Fischerella thermalis : a model organism to study thermophilic diazotrophy, photosynthesis and multicellularity in cyanobacteria Abstract The true-branching cyanobacterium Fischerella thermalis (also known as Mastigocladus laminosus) is widely distributed in hot springs around the world. Morphologically, it has been described as early as 1837. However, its taxonomic placement remains controversial. F. thermalis belongs to the same genus as mesophilic Fischerella species but forms a monophyletic clade of thermophilic Fischerella strains and sequences from hot springs. Their recent divergence from freshwater or soil true-branching species and the ongoing process of specialization inside the thermal gradient make them an interesting evolutionary model to study. F. thermalis is one of the most complex prokaryotes. It forms a cellular network in which the main trichome and branches exchange metabolites and regulators via septal junctions. This species can adapt to a variety of environmental conditions, with its photosynthetic apparatus remaining active in a temperature range from 15 to 58 °C. Together with its nitrogen-fixing ability, this allows it to dominate in hot spring microbial mats and contribute significantly to the de novo carbon and nitrogen input. Here, we review the current knowledge on the taxonomy and distribution of F. thermalis, its morphological complexity, and its physiological adaptations to an extreme environment.

Colombian Andean thermal springs: reservoir of thermophilic anaerobic bacteria producing hydrolytic enzymes Abstract Anaerobic cultivable microbial communities in thermal springs producing hydrolytic enzymes were studied. Thermal water samples from seven thermal springs located in the Andean volcanic belt, in the eastern and central mountain ranges of the Colombian Andes were used as inocula for the growth and isolation of thermophilic microorganisms using substrates such as starch, gelatin, xylan, cellulose, Tween 80, olive oil, peptone and casamino acids. These springs differed in temperature (50–70 °C) and pH (6.5–7.5). The predominant ion in eastern mountain range thermal springs was sulphate, whereas that in central mountain range springs was bicarbonate. A total of 40 anaerobic thermophilic bacterial strains that belonged to the genera Thermoanaerobacter, Caloramator, Anoxybacillus, Caloranaerobacter, Desulfomicrobium, Geotoga, Hydrogenophilus, Desulfacinum and Thermoanaerobacterium were isolated. To investigate the metabolic potential of these isolates, selected strains were analysed for enzymatic activities to identify strains than can produce hydrolytic enzymes. We demonstrated that these thermal springs contained diverse microbial populations of anaerobic thermophilic comprising different metabolic groups of bacteria including strains belonging to the genera Thermoanaerobacter, Caloramator, Anoxybacillus, Caloranaerobacter, Desulfomicrobium, Geotoga, Hydrogenophilus, Desulfacinum and Thermoanaerobacterium with amylases, proteases, lipases, esterases, xylanases and pectinases; therefore, the strains represent a promising source of enzymes with biotechnological potential.

Comparative evaluation of three archaeal primer pairs for exploring archaeal communities in deep-sea sediments and permafrost soils Abstract 16S rRNA gene profiling is a powerful method for characterizing microbial communities; however, no universal primer pair can target all bacteria and archaea, resulting in different primer pairs which may impact the diversity profile obtained. Here, we evaluated three pairs of high-throughput sequencing primers for characterizing archaeal communities from deep-sea sediments and permafrost soils. The results show that primer pair Arch519/Arch915 (V4–V5 regions) produced the highest alpha diversity estimates, followed by Arch349f/Arch806r (V3–V4 regions) and A751f/AU1204r (V5–V7 regions) in both sample types. The archaeal taxonomic compositions and the relative abundance estimates of archaeal communities are influenced by the primer pairs. Beta diversity of the archaeal community detected by the three primer pairs reveals that primer pairs Arch349f/Arch806r and Arch519f/Arch915r are biased toward detection of Halobacteriales, Methanobacteriales and MBG-E/Hydrothermarchaeota, whereas the primer pairs Arch519f/Arch915r and A751f/UA1204r are biased to detect MBG-B/Lokiarchaeota, and the primers pairs Arch349f/Arch806r and A751f/UA1204r are biased to detect Methanomicrobiales and Methanosarcinales. The data suggest that the alpha and beta diversities of archaeal communities as well as the community compositions are influenced by the primer pair choice. This finding provides researchers with valuable experimental insight for selection of appropriate archaeal primer pairs to characterize archaeal communities.

Proto-dolomite formation in microbial consortia dominated by Halomonas strains Abstract Microbes can be found in hypersaline environments forming diverse populations with complex ecological interactions. Microbes in such environments were found to be involved in the formation of minerals including dolomite, a mineral of economic importance and whose origin has been long-debated. Various reports on in vitro experiments using pure cultures provided evidence for the microbial role in dolomite formation. However, culturing experiments have been limited in scope and do not fully address the possible interactions of the naturally occurring microbial communities; consequently, the ability of microbes as a community to form dolomite has been investigated in this study. Our experiments focused on examining the microbial composition by culturing aerobic heterotrophs from the top hypersaline sediments of Al-Khiran sabkha in Kuwait, a modern dolomite-forming environment. The objectives of this study were to assess the ability of two microbial consortia to form dolomite using enrichment culture experiments, mineralogy, and metagenomics. Proto-dolomite was formed by a microbial community dominated by Halomonas strains whereby degradation of the extracellular polymeric substances (EPS) was observed and the pH changed from 7.00 to 8.58. Conversely, proto-dolomite was not observed within a microbial community dominated by Clostridiisalibacter in which EPS continuously accumulated and the pH slightly changed from 7.00 to 7.29.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive Abstract A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L. Graphic Abstract

Diversity and functional profile of bacterial communities at Lancaster acid mine drainage dam, South Africa as revealed by 16S rRNA gene high-throughput sequencing analysis Abstract This study surveyed physicochemical properties and bacterial community structure of water and sediments from an acid mine drainage (AMD) dam in South Africa. High-throughput sequence analysis revealed low diversity bacterial communities affiliated within 8 dominant phyla; Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Nitrospirae, Proteobacteria, Saccharibacteria, and ca. TM6_(Dependentiae). Acidiphilium spp. which are common AMD inhabitants but rarely occur as dominant taxa, were the most abundant in both AMD water and sediments. Other groups making up the community are less common AMD inhabitants; Acidibacillus, Acidibacter, Acidobacterium, Acidothermus, Legionella, Metallibacterium, Mycobacterium, as well as elusive taxa (Saccharibacteria, ca. TM6_(Dependentiae) and ca. JG37-AG-4). Although most of the taxa are shared between sediment and water communities, alpha diversity indices indicate a higher species richness in the sediments. From canonical correspondence analysis, DOC, Mn, Cu, Cr, Al, Fe, Ca were identified as important determinants of community structure in water, compared to DOC, Ca, Cu, Fe, Zn, Mg, K, Mn, Al, sulfates, and nitrates in sediments. Predictive functional profiling recovered genes associated with bacterial growth and those related to survival and adaptation to the harsh environmental conditions. Overall, the study reports on a distinct AMD bacterial community and highlights sediments as microhabitats with higher species richness than water.

Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application Abstract Intracellular β-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC–MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0 kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular β-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0–5.5 and at 70 °C. The enzyme retained > 95% activity after heating to 70 °C for 1 h, or after incubation at pH 5.5 for 1 h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the β-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase Abstract Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In the present study, a novel glycoside hydrolase from Paenibacillus mucilaginosus (PmGH) was expressed in E. coli, purified and characterised. Functional analysis indicated that PmGH is a 130 kDa thermophilic multi-modular and multi-functional enzyme, comprising a GH5, a GH6 and two CBM3 domains and exhibiting cellulase, mannanase and xylanase activities. The enzyme displayed optimum hydrolytic activities at pH 6 and 60 °C and moderate thermostability. Homology modelling of the full-length protein highlighted the structural and functional novelty of native PmGH, with no close structural homologs identified. However, homology modelling of the individual GH5, GH6 and the two CBM3 domains yielded excellent models based on related structures from the Protein Data Bank. The catalytic GH5 and GH6 domains displayed a (β/α)8 and a distorted seven stranded (β/α) fold, respectively. The distinct homology at the domain level but low homology of the full-length protein suggests that this protein evolved by exogenous gene acquisition and recombination.

Cultivation technology development of Rhodothermus marinus DSM 16675 Abstract This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site Abstract Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.