Secretion of the human parathyroid hormone through a microcin type I secretion system in Escherichia coli.

BACKGROUND: Gram negative bacteria possess different secretion systems to export proteins to the extracellular medium. The simplest one, type I secretion system (T1SS), forms a channel across the cell envelope to export proteins in a single step. Peptides secreted by the T1SSs comprise a group of antibiotics, called class II microcins, which carry an amino terminal secretion domain that is processed concomitantly with export. Mature microcins range in size from 60 to 90 amino acids and differ in their sequences. Microcin T1SSs show a high versatility in relation to the peptides they are able to secrete, being mainly limited by the length of the substrates. Different bioactive peptides unrelated to bacteriocins could be secreted by microcin V (MccV) T1SS, while retaining their biological activity. RESULTS: In this work heterologous secretion of two variants of human parathyroid hormone (PTH) by MccV T1SS was evaluated. PTH is a bioactive peptide of 84 amino acids (PTH84), which is involved in the maintenance of bone homeostasis. Currently, a drug corresponding to the active fraction of the hormone, which resides in its first 34 amino acids (PTH34), is commercially produced as a recombinant peptide in Escherichia coli. However, research continues to improve this recombinant production. Here, gene fusions encoding hybrid peptides composed of the MccV secretion domain attached to each hormone variant were constructed and expressed in the presence of microcin T1SS in E. coli cells. Both PTH peptides (PTH34 and PTH84) were recovered from the culture supernatants and could be confirmed to lack the MccV secretion domain, i.e. microcin T1SS efficiently recognised, processed and secreted both PTH variants. Furthermore, the secreted peptides were stable in the extracellular medium unlike their unprocessed counterparts present in the intracellular space. CONCLUSION: The successful secretion of PTH variants using MccV T1SS could be considered as a new alternative for their production, since they would be recovered directly from the extracellular space without additional sequences. Furthermore, it would be a new example revealing the potential of microcin type I secretion systems to be conceived as a novel strategy for the production of recombinant peptides in E. coli.

A bacterial cold-active dye-decolorizing peroxidase from an Antarctic Pseudomonas strain.

DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: • An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. • The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. • rDyP-AU10 showed high activity at low temperatures. • rDyP-AU10 is potentially useful for biotechnological applications.

Photo-repair effect of a bacterial Antarctic CPD-photolyase on UVC-induced DNA lesions in human keratinocytes.

Exposure to ultraviolet radiation from sunlight induces oxidative DNA lesions and bipyrimidine photoproducts that can lead to photo-aging and skin carcinogenesis. CPD-photolyases are flavoproteins that repair cyclobutane pyrimidine dimers using blue light as an energy source. In the present work, we evaluated the photo-repair effect of the recombinant CPD-photolyase PhrAHym from the Antarctic bacterium Hymenobacter sp. UV11 on DNA lesions in human keratinocytes induced by UVC light. By performing immunochemistry assays we observed that PhrAHym repairs in a highly efficient way the CPD-photoproducts and reduces the γH2AX formation. Since this enzyme is non-cytotoxic and repairs UVC-induced DNA lesions in human keratinocytes, we propose that PhrAHym could be used as a biotherapeutic agent against UV-induced skin cancer, photoaging, and related diseases.

A (6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9: recombinant production and in silico features.

Photolyases are proteins that enzymatically repair the UV-induced DNA damage by a protein-DNA electron transfer mechanism. They repair either cyclobutane pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts or just (6-4)-photoproducts. In this work, we report the production and partial characterization of a recombinant (6-4)-photolyase (SphPhrB97) from a bacterial psychrotolerant Antarctic isolate identified as Sphingomonas sp. strain UV9. The spectrum analysis and the in silico study of SphPhrB97 suggest that this enzyme has similar features as compared to the (6-4)-photolyase from Agrobacterium tumefaciens (4DJA; PhrB), including the presence of three cofactors: FAD, DMRL (6,7-dimethyl-8-(1'-D-ribityl) lumazine), and an Fe-S cluster. The homology model of SphPhrB97 predicts that the DNA-binding pocket (area and volume) is larger as compared to (6-4)-photolyases from mesophilic microbes. Based on sequence comparison and on the homology model, we propose an electron transfer pathway towards the FAD cofactor involving the residues Trp342, Trp390, Tyr40, Tyr391, and Tyr399. The phylogenetic tree performed using curated and well-characterized prokaryotic (6-4)-photolyases suggests that SphPhrB97 may have an ancient evolutionary origin. The results suggest that SphPhrB97 is a cold-adapted enzyme, ready to cope with the UV irradiation stress found in a hostile environment, such as Antarctica.

Production and antiproliferative effect of violacein, a purple pigment produced by an Antarctic bacterial isolate.

We studied the production and the potential use of a purple-pigment produced by an Antarctic bacterial isolate. This pigment was identified as violacein, a metabolite produced by many bacterial strains and reported that it has antiproliferative activity in many cell lines. We analyzed the effect of temperature and the composition of the growth medium on pigment production, achieving the highest yield at 20 °C in Tryptic Soy Broth medium supplemented with 3.6 g/L glucose. We doubled the yield of the pigment production when the process was scaled up in a 5 L bioreactor (77 mg/L of crude pigment). The pigment was purified and identified by mass spectrometry (DI-EI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy as violacein. We performed survival assays that showed that the pure pigment has antiproliferative activity and sensitize HeLa cells (cervix cell carcinoma) to cisplatin. Besides, the pigment did not show genotoxic activity in HeLa cells as found performing micronucleus assays. These results suggest that this pigment may be used as anticancer or sensitizer to cisplatin drug in cervix cancer.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Validating biochemical features at the genome level in the Antarctic bacterium Hymenobacter sp. strain UV11.

We present experimental data that complement and validate some biochemical features at the genome level in the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11 strain. The genome was sequenced, assembled and annotated. It has 6 096 246 bp, a GC content of 60.6% and 5155 predicted genes. The secretome analysis, by combining in silico predictions with shotgun proteomics data, showed that UV11 strain produces extracellular proteases and carbohydrases with potential biotechnological uses. We observed the formation of outer membrane vesicles, mesosomes and carbon-storage compounds by using transmission electron microscopy. The in silico analysis of the genome revealed the presence of genes involved in the metabolism of glycogen-like molecules and starch. By HPLC-UV-Vis analysis and 1H-NMR spectra, we verified that strain UV11 produces xanthophyll-like carotenoids such as 2'-hydroxyflexixanthin, and the in silico analysis showed that this bacterium has genes involved in the biosynthesis of cathaxanthin, zeaxanthin and astaxanthin. We also found genes involved in the repair of UV-damaged DNA such as a photolyase, the nucleotide excision repair system and the production of ATP-dependent proteases that are important cellular components involved in the endurance to physiological stresses. This information will help us to better understand the ecological role played by Hymenobacter strains in the extreme Antarctic environment.

Draft Genome Sequence of the UV-Resistant Antarctic Bacterium Sphingomonas sp. Strain UV9.

We report the draft genome sequence of the Antarctic UV-resistant bacterium Sphingomonas sp. strain UV9. The strain has a genome size of 4.25 Mb, a 65.62% GC content, and 3,879 protein-coding sequences. Among others, genes encoding the resolving of the DNA damage produced by the UV irradiation were identified.

A highly efficient and cost-effective recombinant production of a bacterial photolyase from the Antarctic isolate Hymenobacter sp. UV11.

Photolyases are DNA-repairing flavoproteins that are represented in most phylogenetic taxa with the exception of placental mammals. These enzymes reduce the ultraviolet-induced DNA damage; thus, they have features that make them very attractive for dermatological or other medical uses, such as the prevention of human skin cancer and actinic keratosis. In this work, we identified a 50.8 kDa photolyase from the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and its activity was analyzed using different approaches: detection of cyclobutane pyrimidine dimers (CPDs) by immunochemistry, high-performance liquid chromatography and comet assays using Chinese Hamster Ovary (CHO) and immortalized nontumorigenic human epidermal (HaCat) cells. The information supports that the recombinant protein has the ability to repair the formation of CPDs, on both double- and single-stranded DNA. This CPD-photolyase was fully active on CHO and HaCat cell lines, suggesting that this enzyme could be used for medical or cosmetic purposes. Results also suggest that the UV11 CPD-photolyase uses MTHF as chromophore in the antenna domain. The potential use of this recombinant enzyme in the development of new inventions with pharmaceutical and cosmetic applications is discussed during this work.

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m- 2) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.