Bacterium Pseudoalteromonas Haloplanktis TAC125 Research Articles

Recombinant protein production in Pseudoalteromonas haloplanktis TAC125 biofilm

Biofilms have great potential for producing valuable products, and recent research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. However, the production of recombinant proteins using this system is still limited. The recombinant protein production in microbial hosts is a well-established technology and a variety of expression systems are available. Nevertheless, the production of some recombinant proteins can result in proteolyzed, insoluble, and non-functional forms, therefore it is necessary to start the exploration of non-conventional production systems that, in the future, could be helpful to produce some “difficult” proteins. Non-conventional production systems can be based on the use of alternative hosts and/or on non-conventional ways to grow recombinant cells. In this paper, the use of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 grown in biofilm conditions was explored to produce two fluorescent proteins, GFP and mScarlet. The best conditions for the production were identified by working on media composition, and induction conditions, and by building a new expression vector suitable for the biofilm conditions. Results reported demonstrated that the optimized system for the recombinant protein production in biofilm, although it takes longer than planktonic production, has the same potentiality as the classical planktonic approach with additional advantages since it needs a lower concentration of the carbon sources and doesn't require antibiotic addition. Moreover, in the case of mScarlet, the production in biofilm outperforms the planktonic system in terms of a better quality of the recombinant product.

Shaping the phycosphere: Analysis of the EPS in diatom-bacterial co-cultures.

The phycosphere is a unique niche that fosters complex interactions between microalgae and associated bacteria. The formation of this extracellular environment, and the associated bacterial biodiversity, is heavily influenced by the secretion of extracellular polymers, primarily driven by phototrophic organisms. The exopolysaccharides (EPS) represent the largest fraction of the microalgae-derived exudates, which can be specifically used by heterotrophic bacteria as substrates for metabolic processes. Furthermore, it has been proposed that bacteria and their extracellular factors play a role in both the release and composition of the EPS. In this study, two model microorganisms, the diatom Phaeodactylum tricornutum CCAP 1055/15 and the bacterium Pseudoalteromonas haloplanktis TAC125, were co-cultured in a dual system to assess how their interactions modify the phycosphere chemical composition by analyzing the EPS monosaccharide profile released in the culture media by the two partners. We demonstrate that microalgal-bacterial interactions in this simplified model significantly influenced the architecture of their extracellular environment. We observed that the composition of the exo-environment, as described by the EPS monosaccharide profiles, varied under different culture conditions and times of incubation. This study reports an initial characterization of the molecular modifications occurring in the extracellular environment surrounding two relevant representatives of marine systems.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future.Key points• Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125.• Two order of magnitude improvement of OriR-derived psychrophilic expression systems.• Almost twenty times enhancement in Green fluorescent protein production.

Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

BackgroundA significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues.ResultsUsing a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology.ConclusionsCollectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions.

Conditional gene silencing in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Since the release in 2005 of the genome sequence and annotation of the first Antarctic marine bacterium, the number of genomes of psychrophilic microorganisms in public databases has steadily increased. Unfortunately, the lack of effective molecular tools for the manipulation of these environmental strains still hampers our understanding of their peculiar strategies to thrive in freezing conditions, limiting the functional genomics approaches to differential analyses only. Over the past two decades, our research group established the first effective gene cloning/expression technology in the Antarctic Gram-negative marine bacterium Pseudoalteromonas haloplanktis TAC125. The setup of a genome mutagenesis technique (based on homologous recombination and counterselection events) further supported the use of this strain, which became an attractive model for studying microbial adaptations to freezing lifestyle. Moreover, to further extend the functional analyses to its essential genes, the set-up of a conditional gene silencing approach is desirable. In this paper, we report the development of an asRNA regulatory system in the Antarctic bacterium, testing the feasibility of Hfq-dependent and PTasRNA strategies previously developed in Escherichia coli. Stable and efficient silencing of two chromosomal genes was obtained by using PTasRNAs, reaching very high levels of downregulation.

Soluble Recombinant Protein Production in Pseudoalteromonas haloplanktis TAC125: The Case Study of the Full-Length Human CDKL5 Protein.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is an unconventional protein production host displaying a notable proficiency in the soluble production of difficult proteins, especially of human origin. Furthermore, the accumulation of recombinant products in insoluble aggregates has never been observed in this bacterium, indicating that its cellular physicochemical conditions and/or folding processes are rather different from those observed in mesophilic bacteria. The ability of this cell factory was challenged by producing a human protein, the cyclin-dependent kinase-like 5 (hCDKL5) in the bacterium cytoplasm at 0°C. Human CDKL5 is a serine/threonine protein kinase characterized by the absence of a defined structure for the last two/third of its sequence, one of the largest intrinsically disordered regions so far observed in a human protein. This large unstructured domain makes difficult its production in most of the conventional hosts since the recombinant product accumulates as insoluble aggregates and/or is heavily proteolyzed. As the full-length hCDKL5 production is of great interest both for basic science and as protein drug for an enzyme replacement therapy, its production in the Antarctic bacterium was tested by combining the use of a regulated psychrophilic gene expression system with the use of a defined growth medium optimized for the host growth at subzero temperature. This is the first report of soluble and full-length recombinant production of hCDKL5 protein in a bacterium.

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

SummaryAn intricate set of interactions characterizes marine ecosystems. One of the most important is represented by the 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. This interaction is one of the foundations of the entire ocean food‐web. The carbon fixed by phytoplankton can be redirected to bacteria in two main ways; either (i) bacteria feed on dead phytoplankton cells or (ii) DOM is actively released by phytoplankton (a process resulting in up to 50% of the fixed carbon leaving the cell). Here, we have set up a co‐culture of the diatom Phaeodactylum tricornutum and the 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 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 model that reproduces the observed co‐culture dynamics, revealing an overall accuracy of our hypotheses in explaining the experimental data.

Modelling hCDKL5 Heterologous Expression in Bacteria.

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production.

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.

Pentadecanal and pentadecanoic acid coatings reduce biofilm formation of Staphylococcus epidermidis on PDMS

Staphylococcus epidermidis is well known to be one of the major causes of infections related to medical devices, mostly due to its strong capacity to form device-associated biofilms. Nowadays, these infections represent a severe burden to the public health system and the necessity of novel antibacterial strategies for the treatment of these difficult-to-eradicate infections is urgent. The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was found to be able to produce an anti-biofilm molecule, the pentadecanal, active against S. epidermidis. In this work, we modified one of the most widely used silicone-based polymers, polydimethylsiloxane (PDMS), by adsorption of pentadecanal and its most promising derivative, pentadecanoic acid, on the PDMS surface. The biofilm formation of S. epidermidis RP62A on both untreated and modified PDMS was performed in a parallel plate flow chamber system, demonstrating the capability of the proposed anti-biofilm coatings to strongly reduce the biofilm formation. Furthermore, drug-release capacity and long-term efficacy (21 days) were also proven for the pentadecanoic acid coating.

Pentadecanal inspired molecules as new anti-biofilm agents against Staphylococcus epidermidis

Staphylococcus epidermidis, a harmless human skin colonizer, is a significant nosocomial pathogen in predisposed hosts because of its capability to form a biofilm on indwelling medical devices. In a recent paper, the purification and identification of the pentadecanal produced by the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, able to impair S. epidermidis biofilm formation, were reported. Here the authors report on the chemical synthesis of pentadecanal derivatives, their anti-biofilm activity on S. epidermidis, and their action in combination with antibiotics. The results clearly indicate that the pentadecanal derivatives were able to prevent, to a different extent, biofilm formation and that pentadecanoic acid positively modulated the antimicrobial activity of the vancomycin. The cytotoxicity of these new anti-biofilm molecules was tested on two different immortalized eukaryotic cell lines in view of their potential applications.

Cold-Adaptation Signatures in the Ligand Rebinding Kinetics to the Truncated Hemoglobin of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125.

Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on truncated hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of the unusual geminate rebinding phase. Water molecules are present in the distal pocket, but their stabilization by TrpG8, TyrB10, and HisCD1 is much weaker than in thermophilic Thermobifida fusca truncated hemoglobin, resulting in a faster geminate rebinding. Our results support the hypothesis that weaker water-molecule interactions at the reaction site are associated with cold adaptation.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins.The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

Ecology of cold environments: new insights of bacterial metabolic adaptation through an integrated genomic-phenomic approach

Cold environments dominate Earth’s biosphere, hosting complex microbial communities with the ability to thrive at low temperatures. However, the underlying molecular mechanisms and the metabolic pathways involved in bacterial cold-adaptation mechanisms are still not fully understood. Herein, we assessed the metabolic features of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), a model organism for cold-adaptation, at both 4 °C and 15 °C, by integrating genomic and phenomic (high-throughput phenotyping) data and comparing the obtained results to the taxonomically related Antarctic bacterium Pseudoalteromonas sp. TB41 (PspTB41). Although the genome size of PspTB41 is considerably larger than PhTAC125, the higher number of genes did not reflect any higher metabolic versatility at 4 °C as compared to PhTAC125. Remarkably, protein S-thiolation regulated by glutathione and glutathionylspermidine appeared to be a new possible mechanism for cold adaptation in PhTAC125. More in general, this study represents an example of how ‘multi-omic’ information might potentially contribute in filling the gap between genotypic and phenotypic features related to cold-adaptation mechanisms in bacteria.

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.

Pseudoalteromonas haloplanktis produces methylamine, a volatile compound active against Burkholderia cepacia complex strains

The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 has been reported to produce several Volatile Organic Compounds (VOCs), which are able to inhibit the growth of Burkholderia cepacia complex (Bcc) strains, opportunistic pathogens responsible for the infection of immune-compromised patients. However, no specific antibacterial VOCs have been identified to date. The purpose of the present study was to identify specific VOCs that contribute to Bcc inhibition by the Antarctic strain. When grown on defined medium containing D-gluconate and L-glutamate as carbon, nitrogen and energy sources, P. haloplanktis TAC125 is unable to inhibit the growth of Bcc strains. However, single addition of several amino acids to the defined medium restores the P. haloplanktis TAC125 inhibition ability. With the aim of identifying specific volatile compound/s responsible for Bcc inhibition, we set up an apparatus for VOC capture, accumulation, and storage. P. haloplanktis TAC125 was grown in an automatic fermenter which was connected to a cooling system to condense VOCs present in the exhaust air outlet. Upon addition of methionine to the growth medium, the VOC methylamine was produced by P. haloplanktis TAC125. Methylamine was found to inhibit the growth of several Bcc strains in a dose-dependent way. Although it was reported that P. haloplanktis TAC125 produces VOCs endowed with antimicrobial activity, this is the first demonstration that methylamine probably contributes to the anti-Bcc activity of P. haloplanktis TAC125 VOCs.

A novel synthetic medium and expression system for subzero growth and recombinant protein production in Pseudoalteromonas haloplanktis TAC125.

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a model organism of cold-adapted bacteria. The interest in the study of this psychrophilic bacterium stems from its capability either as a non-conventional system for production of recombinant protein and as a rich source of bioactive compounds. To further explore the biotechnological ability of P. haloplanktis TAC125, we have developed a synthetic medium, containing D-gluconate and L-glutamate (GG), which allows the bacterium to grow even at subzero temperatures. P. haloplanktis TAC125 growing in GG medium at low temperature displays growth kinetic parameters which confirm its spectacular adaptation to cold environment and subzero lifestyle, paving the way to the definition of the underlying molecular strategies. Moreover, in this paper, we report the setup of a finely regulated gene expression system inducible by D-galactose to produce recombinant protein in GG synthetic medium at temperatures as low as -2.5°C. Thanks to the combination of the novel medium and the new expression system, we obtained for the first time the production of a recombinant protein at subzero temperature, thus providing an innovative strategy for the recombinant production of "difficult" proteins.

Large-scale biofilm cultivation of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 for physiologic studies and drug discovery.

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air-liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.

Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 Å resolution) shows typical features of group-II truncated hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.

Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.