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.

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.

Pentadecanoic acid against Candida albicans-Klebsiella pneumoniae biofilm: towards the development of an anti-biofilm coating to prevent polymicrobial infections.

The ability to form biofilms is a common feature of microorganisms, which can colonize a variety of surfaces, such as host tissues and medical devices, resulting in infections highly resistant to conventional drugs. This aspect is particularly critical in polymicrobial biofilms involving both fungi and bacteria, therefore, to eradicate such severe infections, new and effective anti-biofilm strategies are needed. The efficacy of pentadecanal and pentadecanoic acid as anti-biofilm agents has been recently reported against different bacterial strains. Their chemical similarity with diffusible signal factors (DSFs), plus the already known ability of fatty acids to act as anti-biofilm agents, suggested to explore their use against Candida albicans and Klebsiella pneumoniae mixed biofilm. In this work, we demonstrated the ability of both molecules to prevent the formation and destabilize the structure of the dual-species biofilm. Moreover, the pentadecanoic acid anti-biofilm coating, previously developed through the adsorption of the fatty acid on polydimethylsiloxane (PDMS), was proved to prevent the polymicrobial biofilm formation in dynamic conditions by confocal laser scanning microscopy analysis. Finally, the evaluation of the expression levels of some biofilm-related genes of C. albicans and K. pneumoniae treated with pentadecanoic acid provided some insights into the molecular mechanisms underpinning its anti-biofilm effect.

Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes.

Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized. The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli. The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum. From this analysis it turned out that indole-3-glycerol-phosphate synthase from S. solfataricus can be considered a model for studying thermostable TIM-barrel enzymes. Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S. solfataricus are discussed in relation to the thermostability of the enzyme.

Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp. TA144.

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.

Expression of Sulfolobus solfataricus trpE and trpG genes in E. coli.

The genes trpE and trpG of the hyperthermophilic archaeon Sulfolobus solfataricus, encoding the components I and II of anthranilate synthase, were cloned and co-expressed in Escherichia coli. The properties of the recombinant protein were determined and compared to those of the wild type complex. Gel filtration chromatography revealed an alpha2beta2 composition. The heteromeric enzyme is fully active above 85 degrees C and can be considered to be an "extremozyme" according to Adams et al.[1]. Sulfolobus solfataricus anthranilate synthase is subject to feedback inhibition by L-tryptophan even if it lacks the co-operativity that has been observed for all the other tetrameric anthranilate synthases.

A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature.

Genetic manipulation of Antarctic bacteria has been very limited so far. This article reports the isolation and molecular characterization of a novel plasmid, pMtBL, from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAC 125. This genetic element, 4,081 bp long, appeared to be a multicopy cryptic replicon with no detectable transcriptional activity. By an in vivo assay, the pMtBL autonomous replication sequence was functionally limited to an AluI plasmid fragment of about 850 bp. This novel cold-adapted replication element showed quite a broad host range profile: it was cloned into a mesophilic genetic construction, obtaining a cold-adapted expression vector that was able to promote the production of P. haloplanktis A23 alpha-amylase in a psychrophilic bacterium. This study represents the first report of successful recombinant production of a cold-adapted protein in an Antarctic host.

Structural investigation on the lipooligosaccharide fraction of psychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium.

The core structure of the cell-wall lipooligosaccharide (LOS) fraction of an Antarctic Gram-negative bacterium, Pseudoalteromonas haloplanktis TAC 125 strain, was determined to be deacetylated alditols. These were obtained from native LOS fraction by O-deacylation, dephosphorylation, reduction and finally N-deacylation. Two novel structures were detected, the more highly represented molecule consisting of the following hexasaccharide chain: alpha-D-ManpNH(2)-(1-->3)-beta-D-Galp-(1-->4)-alpha-L-glycero-D-manno-Hepp-(1-->5)-alpha-D-Kdo-(2-->6)-beta-D-GlcpNH(2)-(1-->6)-D-GlcNH(2)(ol) while the corresponding pentasaccharide, lacking the ManpNH(2) residue, was less abundant. To the best of our knowledge, the structural investigation presented here, mainly performed by NMR and MS methods, is the first report of the lipopolysaccharide fraction of a psychrophilic bacterium.

A rolling-circle plasmid from Psychrobacter sp. TA144: evidence for a novel rep subfamily.

In this paper we report the cloning and sequencing of two small plasmids, pTAUp and pTADw, from the Antarctic Gram-negative Psychrobacter sp strain TA144. The observation that pTAUp contains a putative Rep-coding gene (Psyrep) suggested that its duplication occurs via a rolling-circle replication mechanism. This hypothesis was confirmed by the identification of the pTAUp single-stranded DNA form. The putative pTAUp plus origin of replication was found at the 3' end of the Psyrep by using an in vivo complementation assay. Structural similarities at the level of (i) gene organization, (ii) protein sequence, and (iii) nick site sequences strongly suggest that the psychrophilic enzyme belongs to a new subfamily of replication enzymes.

Tryptophan biosynthesis genes trpEGC in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

A DNA fragment containing the trpEGC gene cluster was isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The products of trpE, trpG, and trpC from S. solfataricus were compared to the homologous products from a eukaryote, a eubacterium, and two archaebacteria, namely, a methanogen and an extreme halophile. They appeared to be equally related to the proteins from Escherichia coli and Saccharomyces cerevisiae, the percentages of conserved amino acids being roughly the same as those measured when comparing the eubacterial and eukaryotic sequences directly. These percentages did not rise significantly when a comparison with the proteins from Haloferax volcanii was drawn, while a slightly closer relationship with the proteins from Methanococcus thermoautotrophicum was found.

Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling.

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is approximately 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at approximately 50 degrees C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both kcat and kcat/Km of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature. However, binding of maleate and 2-methyl-L-aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed.