Prediction and validation of novel SigB regulon members in Bacillus subtilis and regulon structure comparison to Bacillales members.

BACKGROUND: Sigma factor B (SigB) is the central regulator of the general stress response in Bacillus subtilis and regulates a group of genes in response to various stressors, known as the SigB regulon members. Genes that are directly regulated by SigB contain a promotor binding motif (PBM) with a previously identified consensus sequence. RESULTS: In this study, refined SigB PBMs were derived and different spacer compositions and lengths (N12-N17) were taken into account. These were used to identify putative SigB-regulated genes in the B. subtilis genome, revealing 255 genes: 99 had been described in the literature and 156 genes were newly identified, increasing the number of SigB putative regulon members (with and without a SigB PBM) to > 500 in B. subtilis. The 255 genes were assigned to five categories (I-V) based on their similarity to the original SigB consensus sequences. The functionalities of selected representatives per category were assessed using promoter-reporter fusions in wt and ΔsigB mutants upon exposure to heat, ethanol, and salt stress. The activity of the PrsbV (I) positive control was induced upon exposure to all three stressors. PytoQ (II) showed SigB-dependent activity only upon exposure to ethanol, whereas PpucI (II) with a N17 spacer and PylaL (III) with a N16 spacer showed mild induction regardless of heat/ethanol/salt stress. PywzA (III) and PyaaI (IV) displayed ethanol-specific SigB-dependent activities despite a lower-level conserved - 10 binding motif. PgtaB (V) was SigB-induced under ethanol and salt stress while lacking a conserved - 10 binding region. The activities of PygaO and PykaA (III) did not show evident changes under the conditions tested despite having a SigB PBM that highly resembled the consensus. The identified extended SigB regulon candidates in B. subtilis are mainly involved in coping with stress but are also engaged in other cellular processes. Orthologs of SigB regulon candidates with SigB PBMs were identified in other Bacillales genomes, but not all showed a SigB PBM. Additionally, genes involved in the integration of stress signals to activate SigB were predicted in these genomes, indicating that SigB signaling and regulon genes are species-specific. CONCLUSION: The entire SigB regulatory network is sophisticated and not yet fully understood even for the well-characterized organism B. subtilis 168. Knowledge and information gained in this study can be used in further SigB studies to uncover a complete picture of the role of SigB in B. subtilis and other species.

Development of a database and standardized approach for rpoB sequence-based subtyping and identification of aerobic spore-forming Bacillales.

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB-based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy, the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.

A new role for SR1 from Bacillus subtilis: regulation of sporulation by inhibition of kinA translation.

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.

Genome analysis of the salt-resistant Paludifilum halophilum DSM 102817T reveals genes involved in flux-tuning of ectoines and unexplored bioactive secondary metabolites.

Paludifilum halophilum DSM 102817T is the first member of the genus Paludifilum in the Thermoactinomycetaceae family. The thermohalophilic bacterium was isolated from the solar saltern of Sfax, Tunisia and was shown to be able to produce ectoines with a relatively high-yield and to cope with salt stress conditions. In this study, the whole genome of P. halophilum was sequenced and analysed. Analysis revealed 3,789,765 base pairs with an average GC% content of 51.5%. A total of 3775 genes were predicted of which 3616 were protein-coding genes and 73 were RNA genes. The genes encoding key enzymes for ectoines (ectoine and hydroxyectoine) synthesis (ectABCD) were identified from the bacterial genome next to a gene cluster (ehuABCD) encoding a binding-protein-dependent ABC transport system responsible for ectoines mobility through the cell membrane. With the aid of KEGG analysis, we found that the central catabolic network of P. halophilum comprises the pathways of glycolysis, tricarboxylic acid cycle, and pentose phosphate. In addition, anaplerotic pathways replenishing oxaloacetate and glutamate synthesis from central metabolism needed for high ectoines biosynthetic fluxes were identified through several key enzymes. Furthermore, a total of 18 antiSMASH-predicted putative biosynthetic gene clusters for secondary metabolites with high novelty and diversity were identified in P. halophilum genome, including biosynthesis of colabomycine-A, fusaricidin-E, zwittermycin A, streptomycin, mycosubtilin and meilingmycin. Based on these data, P. halophilum emerged as a promising source for ectoines and antimicrobials with the potential to be scaled up for industrial production, which could benefit the pharmaceutical and cosmetic industries.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol-1, -76.8 kJ.mol-1.°K-1, and 84.1 kJ.mol-1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

Draft genome sequencing of Sporolactobacillus terrae SBT-1, an efficient bacterium to ferment concentrated sugar to D-lactic acid.

Recently, the industrial-scale development of microbial D-lactic acid production has been discussed. In this study, the efficiency of the new isolate Sporolactobacillus terrae SBT-1 for producing D-lactic acid under challenge conditions was investigated. The isolate SBT-1 exhibited superior activity in fermenting a very high glucose or sucrose concentration to D-lactic acid compared to the other S. terrae isolates previously reported in the literature; therefore, SBT-1 could overcome the limitations of effective lactic acid production. In batch cultivation using 360 g/L glucose, SBT-1 produced 290.30 g/L D-lactate with a sufficiently high glucose conversion yield of 86%, volumetric productivity of 3.02 g/L h, and optical purity of 96.80% enantiomer excess. SBT-1 could also effectively utilize 440 g/L sucrose as a sole carbon source to produce 276.50 g/L lactic acid with a conversion yield of 90%, a production rate of 2.88 g/L h, and an optical purity of 98%. D-Lactic acid fermentation by two other related producers, S. inulinus NRIC1133T and S. terrae NRIC0357T, was compared with fermentation by isolate SBT-1. The experimental data revealed that SBT-1 possessed the ability to ferment relatively high glucose or sucrose concentrations to D-lactic acid without obvious catabolite repression and byproduct formation compared to the two reference strains. In draft genome sequencing of S. terrae SBT-1, the results provided here can promote further study to overcome the current limitations for the industrial-scale production of D-lactic acid.

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Characterization of extracellular and cell bound biosurfactants produced by Aneurinibacillus aneurinilyticus isolated from commercial corn steep liquor.

The presence of biosurfactants produced by a Bacillus strain in corn steep liquor (CSL), a wastewater stream of the corn milling process, has been recently discovered. However, the species responsible for their production has not been identified at the moment. Therefore, in this work, the Bacillus strain isolated from CSL, with capacity to produce biosurfactants, was subjected to amplification and sequence analysis of the 16S rRNA, being identified as Aneurinibacillus aneurinilyticus. This strain has been proved to be endospore forming and thermophile, what would explain its presence in the commercial CSL. It was observed that the strain under evaluation has the ability to produce both cell-bound and extracellular biosurfactant extracts, which were characterized in this work. The electrospray ionization mass spectrometry (ESI) analysis of the biosurfactant extracts revealed that the extracellular biosurfactant produced by Aneurinibacillus aneurinilyticus is composed by a mixture of lipopeptides, containing C16 and C18 fatty acids and amino acids, including valine, phenylalanine, proline, cysteine, histidine, aspartic acid/asparagine, alanine, glycine, leucine/isoleucine, with biomarkers between 1025-458 m/z. Conversely, the cell-bound biosurfactant extract produced by Aneurinibacillus aneurinilyticus was composed by the cyclic decapeptide gramicidin S, with a characteristic peak at 571 m/z, and lipopeptides with characteristic peaks between 1034-705 m/z, containing alanine, glycine, cysteine, serine, proline, aspartic acid/asparagine, similarly to the amino acid sequence of the extracellular biosurfactant extract.

Discovery of thermophilic Bacillales using reduced-representation genotyping for identification.

BACKGROUND: This study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome. RESULTS: Based on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases. CONCLUSIONS: Previously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.

Utilization of methyltrioctylammonium chloride as new ionic liquid in pretreatment of sugarcane bagasse for production of cellulase by novel thermophilic bacteria.

Fermentation of carbohydrates present in lignocellulosic (LC) biomass is facilitated by lignin removal, which is usually achieved by adopting various pretreatment methods to provide the enzymes proper access to their respective substrates. Pretreatment using ionic liquid (IL) is relatively recent advancement and considered as mild and green process. ILs can dissolve extensive quantities of biomass and depolymerize the cellulose. In this context, an abundantly available LC biomass, sugarcane bagasse (SB), was pretreated using alkali or with an IL, methyltrioctylammonium chloride, and was used for cellulase production from thermophilic bacteria. In all, 26 indigenously isolated thermophilic bacterial strains were quantitatively screened for cellulase production. 16S rDNA sequences of the promising isolates UE10 and UE27 revealed relatedness with Brevibacillus borstelensis, while the strain UE1 belonged to Aneurinibacillus thermoaerophilus. Cellulase production was compared by utilizing alkali pretreated and IL pretreated SB and the later was found more appropriate. UE1, UE10 and UE27 yielded 22.2, 22.18 and 33.3 IU mL-1 of endoglucanase, respectively, by fermenting IL pretreated SB. The changes in SB structure after pretreatment were evaluated by scanning electron microscopy. This study demonstrated the potential of novel thermophilic bacterial strains to utilize IL pretreated SB for production of industrially important enzyme, cellulase.

Shimazuella alba sp. nov. isolated from desert soil and emended description of the genus Shimazuella Park et al. 2007.

A novel, Gram-stain-positive bacterium, designated KC615T, was isolated from desert soil which was collected from the Karakum Desert, Turkmenistan. Phylogenetic analysis based on an almost-complete 16S rRNA gene sequence showed that isolate KC615T formed a monophyletic clade with Shimazuella kribbensis KCTC 9933T, sharing 98.2% similarity and polyphasic taxonomic studies confirmed the affiliation of the strain to the genus Shimazuella. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Whole-cell hydrolysates contained ribose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and hydroxy-phosphatidylethanolamine. The predominant menaquinones (> 10%) were MK-9(H4) and MK-10(H4). Major fatty acids were anteiso-C15:0, C20:0 and C18:0. The genomic DNA G + C content observed for strain KC615T was 38.5 mol%. Based on 16S rRNA gene similarity, DNA-DNA hybridization value, chemotaxonomic characteristics and differential physiological properties, strain KC615T is considered to represent a novel species within the genus Shimazuella, for which the name Shimazuella alba sp. nov. is proposed. The type strain is KC615T (= JCM 33532T = CGMCC 4.7616T).

Regulatory relationship between quality variation and environment of Cistanche deserticola in three ecotypes based on soil microbiome analysis.

The environment affects the composition and function of soil microbiome, which indirectly influences the quality of plants. In this study, 16S amplicon sequencing was used to reveal the differences in soil microbial community composition of Cistanche deserticola in three ecotypes (saline-alkali land, grassland and sandy land). Through the correlation analysis of microbial community abundance, phenylethanoid glycoside contents and ecological factors, the regulatory relationship between microbial community and the quality variation of C. deserticola was expounded. The metabolic function profile of soil microbiome was predicted using Tax4Fun. Data showed that the soil microbial communities of the three ecotypes were significantly different (AMOVA, P < 0.001), and the alpha diversity of grassland soil microbial community was the highest. Core microbiome analysis demonstrated that the soil microbial communities of C. deserticola were mostly have drought, salt tolerance, alkali resistance and stress resistance, such as Micrococcales and Bacillales. The biomarkers, namely, Oceanospirillales (saline-alkali land), Sphingomonadales (grassland) and Propionibacteriales (sandy land), which can distinguish three ecotype microbial communities, were excavated through LEfSe and random forest. Correlation analysis results demonstrated that 2'-acetylacteoside is positively correlated with Oceanospirillales in saline-alkali land soil. The metabolic function profiles displayed highly enriched metabolism (carbohydrate and amino acid metabolisms) and environmental information processing (membrane transport and signal transduction) pathways. Overall, the composition and function of soil microbiomes were found to be important factors to the quality variation of C. deserticola in different ecotypes. This work provided new insight into the regulatory relationship amongst the environment, soil microbial community and plant quality variation.

Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination.

Biosynthesizing unnatural chiral amino acids is challenging due to the limited reductive amination activity of amino acid dehydrogenase (AADH). Here, for the asymmetric synthesis of l-phosphinothricin from 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), a glutamate dehydrogenase gene (named GluDH3) from Pseudomonas monteilii was selected, cloned and expressed in Escherichia coli (E. coli). To boost its activity, a "two-step"-based computational approach was developed and applied to select the potential beneficial amino acid positions on GluDH3. l-phosphinothricin was synthesized by GluDH-catalyzed asymmetric amination using the d-glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration. Using lyophilized E. coli cells that co-expressed GluDH3_V375S and EsGDH, up to 89.04 g L-1 PPO loading was completely converted to l-phosphinothricin within 30 min at 35 °C with a space-time yield of up to 4.752 kg·L-1·d-1. The beneficial substitution V375S with increased polar interactions between K90, T193, and substrate PPO exhibited 168.2-fold improved catalytic efficiency (kcat/KM) and 344.8-fold enhanced specific activity. After the introduction of serine residues into other GluDHs at specific positions, forty engineered GluDHs exhibited the catalytic functions of "glufosinate dehydrogenase" towards PPO.

Gorillibacterium timonense sp. nov., isolated from an obese patient.

A Gram-negative and facultative anaerobic bacterium, designated strain SN4T, was isolated from the stool sample of an obese Amazonian patient. The new isolate was characterized by the taxonogenomics approach. The strain SN4T was beige-colored, circular and not haemolytic. Cells are rod shaped and motile with several flagella. Strain SN4T grows optimally at pH 7 and can survive in the presence of a saline concentration of up to 75 g/l NaCl. The 16S ribosomal RNA gene sequence analysis of the novel strain SN4T showed 95.28% similarity in nucleotide sequence with Gorillibacterium massiliense G5T, the phylogenetically closest neighbor and the type species of this genus. Anteiso-C15:0, iso-C15:0 and C16:0 were found as the major components in the cellular fatty acid analysis of this isolate. The genomic draft of strain SN4T is 5,263,742 bp long with 53.33% of G+C content. The differences in physiological, biochemical characteristics and phylogenetic and genomic data make it possible to clearly distinguish the strain SN4T from G. massiliense G5T. Based on the taxonogenomic description and the phenotypic and biochemical characteristics of this bacterium presented in this article, we propose the SN4T strain (= CSUR P2011 = DSM 100,698) as a new species, Gorillibacterium timonense sp. nov.

Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.

Phylogenetic diversity of aerobic spore-forming Bacillalles isolated from Brazilian soils

The phylum Firmicutes comprises seven classes where most species are either aerobic or anaerobic endospore former. Inside Firmicutes, species allocated in the genus Bacillus and related genera are collectively named aerobic endospore-forming bacteria (AEFB), and the soil is their major reservoir. AEFB have great importance in health, agriculture, and biotechnology although the more studied species are Bacillus subtilis and the human pathogens Bacillus cereus and Bacillus anthracis. AEFB have great importance in health, agriculture, and biotechnology; although the knowledge about these organisms is based on few species, notably, Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. In this work, we generated partial 16S rRNA gene sequences of both strands of 192 AEFB strains isolated from soils of Distrito Federal, Brazil (SDF strains). The resulting consensus sequences were used to obtain taxonomic assignment and establish the phylogenetic relationships among these strains. Through this approach, we could observe that classified SDF strains were distributed among genera Bacillus (169 strains; 88.02%), Paenibacillus (11; 5.73%), Lysinibacillus (6; 3.13%), Brevibacillus (4; 2.08%), Terribacillus (1; 0.52%), and Rummeliibacillus (1; 0.52%). Phylogenetic trees revealed these 192 SDF strains can be segregated into eight groups spanning families Bacillaceae and Paenibacillaceae belonging to the order Bacillales. To expand the knowledge about the diversity of these SDF strains, further studies regarding characterization with different methodologies are underway

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Cohnella abietis sp. nov., isolated from Korean fir (Abies koreana) rhizospheric soil of Halla mountain.

A strictly aerobic, motile, endospore-forming, rod-shaped bacterium, designated HS21T, was isolated from rhizospheric soil of the Korean fir tree (Abies koreana) from Halla mountain on Jeju island, Korea. Growth of strain HS21T was observed at pH 6.0-8.0 (optimum: pH 7.0), 0-2% (w/v) NaCl and 4-30°C (optimum: 25°C). A comparative analysis of 16S rRNA gene sequences showed that strain HS21T was most closely related to Cohnella luojiensis HY-22RT (97.6%), followed by C. lupini RLAHU4BT (97.4%) and C. collisoli NKM-5T (97.2%). The genome of strain HS21T comprised a circular chromosome of 7,059,027 bp with 44.8% G + C content. The DNA-DNA relatedness values between strain HS21T and C. luojiensis HY-22RT and C. lupini RLAHU4BT were 18.1% and 13.8%, respectively. The major cellular fatty acids (> 5%) of the isolate were anteiso-C15:0, iso-C16:0, C16:0, and iso-C15:0. The polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, and three unidentified aminophospholipids. Based on its phenotypic, phylogenetic, genomic, and chemotaxonomic properties, strain HS21T represents a novel species of the genus Cohnella, for which the name Cohnella abietis sp. nov. is proposed. The type strain is HS21T (= KCTC 43028T = CCTCC AB 2019010T).

Phylogenetic diversity of aerobic spore-forming Bacillalles isolated from Brazilian soils.

The phylum Firmicutes comprises seven classes where most species are either aerobic or anaerobic endospore former. Inside Firmicutes, species allocated in the genus Bacillus and related genera are collectively named aerobic endospore-forming bacteria (AEFB), and the soil is their major reservoir. AEFB have great importance in health, agriculture, and biotechnology although the more studied species are Bacillus subtilis and the human pathogens Bacillus cereus and Bacillus anthracis. AEFB have great importance in health, agriculture, and biotechnology; although the knowledge about these organisms is based on few species, notably, Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. In this work, we generated partial 16S rRNA gene sequences of both strands of 192 AEFB strains isolated from soils of Distrito Federal, Brazil (SDF strains). The resulting consensus sequences were used to obtain taxonomic assignment and establish the phylogenetic relationships among these strains. Through this approach, we could observe that classified SDF strains were distributed among genera Bacillus (169 strains; 88.02%), Paenibacillus (11; 5.73%), Lysinibacillus (6; 3.13%), Brevibacillus (4; 2.08%), Terribacillus (1; 0.52%), and Rummeliibacillus (1; 0.52%). Phylogenetic trees revealed these 192 SDF strains can be segregated into eight groups spanning families Bacillaceae and Paenibacillaceae belonging to the order Bacillales. To expand the knowledge about the diversity of these SDF strains, further studies regarding characterization with different methodologies are underway.

Cohnella candidum sp. nov., radiation-resistant bacterium from soil.

A Gram-stain positive, endospore-forming, circular, convex, cream colored, designated strain 18JY8-7T, was isolated from soil collected in Jeju Island, South Korea. Phylogenetic analysis using 16S rRNA gene sequences showed that strain 18JY8-7T formed a distinct lineage within the family Paenibacillaceae (order Bacillales, class Bacilli), and is closely related to Cohnella rhizosphaerae (96.1%, sequence similarity) and Cohnella xylanilytica (96.0%). Optimal growth occurred at 30 °C, pH 6.5 and in the absence of NaCl. The predominant cellular fatty acids were anteiso-C15:0 and iso-C16:0. The major respiratory quinone was MK-7. The polar lipids profile comprised of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The DNA G + C content was 57.0 mol %. The genotypic and phenotypic analyses revealed the differentiation of strain 18JY8-7T from all recognized Cohenella species. The strain 18JY8-7T, therefore represents a novel bacterial species within the family Paenibacillaceae, for which the name Cohnella candidum sp. nov. is proposed. The type strain is 18JY8-7T (= KCTC 33969T = JCM 33199T).