Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Isolation of Saccharibacillus WB17 strain from wheat bran phyllosphere and genomic insight into the cellulolytic and hemicellulolytic complex of the Saccharibacillus genus.

The microorganisms living on the phyllosphere (the aerial part of the plants) are in contact with the lignocellulosic plant cell wall and might have a lignocellulolytic potential. We isolated a Saccharibacillus strain (Saccharibacillus WB17) from wheat bran phyllosphere and its cellulolytic and hemicellulolytic potential was investigated during growth onto wheat bran. Five other type strains from that genus selected from databases were also cultivated onto wheat bran and glucose. Studying the chemical composition of wheat bran residues by FTIR after growth of the six strains showed an important attack of the stretching C-O vibrations assigned to polysaccharides for all the strains, whereas the C = O bond/esterified carboxyl groups were not impacted. The genomic content of the strains showed that they harbored several CAZymes (comprised between 196 and 276) and possessed four of the fifth modules reflecting the presence of a high diversity of enzymes families. Xylanase and amylase activities were the most active enzymes with values reaching more than 4746 ± 1400 mIU/mg protein for the xylanase activity in case of Saccharibacillus deserti KCTC 33693 T and 452 ± 110 mIU/mg protein for the amylase activity of Saccharibacillus WB17. The total enzymatic activities obtained was not correlated to the total abundance of CAZyme along that genus. The Saccharibacillus strains harbor also some promising proteins in the GH30 and GH109 modules with potential arabinofuranosidase and oxidoreductase activities. Overall, the genus Saccharibacillus and more specifically the Saccharibacillus WB17 strain represent biological tools of interest for further biotechnological applications.

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.

Cohnella terricola sp. nov., isolated from soil.

A Gram-positive, aerobic, flagellated, endospore-forming, rod-shaped strain, designated as G13T, was isolated from soil. The results of 16S rRNA gene sequence analysis led to the conclusion that strain G13T was phylogenetically related to Cohnella boryungensis BR29T (97.5 %) and Cohnella phaseoli CECT 7287T (96.9 %) with digital DNA-DNA hybridization values of 21.0 and 21.4 %, and distantly related to Cohnella thermotolerans CCUG 47242T (94.8 %), type species of the genus Cohnella, at 19.0 %. The genome size of strain G13T was 5 387 258 bp, with 51.3 mol% G+C content. The predominant fatty acids were summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), anteiso-C17 : 0, iso-C17 : 0 and iso-C15 : 0. The predominant quinone was menaquinone-7 and the major polar lipids were diphosphatidyglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysylphosphatidylglycerol, three aminophospholipids, two phosphoglycolipids, three aminolipids and two unidentified lipids. Based on the data from phenotypic tests and the genotypic differences between strain G13T and its close phylogenetic relatives, strain G13T represents a new species belonging to the genus Cohnella, for which the name Cohnella terricola sp. nov. (=KACC 19905T=NBRC 113748T) is proposed.

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.

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.

Cohnella fermenti sp. nov., isolated from a fermentation process.

A novel Gram-stain-positive, aerobic, motile with peritrichous flagella, rod-shaped bacterium, designated CC-MHH1044T, was isolated from a preserved vegetable sample. A polyphasic taxonomic approach was applied to the isolates in order to clarify its taxonomic position. Growth of the strain CC-MHH1044T occurred at 15-50 °C (optimum, 30 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-2.0 % (w/v) NaCl (optimum, 1 %, w/v). The genome of strain CC-MHH1044T consisted of 8.5 Mb and the genomic DNA G+C content was 58.5 mol%. Comparison of the 16S rRNA gene sequences showed that CC-MHH1044T belonged to the genus Cohnella and showed a close relationship with the type strains of Cohnella damuensis (96.2 %) and Cohnella panacarvi (95.9 %), and lower sequence similarity to other species. Average nucleotide identity values calculated from whole-genome sequencing data proved that CC-MHH1044T represents a distinct Cohnella species. The dominant cellular fatty acids (>5 %) included iso-C14 : 0(7.4 %), iso-C15 : 0 (6.4 %), anteiso-C15 : 0(40.3 %), C16 : 0 (6.6 %) and iso-C16 : 0 (27.0 %). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids, one unidentified phospholipid and glycolipid. The major polyamine was spermidine. The predominant isoprenoid quinone was menaqinone 7 (MK-7). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits, together with results of comparative 16S rRNA gene sequence, average nucleotide identity and digital DNA-DNA hybridization analyses, we conclude that strain CC-MHH1044T represents a novel member of the genus Cohnella, for which the name Cohnella fermenti sp. nov. is proposed. The type strain is CC-MHH1044T (=BCRC 81147T=JCM 32834T).

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.

Saccharibacillus brassicae sp. nov., an endophytic bacterium isolated from kimchi cabbage (Brassica rapa subsp. pekinensis) seeds.

Strain ATSA2T was isolated from surface-sterilized kimchi cabbage (Brassica rapa subsp. pekinensis) seeds and represents a novel bacterium based on the polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ATSA2T formed a lineage within genus Saccharibacillus and was most closely to Saccharibacillus deserti WLG055T (98.1%) and Saccharibacillus qing-shengii H6T (97.9%). The whole-genome of ATSA2T comprised a 5,619,468 bp of circular chromosome with 58.4% G + C content. The DNA-DNA relatedness values between strain ATSA2T and its closely related type strains S. deserti WLJ055 and S. qingshengii H6T were 26.0% and 24.0%, respectively. Multiple gene clusters associated with plant growth promotion activities (stress response, nitrogen and phosphorus metabolism, and auxin biosynthesis) were annotated in the genome. Strain ATSA2T was Gram-positive, endospore-forming, facultatively anaerobic, and rod-shaped It grew at 15-37°C (optimum 25°C), pH 6.0-10.0 (optimum pH 8.0), and in the presence of 0-5% (w/v) NaCl (optimum 1%). The major cellular fatty acids (> 10%) of strain ATSA2T were anteiso-C15:0 and C16:0. MK-7 was the major isoprenoid quinone. The major polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, and three unknown glycolipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxo-nomic features, strain ATSA2T is proposed to represent a novel species of genus Saccharibacillus, for which the name is Saccharibacillus brassicae sp. nov. The type strain is ATSA2T (KCTC 43072T = CCTCC AB 2019223T).

Application of osmotic challenge for enrichment of microbial consortia in polyhydroxyalkanoates producing thermophilic and thermotolerant bacteria and their subsequent isolation.

Polyhydroxyalkanoates are microbial polyesters which are considered being biological alternatives to petrochemical polymers. Extremophiles, such as thermophilic PHA producers, hold promise to improve competitiveness of PHA production process. Therefore, this work aimed at isolation of new strains, which could produce PHA under elevated temperature. Since traditional Nile red staining of colonies provided false positive results in thermophiles, we developed a novel strategy of enriching microbial consortia by PHA producers. This so called "osmoselective strategy" is based on application of osmotic challenge by sudden exposition of the mixed microbial culture to hypertonic and subsequently to hypotonic conditions; moreover, this strategy relies on the fact that PHA protect bacteria from negative effects of rapid fluctuations in osmotic pressure. In combination with fast and reliable ATR-FTIR inspection of selected colonies for presence of PHA, we were able to isolate several promising thermophilic or thermotolerant PHA producing strains belonging to the genera Bacillus, Aneurinibacillus and Chelatococcus, which indeed deserves further investigation to evaluate their potential for industrial production of PHA.

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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Longirhabdus pacifica gen. nov., sp. nov., isolated from a deep-sea hydrothermal sediment in the West Pacific Ocean.

A novel Gram-stain-negative bacterium, designated as SCSIO 06110T, was isolated from a deep-sea sediment of the West Pacific Ocean. Cells were 0.5-0.8 µm in width and 3.0-4.0 µm in length, spore-forming, rod-shaped with peritrichous flagella. Positive for catalase and urease, negative for oxidase and nitrate reduction. Growth occurred at 15-37 °C, pH 6-9 and 1-5 % (w/v) NaCl, with optimum growth at 28 °C, pH 7 and 3 % (w/v) NaCl. MK-7 was the only menaquinone. The strain possessed diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids. Iso-C16 : 0, iso-C15 : 0 and iso-C14 : 0 were the major fatty acids. The novel isolate clustered with genera in the family Paenibacillaceae, but formed a separated branch with the closest relative Chengkuizengella sediminis J15A17T (91.1 % sequence similarity) when compared in a phylogenetic analysis of 16S rRNA gene sequences. The DNA G+C content of strain SCSIO 06110T was 38.5 mol%. Based on the polyphasic data presented, a new genus, Longirhabdus gen. nov., is proposed in the family Paenibacillaceae with the type species Longirhabdus pacifica sp. nov. and the type strain SCSIO 06110T (=DSM 105158T=CGMCC 1.16550T).

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).

Thermoflavimicrobium daqui sp. nov., a thermophilic microbe isolated from Moutai-flavour Daqu.

Moutai-flavour Daqu is an important starter to support growth of microorganisms in the fermented process of Moutai-flavour liquor. A novel thermophilic microorganism, designated strain FBKL4.011T, was isolated from Moutai-flavour Daqu samples collected from Guotai distillery in Renhuai, Guizhou province, south-west China. The strain could grow at 45-65 °C (optimum 45 °C). Based on polyphasic analysis, strain FBKL4.011T was affiliated to the genus Thermoflavimicrobium. It formed abundant pale-yellow aerial and substrate mycelium, bearing single endospores (7.0-10.0 µm diameter) on branched long sporophores (5.0 µm diameter). The cell-wall peptidoglycan contained meso-diaminopimelic acid; ribose, glucose and mannose were the primary whole-cell sugars. The major fatty acids were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. The predominant menaquinone were MK-8 and MK-9. The polar phospholipids contained diphosphatidyl glycerol, phosphatidyl ethanolamine, one unidentified phospholipid and one unidentified lipid. The G+C content of the genome was 43.1 mol%. According to the 16S rRNA gene sequence analysis, strain FBKL4.011T was closely related to Thermoflavimicrobium dichotomicum JCM 9688T (95.3 % sequence similarity), and other members within the family Thermoactinomycetaceae (less than 93.0 % sequence similarity). The DNA-DNA hybridisation data showed low relatedness between strain FBKL4.011T and T. dichotomicum JCM 9688T, Laceyella sacchari KCTC 9790T, Laceyella tengchongensis YIM 10002T, Laceyella sediminis RHA1T(36.7±1.1 %, 30.0±1.2 %, 21.3±2.1 % % and 37.6±0.9 %, respectively). Based on data from the polyphasic analysis, strain FBKL4.011T is considered to represent a novel species of the genus Thermoflavimicrobium, for which the name Thermoflavimicrobium daqui sp. nov. is proposed. The type strain is FBKL4.011T(=KCTC 43036T=CICC 24504T).

Chengkuizengella marina sp. nov., isolated from deep-sea sediment of the Pacific Ocean.

A taxonomic study was carried out on strain YPA3-1-1T, which was isolated from deep-sea sediment of the Pacific Ocean. The bacterium was Gram-stain-positive, oxidase-positive, catalase-negative, rod-shaped and spore-forming. Growth was observed at salinities of 1.0-6.0 % and at temperatures of 10-40 °C. The isolate could degrade gelatin and aesculin. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YPA3-1-1T belonged to the genus Chengkuizengella, with the highest sequence similarity to the only typespecies, Chengkuizengella sediminis J15A17T (98.5 %). The estimated average nucleotide identity and DNA-DNA hybridization values between strain YPA3-1-1T and C. sediminis J15A17T were 88.1 and 35.0 %, respectively. The cell wall of strain YPA3-1-1T contained meso-diaminopimelic acid. The principal fatty acids (>10 %) were iso-C16 : 0 (35.5 %) and anteiso-C15 : 0 (17.5 %). The G+C content of the chromosomal DNA was 33.1 mol%. The respiratory quinone was determined to be MK-7 (100 %). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, glycolipid and three unidentified phospholipids. The combined genotypic and phenotypic data show that strain YPA3-1-1T represents a novel species within the genus Chengkuizengella, for which the name Chengkuizengella marina sp. nov. is proposed, with the type strain YPA3-1-1T (=MCCC 1A14042T=KCTC 43019T).

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 endophytica sp. nov., a novel endophytic bacterium isolated from bark of Sonneratia apetala.

A Gram-stain-positive, aerobic, rod-shaped, endospore-forming bacterium, designated strain M2MS4P-1T, was isolated from surface-sterilized bark of Sonneratiaapetala sampled in Guangxi, China. The bacterium was characterized by a polyphasic approach to determine its taxonomic position. 16S rRNA gene sequence comparisons revealed that strain M2MS4P-1T belonged to the genus Cohnella and was most closely to Cohnella luojiensis HY-22RT (98.4 % similarity). The average nucleotide identity value and estimated DDH value between strain M2MS4P-1T and the type strain of C. luojiensis HY-22RT were 79.2 and 20.1 %, respectively. Neither substrate nor aerial mycelia were formed, and no diffusible pigments were observed on the media tested. Strain M2MS4P-1T grew in the pH range 6.0-9.0 (optimum, pH 7.0-8.0), at temperatures between 10-37 °C (30 °C) and in 0-1 % (w/v) NaCl (0 %). The predominant isoprenoid quinone in strain M2MS4P-1T was menaquinone-7. The major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysyl-phosphatidylglycerol, four unidentified aminophospholipids and two unidentified phospholipids. The DNA G+C content was 51.5 mol%. According to the phylogenetic, phenotypic and chemotaxonomic evidence, strain M2MS4P-1T was clearly distinguishable from other species with validly published names in the genus Cohnella and should therefore be classified as a novel species, for which we suggest the name Cohnellaendophytica sp. nov. The type strain is M2MS4P-1T (=KCTC 43011T=CGMCC 1.13745T).

Tracking the sources of psychrotrophic bacteria contaminating chicken cuts during processing.

The major aim of the study was to establish the routes via which spoilage associated psychrotrophic bacteria contaminate poultry products at a large processing plant located in Belgium. Environmental samples were collected consisting of samples of air and swabs of food contact surfaces. Product samples were also collected consisting of modified atmosphere packaged (MAP) chicken wings and legs, which were analyzed microbiologically on the same day they were produced as well as after their sell-by date. Psychrotrophic bacteria from these samples were subsequently clustered and identified by means of MALDI-TOF MS and 16S rRNA gene sequencing. Carnobacterium maltaromaticum was determined to dominate the spoilage flora of both wings and legs. Other psychrotrophic bacteria able to grow on MRS which were identified on expired wings and legs included Carnobacterium divergens, Brocothrix thermosphacta, Lactobacillus curvatus, and Lactobacillus brevis. These were determined to arise from food contact surfaces such as cutting blades, leg hooks, Ertalon and polyurethane conveyor belts, working tables, and the hands of the operators. Importantly, it was determined that cleaning and disinfection was largely inadequate. Air was also determined to be an important vector of psychrotrophic bacteria in the processing environment, potentially contaminating the products directly or indirectly.