic models for the investigation of photosynthetic and metabolic pathways in organisms adapted to live in cold seawater. PhaCA was much more sensitive to activation by these compounds compared to the genetically related enzyme CpsCA. The most effective PhaCA activators were d-Phe, l-/d-DOPA, l-Tyr and 2-pyridyl-methylamine, with the activation constant KA values of 0.72–3.27 µM. d-His, l-Trp, d-Tyr, histamine, dopamine, serotonin anddicarboxylic amino acids were also effective activators of PhaCA, with KA values of 6.48–9.85 µM. CpsCA was activated by d-Phe, d-DOPA, l-Trp, l-/d-Tyr, 4-amino-l-Phe, histamine, 2-pyridyl-methylamine and l-/d-Glu with KA values of 11.2–24.4 µM. The most effective CpsCA activator was l-DOPA (KA of 4.79 µM). Given that modulators of CAs from Antarctic bacteria have not been identified and investigated in detail for their metabolic roles to date, this research sheds some light on these poorly understood processes.