Loreto Salas

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

ABSTRACT Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.

Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese.

A novel subaerial Dunaliella species growing on cave spiderwebs in the Atacama Desert

Strategies for life adaptation to extreme environments often lead to novel solutions. As an example of this assertion, here we describe the first species of the well-known genus of green unicellular alga Dunaliella able to thrive in a subaerial habitat. All previously reported members of this microalga are found in extremely saline aquatic environments. Strikingly, the new species was found on the walls of a cave located in the Atacama Desert (Chile). Moreover, on further inspection we noticed that it grows upon spiderwebs attached to the walls of the entrance-twilight transition zone of the cave. This peculiar growth habitat suggests that this Dunaliella species uses air moisture condensing on the spiderweb silk threads as a source of water for doing photosynthesis in the driest desert of the world. This process of adaptation recapitulates the transition that allowed land colonization by primitive plants and shows an unexpected way of expansion of the life habitability range by a microbial species.

Ancient Photosynthetic Eukaryote Biofilms in an Atacama Desert Coastal Cave

Caves offer a stable and protected environment from harsh and changing outside prevailing conditions. Hence, they represent an interesting habitat for studying life in extreme environments. Here, we report the presence of a member of the ancient eukaryote red algae Cyanidium group in a coastal cave of the hyperarid Atacama Desert. This microorganism was found to form a seemingly monospecific biofilm growing under extremely low photon flux levels. Our work suggests that this species, Cyanidium sp. Atacama, is a new member of a recently proposed novel monophyletic lineage of mesophilic “cave” Cyanidium sp., distinct from the remaining three other lineages which are all thermo-acidophilic. The cave described in this work may represent an evolutionary island for life in the midst of the Atacama Desert.

Gloeocapsopsis AAB1, an extremely desiccation-tolerant cyanobacterium isolated from the Atacama Desert

The comprehensive study of microorganisms that evolved in the Atacama Desert, the driest and oldest on earth, may help to understand the key role of water for life. In this context, we previously characterized the microenvironment that allows colonization of the underside of quartzes in the Coastal Range of this desert by hypolithic microorganisms (Azua-Bustos et al. Microb Ecol 58:568–581, 2011). Now, we describe the biodiversity composition of these biofilms and the isolation from it of a new cyanobacterial strain. Based on morphologic and phylogenetic analyses, this isolate (AAB1) was classified as a new member of the Gloeocapsopsis genus. Physiological, morphological and molecular responses by isolate AAB1 show that this strain is extremely tolerant to desiccation. Our results also indicate that the isolate biosynthesizes sucrose and trehalose in response to this stressful condition. We identified two candidate genes involved in sucrose synthesis, namely sucrose 6-phosphate synthase and sucrose 6-phosphate phosphatase. Thus, the Gloeocapsopsis isolate AAB1 may represent a suitable model for understanding tolerance to low water availability.

Microbial and biochemical characterization of a bacterial consortium isolated from decaying wood by growth on a β-O-4 lignin-related dimeric compound

As an approach to evaluate the contribution of bacteria to lignin degradation in wood, we have chosen to study these microorganisms in the natural wood decay ecosystem known as Palo Podrido. Initially, the characterization of bacteria able to metabolize lignin-related compounds present in samples of Palo Podrido was undertaken. For their isolation, minimal salt media containing lignin dimers of either the arylglycerol-β-aryl ether (β-O-4) or 1,2-diarylpropane (β-1) types as the only source of carbon and energy were inoculated with various wood samples exhibiting different degrees of decay. The β-1 dimers used failed to support bacterial growth. However, three bacterial consortia able to consume quantitatively the β-O-4 model 1-[3,4-dimethoxyphenyl]-2-[2-methoxyphenoxy]-3-hydroxypropanone (compound 1) were isolated. One of these was further characterized. It is composed of eight strains belonging to the families of Streptomycetaceae, Dermatophilaceae and Actinoplanaceae. HPLC and GC-MS analyses revealed that the consortium utilizes two pathways to degrade β-O-4 dimers, both involving direct cleavage of the ether linkage. The formation of a novel C6−C3 degradation intermediate is described. Some metabolic properties of each strain, as well as those of the intact consortium, are also reported.