Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.

The aerobiology of caves in Southern Spain possesses special characteristics, different from caves located in Northern Spain. Previous studies demonstrated the influence of outdoor air on caves in the north and the existence of two different patterns, depending on the season. In summer there is an abundance of Ascomycota, whereas in winter Basidiomycota predominates, which are related to the periods of stagnation and ventilation, respectively. In caves in Southern Spain the presence of airborne Basidiomycota is scarce and Ascomycota represents the main group of fungi widely distributed across the caves in all seasons. The most characteristic features were the abundant presence of entomopathogenic fungi (Beauveria bassiana, Parengyodontium album, Pochonia chlamydosporia, Leptobacillium symbioticum, Leptobacillium leptobactrum) and Cladosporium cladosporioides in Cueva del Tesoro, Cueva de Ardales and Gruta de las Maravillas. However, the presence of yeasts of the genera Cutaneotrichosporon, Trichosporon, Cryptococcus, Naganishia, Cystobasidium, Microstroma and Phragmotaenium was exclusive to Gruta de las Maravillas. Fungal hazard in the three show caves were determined using an ecological indicator based on the concentration of spores in cave air.

The Circular Mausoleum tomb (Roman Necropolis of Carmona, Spain) dates back from the first century AD and is characterized by a dense microbial (phototrophic) colonization on the walls and ceiling. However, some walls exhibited an important number of violet stains of unknown origin. The microbial communities of these violet stains are mainly composed of cyanobacteria, streptomycetes and fungi. A strain of Streptomyces parvus, isolated from the walls, produces a violet pigment in culture media. High performance liquid chromatography-mass spectrometry of the culture extracts obtained from this Streptomyces revealed the presence of a few granaticins, pigments with a benzoisochromanequinone structure. When metabolically active in the tomb, S. parvus synthesizes the pigments that diffuse into the mortar. During rain and/or wetting periods, the pigments are solubilized by alkaline waters and elute from the starting position to the surrounding mortar, enlarging the pigmented area and thus contributing to this exceptional biodeterioration phenomenon.

Of the several critical challenges present in environmental microbiology today, one is the assessment of the contribution of microorganisms in the carbon cycle in the Earth-climate system. Karstic subterranean ecosystems have been overlooked until recently. Covering up to 25% of the land surface and acting as a rapid CH4 sink and alternately as a CO2 source or sink, karstic subterranean ecosystems play a decisive role in the carbon cycle in terms of their contribution to the global balance of greenhouse gases. Recent data indicate that microbiota must play a significant ecological role in the biogeochemical processes that control the composition of the subterranean atmosphere, as well as in the availability of nutrients for the ecosystem. Nevertheless, there are still essential gaps in our knowledge concerning the budgets of greenhouse gases at the ecosystem scale and the possible feedback mechanisms between environmental-microclimatic conditions and the rates and type of activity of microbial communities in subterranean ecosystems. Another challenge is searching for bioactive compounds (antibiotics) used for treating human diseases. At present, there is a global health emergency and a strong need for novel biomolecules. In recent decades, great research efforts have been made to extract antibiotics from marine organisms. More recently, caves have been receiving considerable attention in search of novel antibiotics. Cave methanotrophic and heterotrophic bacteria are producers of bioactive compounds and may be potential sources of metabolites with antibacterial, antifungal or anticancer activities of interest in pharmacological and medical research, as well as enzymes with a further biotechnological use. Here we also show that bacteria isolated from mines, a still unexplored niche for scientists in search of novel compounds, can be a source of novel secondary metabolites.

Two Gram-positive, catalase-positive, oxidase-negative, motile, endospore-forming, rod-shaped bacteria, designated as 0911MAR22V3T and 0911TES10J4, were isolated from air samples collected in two show caves, located in Andalusia, Southern Spain. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both strains were indistinguishable and they were most closely related to Bacillus humi DSM 16318T (98%). DNA-DNA hybridization values of the strain 0911MAR22V3T with respect to strain 0911TES10J4 and B. humi DSM 16318T were 76.8% (73.9%, reciprocal) and 56.9% (63.3%, reciprocal analysis), respectively. Whole genome average nucleotide identity (ANI) values of both strains were in the threshold value for species delineation and less than 85% with B. humi. Strains 0911MAR22V3T and 0911TES10J4 grew at 10-47°C (optimum 37°C), at pH 6-9.5 and with 0-8% (w/v) NaCl (optimum 1%). In both strains the dominant isoprenoid quinone was MK-7, the major cellular polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and two more phospholipids, the predominant fatty acids were iso-C15:0 and anteiso-C15:0 and the DNA G+C content was 38mol%. On the basis of their phylogenetic relatedness and their phenotypic and genotypic features, the strains 0911MAR22V3T and 0911TES10J4 should be attributed to a novel species within the genus Bacillus, for which the name Bacillus onubensis sp. nov. is proposed. The type strain is 0911MAR22V3T (=LMG 27963T=CECT 8479T); and strain 0911TES10J4 (CECT 8478) is a reference strain.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterium, strain 0511ARD5E5 T , was isolated from an air sample collected in Ardales Cave (Malaga, Spain). Strain 0511ARD5E5 T grew at 4-37 C and in the presence of 0-4 % (w/v) NaCl [optimally at 25 C and with 1 % (w/v) NaCl]. Cells were catalase-and oxidase-positive. The major respiratory quinone was ubiquinone-10. The predominant fatty acids were C 18:1 !7c and C 16:0 . The DNA G +C content was 63.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 0511ARD5E5 T was a member of the genus Paracoccus and was related most closely to Paracoccus aminophilus DSM 8538 T and Paracoccus marinus CIP 108500 T (96.93 and 96.92 % similarity, respectively). Strain 0511ARD5E5 T exhibited DNA-DNA relatedness of 47 % to P. aminophilus DSM 8538 T and 31 % to P. marinus CIP 108500 T . Chemotaxonomic, phenotypic and phylogenetic analyses indicated that strain 0511ARD5E5 T represents a novel species of the genus Paracoccus, for which the name Paracoccus cavernae sp. nov. is proposed. The type strain is 0511ARD5E5 T (=LMG 27962 T =CECT 8482 T ).The genus Paracoccus was first described by Davis et al. (1969) and the description was subsequently emended several times (Ludwig et al., 1993;Katayama et al., 1995;Liu et al., 2008). The members of this genus are Gram-negative, non-spore-forming, non-motile cocci or short rods that are oxidase-and catalase-positive, and metabolically versatile. The major cellular fatty acid is C 18:1 !7c, ubiquinone-10 is the sole isoprenoid quinone and the DNA G+C content is between 64 and 70 mol%. At the time of writing, the genus Paracoccus comprises 42 species.In this study, we describe strain 0511ARD5E5 T isolated from an air sample collected in Ardales Cave (Malaga, Spain). Phenotypic and genotypic studies showed that this strain represents a novel species within the genus Paracoccus.Strain 0511ARD5E5 T was isolated using a tryptose soy agar medium (TSA; BD) with cycloheximide (Applichem) (50 µg ml -1 ). Morphological, physiological, biochemical and chemotaxonomic studies of strain 0511ARD5E5 T and the most closely related type strains (Paracoccus aminophilus DSM 8538 T and Paracoccus marinus CIP 108500 T ) were carried out in triplicate on Luria-Bertani (LB; BD) agar at 30 C.The Gram-reaction was performed by conventional Gramstaining and was confirmed with the KOH lysis test (Halebian et al., 1981). Cell morphology, dimensions and motility were examined by phase contrast microscopy. Furthermore, motility was also tested in LB broth containing 0.3 % agar (Tambalo et al., 2010) and the presence of flagella was examined by microscopy after staining with the reagent Flagella Stain Droppers (BD), according to the manufacturer's instructions. Spore production was checked with malachite green staining. Catalase activity was indicated by the production of bubbles after mixing cells from colonies grown on LB plates with a drop of 3 % (v/v) hydrogen peroxide on a glass slide. Oxidase activity was determined by monitor...

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Antimicrobial resistance has become a global problem in recent decades. A gradual reduction in drug discoveries has led to the current antimicrobial resistance crisis. Caves and other subsurface environments are underexplored thus far, and they represent indispensable ecological niches that could offer new molecules of interest to medicine and biotechnology. We explored Spanish show caves to test the bioactivity of the bacteria dwelling in the walls and ceilings, as well as airborne bacteria. We reported the isolation of two strains of the genus Crossiella, likely representing a new species, isolated from Altamira Cave, Spain. In vitro and in silico analyses showed the inhibition of pathogenic Gram-positive and Gram-negative bacteria, and fungi, as well as the taxonomical distance of both strains from their closest relative, Crossiella cryophila. The presence of an exclusive combination of gene clusters involved in the synthesis of lanthipeptides, lasso peptides, nonribosomal peptides and polyketides indicates that species of this genus could represent a source of new compounds. Overall, there is promising evidence for antimicrobial discovery in subterranean environments, which increases the possibility of identifying new bioactive molecules.