Genomic characterization of a novel ureolytic bacteria, Lysinibacillus capsici TSBLM, and its application to the remediation of acidic heavy metal-contaminated soil.

Soil heavy metal contamination is an essential challenge in ecological and environmental management, especially for acidic soils. Microbially induced carbonate precipitation (MICP) is an effective and environmentally friendly remediation technology for heavy metal contaminated sites, and one of the key factors for its realization lies in the microorganisms. In this study, Lysinibacillus capsici TSBLM was isolated from heavy metal contaminated soil around a gold mine, and inferred to be a novel ureolytic bacteria after phylogenomic inference and genome characterization. The urease of L. capsici TSBLM was analyzed by genetic analysis and molecular docking, and further applied this bacteria to the remediation of Cu and Pb in solution and acidic soils to investigate its biomineralization mechanism and practical application. The results revealed L. capsici TSBLM possessed a comprehensive urease gene cluster ureABCEFGD, and the encoded urease docked with urea at the lowest binding energy site (ΔG = -3.43 kcal/mol) connected to three amino acids threonine, aspartic, and alanine. The urease of L. capsici TSBLM is synthesized intracellularly but mainly functions extracellularly. L. capsici TSBLM removes Cu/Pb from the solution by generating heavy metal carbonates or co-precipitating with CaCO3 vaterite. For acidic heavy metal-contaminated soil, the carbonate-bound states of Cu and Pb increased significantly from 7 % to 16 % and from 23 % to 35 % after 30 days by L. capsici TSBLM. Soil pH improved additionally. L. capsici TSBLM maintained the dominant status in the remediated soil after 30 days, demonstrating good environmental adaptability and curing persistence. The results provided new strain resources and practical application references for the remediation of acidic heavy metal contaminated soil based on MICP.

Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications.

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

Short communication: Investigating optimal laboratory growth conditions of Gracilibacillus halotolerans in media supplemented with salt.

Gracilibacillus halotolerans, a new and relatively unstudied extremophile, extracted from the Great Salt Lake USA, survives in an extreme saline environment. Uncovering optimal laboratory growth conditions can be useful to improve treatment strategies against antibiotic resistance and biofilm formation. In the current study, G. halotolerans growth optimization was tested to determine the ideal saline concentration. In addition, a variety of G. halotolerans'-derived survival strategies were reviewed. The major findings of the current study includes the optimal laboratory growth condition for G. halotolerans that requires the supplement of 5% NaCl. In addition, optimal growth was observed up to 72 h in Luria Bertani (LB) broth. Identifying the optimal laboratory growth conditions for G. halotolerans will standardize growth methods, reduce laboratory cost, and can improve future investigations of extremophile bacteria as model organisms to combat antibiotic resistance, biofilm, and other persister cell characteristics that negatively affect research and clinical settings.

Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822T.

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H2 O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2 O2 , suggest it has potential for biotechnological applications.

Genome-based reclassification of Bacillus acidicola, Bacillus pervagus and the genera Heyndrickxia, Margalitia and Weizmannia.

In the present study, the taxonomic positions of Bacillus acidicola, Bacillus pervagus and members of the genera Heyndrickxia, Margalitia and Weizmannia were evaluated. The 16S rRNA gene sequence similarity between Bacillus acidicola DSM 14745T, Bacillus pervagus DSM 23947T and members of the genera Heyndrickxia and Margalitia were above the cut-off level (>95 %) for genus delineation. Amino acid identity (AAI) values and the results of phylogenomic analysis suggested that B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia belong to the same genus. Furthermore, the AAI and phylogenomic results also differentiate B. pervagus from B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia. Based on the results, we propose to transfer Bacillus acidicola, Margalitia and Weizmannia to the genus Heyndrickxia. We also propose the reclassification of B. pervagus into a new genus Oikeobacillus gen. nov., with the type species Oikeobacillus pervagus comb. nov.

Identification and characterization of a novel bacteriocin gene cluster in Lysinibacillus boronitolerans.

Although the antibiotics inhibit or kill pathogens, the abuse leads to the resistance formation and even "Super Bacteria." Therefore, it is urgent to explore the natural and safe alternatives such as bacteriocin. In this study, an uncharacterized bacteriocin gene cluster for Lysinibacillus boronitolerans was first predicted by genome sequencing and bioinformatics analysis, of which including two biosynthetic genes, a regulatory gene, a transport-related gene, and six other genes. Subsequently, the 10.24-kb gene cluster was expressed in Escherichia coli BL21, and the lysate effectively inhibited the growths of pathogenic bacteria containing Bacillus pumilus, Bacillus velezensis, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas axonopodis pv. manihotis. The antibacterial substance was purified by 70% ammonium sulfate precipitation and further identified by liquid chromatography-tandem mass spectrometry. The results showed that the antibacterial substance consisted of 44 amino acids and had 24.1% sequence identity with the cyanobacterin Piricyclamide 7005 E4 PirE4, a bacteriocin analogue. The minimal set of genes required for the biosynthesis of the antibacterial substance was determined by site-directed mutagenesis, suggesting both a transcriptional repressor and a phosphohydroxythreonine transaminase were essential. Subsequently, the evolution and conservation of the two proteins were analyzed among 22 Lysinibacillus species. Among them, the residues responsible for functions were identified. Collectively, our results set a solid foundation for investigation of the biosynthesis and application of bacteriocin.

Proposal to transfer Bacillus dafuensis and Bacillus massiliigabonensis to the genus Cytobacillus as Cytobacillus dafuensis comb. nov., and Cytobacillus massiliigabonensis comb. nov., respectively.

In the present study, the taxonomic position of Bacillus dafuensis and Bacillus massiliigabonensis was evaluated using genome-based comparison. The 16S rRNA gene sequence obtained from the Bacillus dafuensis FJAT-25496T genome showed 99.7% similarity with the type strain of Cytobacillus citreus, while Bacillus massiliigabonensis Marseille-P2639T showed 98.7% similarity with the type species of Cytobacillus solani. The 16S rRNA gene sequence similarity of Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T with Cytobacillus members was above the threshold (94.5%) for genus delineation. In phylogenetic (based on 16S rRNA gene sequences) and phylogenomic (based on 71 bacterial single-copy genes) trees, Bacillus dafuensis and Bacillus massiliigabonensis clustered with Cytobacillus members. The 16S rRNA gene sequence, amino acid identity and percentage of conserved proteins analysis indicated Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T as a member of the genus Cytobacillus. The digital DNA-DNA hybridization and the average nucleotide identity values of Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T with Cytobacillus members was below the cut-off value (70/94%-95%) for species delineation. Based on the results we propose to transfer Bacillus dafuensis and Bacillus massiliigabonensis to the genus Cytobacillus as Cytobacillus dafuensis comb. nov., and Cytobacillus massiliigabonensis comb. nov., respectively.

Effect of Lysinibacillus isolated from environment on probiotic properties and gut microbiota in mice.

Soil microorganisms (SM) are primarily involved in organism degradation, plant nitrogen nutrient immobilization, host microorganisms and oxidation. However, research on the effect of soil-derived Lysinibacillus on the intestinal microbiota spatial disparity of mice is lacking. To test the probiotic properties of Lysinibacillus and the spatial disparity on mice intestinal microorganisms, hemolysis test, molecular phylogenetic analysis, antibiotic sensitivity testing, serum biochemical assays and 16S rRNA profiling were applied. The results showed that Lysinibacillus (LZS1 and LZS2) was resistant to two common antibiotics, Tetracyclines and Rifampin, and sensitive to other antibiotics among the 12 antibiotics tested and negative for hemolysis. In addition, the body weight of group L (treatment of Lysinibacillus, 1.0 × 108 CFU/d for 21days) mice was significantly greater than that of the control group; serum biochemical tests showed that the TG and UREA were significantly lower in group L. The spatial disparity of intestinal microorganisms in mice was significant, treatment of Lysinibacillus (1.0 × 108 CFU/d for 21days) reduced the intestinal microbial diversity and decreased the richness of Proteobacteria, Cyanobacteria and Bacteroidetes in mice. Furthermore, Lysinibacillus treatment enhanced Lactobacillus and Lachnospiraceae richness and significantly reduced 6 bacterial genera in jejunum community, reduced 8 bacterial genera, but increased bacteria at the 4 genera level in cecum microorganisms. In conclusion, this study demonstrated spatial disparity of intestinal microorganisms in mice and probiotic potential of Lysinibacillus isolated from soil.

New robust subtilisins from halotolerant and halophilic Bacillaceae.

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. KEY POINTS: • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications.

Lysinibacillus spp.: an IAA-producing endospore forming-bacteria that promotes plant growth.

Lysinibacillus is a bacterial genus that has generated recent interest for its biotechnological potential in agriculture. Strains belonging to this group are recognized for their mosquitocidal and bioremediation activity. However, in recent years some reports indicate its importance as plant growth promoting rhizobacteria (PGPR). This research sought to provide evidence of the PGP activity of Lysinibacillus spp. and the role of the indole-3-acetic acid (IAA) production associated with this activity. Twelve Lysinibacillus spp. strains were evaluated under greenhouse conditions, six of which increased the biomass and root architecture of corn plants. In most cases, growth stimulation was evident at 108 CFU/mL inoculum concentration. All strains produced IAA with high variation between them (20-70 µg/mL). The bioinformatic identification of predicted genes associated with IAA production allowed the detection of the indole pyruvic acid pathway to synthesize IAA in all strains; additionally, genes for a tryptamine pathway were detected in two strains. Extracellular filtrates from all strain's cultures increased the corn coleoptile length in an IAA-similar concentration pattern, which demonstrates the filtrates had an auxin-like effect on plant tissue. Five of the six strains that previously showed PGPR activity in corn also promoted the growth of Arabidopsis thaliana (col 0). These strains induced changes in root architecture of Arabidopsis mutant plants (aux1-7/axr4-2), the partial reversion of mutant phenotype indicated the role of IAA on plant growth. This work provided solid evidence of the association of Lysinibacillus spp. IAA production with their PGP activity, which constitutes a new approach for this genus. These elements contribute to the biotechnological exploration of this bacterial genus for agricultural biotechnology.

Cytobacillus spongiae sp. nov. isolated from sponge Diacarnus spinipoculum.

A novel Gram-stain-positive, aerobic and motile bacterium, designated strain CY-GT, was isolated from a sponge (Diacarnus spinipoculum) collected from the Red Sea. The strain grew at 13-43 °C (optimum 30 °C), pH 5.5-10.0 (optimum pH 9.0) and with 0-8.0 % (w/v) (0-1.37 M) NaCl (optimum 0 %). The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that CY-GT represents a member of the genus Cytobacillus, with the highest sequence identity to Cytobacillus oceanisediminis H2T (97.05 %), followed by Cytobacillus firmus IAM 12464T (96.76 %). The major cellular fatty acids (>5 % of the total) of CY-GT were C15 : 0iso, C16 : 0iso, C16 : 1ω7c alcohol, C16 : 0, C17 : 1iso ω10c and C17 : 0iso. The major polar lipids were glycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major respiratory quinone is menaquinone-7 (MK-7). The cell-wall peptidoglycan contains meso-diaminopimelic acid. The total genome size of CY-GT is 4 789 051 bp. The DNA G+C content is 38.83 mol%. The average nucleotide identity and DNA-DNA hybridization among CY-GT and type strains of other species of the genus Cytobacillus were 76.79-78.97 % and 20.10-24.90 %, respectively. On the basis of the results of phylogenetic analysis, physiological and biochemical characterization, strain CY-GT represents a novel species of the genus Cytobacillus, for which the name Cytobacillus spongiae sp. nov. is proposed. The type strain is CY-GT (=MCCC 1K06383T=KCTC 43348T).

Lysinibacillus capsici 38,328 isolated from agricultural soils as a promising probiotic candidate for intestinal health.

There is an increasing interest in the use of spore-forming Bacillus spp. as probiotic ingredients on the market. However, probiotics Bacillus species are insufficient, and more safe Bacillus species were required. In the study, traditional fermented foods and soil samples were collected from more than ten provinces in China, and 506 Bacillus were selected from 109 samples. Using the optimized procedure, we screened nine strains, which successfully passed the acid, alkali, bile salt, and trypsin resistance test. Drug sensitivity test results showed that three Bacillus out of the nine isolates exhibited antibiotic sensitivity to more than 29 antibiotics. The three strains sensitive to antibiotics were identified by 16S ribosomal RNA, recA, and gyrB gene analysis, two isolates (38,327 and 38,328) belong to the species Lysinibacillus capsici and one isolate (37,326) belong to Bacillus halotolerans. Moreover, the three strains were confirmed safe through animal experiments. Finally, L. capsici 38,327 and 38,328 showed protections in the Salmonella typhimurium infection mouse model, which slowed down weight loss, reduced bacterial load, and improved antioxidant capacity. Altogether, our data demonstrated that selected L. capsici strains can be used as novel probiotics for intestinal health.

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Identification and characterization of biocontrol agent Lysinibacillus boronitolerans P42 against Cerrena unicolor that causes root rot of arecanut palm.

The arecanut palm is one of the most important industrial crops in tropical area around the world. The root rot of arecanut palm, which is caused by Cerrena unicolor, has led to heavy economic losses and restricted greatly the development of arecanut industry, especially in Hainan province of China. The common use of chemical agents has worsened the problems of the emergence of resistant pathogens and the pollution of agricultural environment. This study aims to screen and identify a more effective and environment friendly biocontrol method for the prevention and treatment of root rot of arecanut palm. The mycelium growth rate is investigated to select antagonistic bacteria from tropical crop rotation fields which show improved resistance against soil-borne pathogens, and the strain P42 is revealed with the strongest antagonistic effects (82.18%). Based on 16 s rDNA sequence analysis, the strain P42 is identified as Lysinibacillus boronitolerans. In vitro antimicrobial activity shows that the strain P42 exhibits broad-spectrum antagonistic activity against a wide variety of tropical agricultural fungal pathogens, including Cerrena unicolor, Magnaporthe oryzea, Botryodiplodia theobromae, Neoscytalidium dimidiatum, Thanatephorus cucumeris, Fusarium oxysporum, and Botrytis cinerea Per.. The antagonistic activity of the culture of P42 is tolerant to common proteases, longer storage time, and temperature range of 40-121 °C; and is significantly influenced by alkaline (7-9) and acidic (1-2) pH, as well as by ultraviolet ray treatment for more than 30 min. The investigation on the antagonistic activity of the crude extract of fermentation filtrate indicates that the active compounds might be lipopeptides, polyketones, or proteins. To our knowledge, this is the first report of L. boronitolerans as potential bio-reagents for controlling root rot of arecanut palm caused by Cerrena unicolor.

Neobacillus terrae sp. nov., isolated from mountain soil.

A Gram-stain-positive, spore-forming and facultative aerobic bacterium, designated C11T, was isolated from mountain soil collected in the Republic of Korea. The cells were motile rods with peritrichous flagella, and positive for catalase and oxidase activities. Strain C11T grew at 15-45 °C (optimum, 30-37 °C) and pH 6.0-8.0 (optimum, pH 6.0) and in the presence of 0-1 % (w/v) NaCl (optimum, 0.5 %). Strain C11T contained menaquinone-7 as the sole isoprenoid quinone and iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. The G+C content of the genomic DNA was 38.8 mol%. Strain C11T was most closely related to Neobacillus drentensis IDA1967T and Mesobacillus foraminis CV53T, with 98.0 and 97.7 %, 71.7 and 69.9 %, and 20.1 and 20.3 % 16S rRNA gene sequence similarity, average nucleotide identity, and digital DNA-DNA hybridization values, respectively. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain C11T was included in a phyletic lineage with members of the genus Neobacillus but was distinct from members of the genus Mesobacillus. Phenotypic, chemotaxonomic and molecular properties suggested that strain C11T represents a novel species of the genus Neobacillus, for which the name Neobacillus terrae sp. nov. is proposed. The type strain is C11T (=KACC 21661T=JCM 33943T).

A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

Infections caused by antibiotic-resistant Streptococcus pneumoniae are of growing concern for healthcare systems, which need new treatment options. Screening microorganisms in terrestrial environments has proved successful for discovering antibiotics, while production of antimicrobials by marine microorganisms remains underexplored. Here we have screened microorganisms sampled from the Oslo Fjord in Norway for production of molecules that prevent the human pathogen S. pneumoniae from growing. A bacterium belonging to the genus Lysinibacillus was identified. We show that this bacterium produces a molecule that kills a wide range of streptococcal species. Genome mining in BAGEL4 and AntiSmash suggested that it was a new antimicrobial compound, and we therefore named it lysinicin OF. The compound was resistant to heat (100 °C) and polymyxin acylase but susceptible to proteinase K, showing that it is of proteinaceous nature, but most probably not a lipopeptide. S. pneumoniae became resistant to lysinicin OF by obtaining suppressor mutations in the ami locus, which encodes the AmiACDEF oligo peptide transporter. We created ΔamiC and ΔamiEF mutants to show that pneumococci expressing a compromised Ami system were resistant to lysinicin OF. Furthermore, by creating mutants expressing an intact but inactive Ami system (AmiED184A and AmiFD175A) we could conclude that the lysinicin OF activity depended on the active form (ATP-hydrolysing) of the Ami system. Microscopic imaging and fluorescent labelling of DNA showed that S. pneumoniae treated with lysinicin OF had an average reduced cell size with condensed DNA nucleoid, while the integrity of the cell membrane remained intact. The characteristics and possible mode of action of lysinicin OF are discussed.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase.

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

Isolation and screening of chromium resistant bacteria from industrial waste for bioremediation purposes / Isolamento e triagem de bactérias resistentes ao cromo de resíduos industriais para fins de biorremediação

Chromium (VI) a highly toxic metal, a major constituent of industrial waste. It is continuously release in soil and water, causes environmental and health related issues, which is increasing public concern in developing countries like Pakistan. The basic aim of this study was isolation and screening of chromium resistant bacteria from industrial waste collected from Korangi and Lyari, Karachi (24˚52ʹ46.0ʺN 66˚59ʹ25.7ʺE and 24˚48ʹ37.5ʺN 67˚06ʹ52.6ʺE). Among total of 53 isolated strains, seven bacterial strains were selected through selective enrichment and identified on the basis of morphological and biochemical characteristics. These strains were designated as S11, S13, S17, S18, S30, S35 and S48, resistance was determined against varying concentrations of chromium (100-1500 mg/l). Two bacterial strains S35 and S48 showed maximum resistance to chromium (1600 mg/l). Bacterial strains S35 and S48 were identified through 16S rRNA sequence and showed 99% similarity to Bacillus paranthracis and Bacillus paramycoides. Furthermore, growth condition including temperature and pH were optimized for both bacterial strains, showed maximum growth at temperature 30ºC and at optimum pH 7.5 and 6.5 respectively. It is concluded that indigenous bacterial strains isolated from metal contaminated industrial effluent use their innate ability to transform toxic heavy metals to less or nontoxic form and can offer an effective tool for monitoring heavy metal contamination in the environment.

O cromo (VI), metal altamente tóxico, é um dos principais constituintes dos resíduos industriais. É liberado no solo e na água, causa problemas ambientais e de saúde de crescente preocupação pública em países em desenvolvimento como o Paquistão. O objetivo básico deste estudo foi o isolamento e a triagem de bactérias resistentes ao cromo de resíduos industriais coletados em Korangi e Lyari, Karachi (24˚52’46,0”N 66˚59’25,7”E e 24˚48’37,5”N 67˚06’52,6”E). Do total de 53 cepas isoladas, sete cepas bacterianas foram selecionadas por enriquecimento seletivo e identificadas com base em características morfológicas e bioquímicas. Essas cepas foram designadas como S11, S13, S17, S18, S30, S35 e S48, apresentaram alta resistência aos metais contra concentrações variáveis (100-1500 mg / l) de cromo. Já as cepas S35 e S48 foram identificadas por meio da sequência 16S rRNA e apresentaram 99% de similaridade com Bacillus paranthracis e Bacillus paramycoides. Além disso, as condições de crescimento incluindo temperatura e pH foram otimizadas e ambas as cepas bacterianas apresentaram crescimento máximo na temperatura de 30ºC, enquanto seu pH ótimo foi observado em 7,5 e 6,5, respectivamente. Conclui-se que o potencial de resistência dessas bactérias resistentes ao cromo pode ser efetivamente utilizado na remoção de cromo de efluentes industriais contaminados. Técnicas de base biológica usando bactérias ajudarão a fornecer métodos mais baratos e ecológicos de remoção, recuperação e desintoxicação de cromo.

Sutcliffiella rhizosphaerae sp. nov. isolated from roots.

An aerobic, Gram-staining-positive, endospore-forming bacterium, isolated from the rhizosphere of roots of maize (Zea mays), was taxonomically studied. On the basis of 16S rRNA gene sequence similarity comparisons, strain JJ-125T clustered together with species of the genus Sutcliffiella and showed the highest similarities with Sutcliffiella zhanjiangensis (98.7 %). The 16S rRNA gene sequence similarities to the sequences of the type strains of other species of the genus Sutcliffiella were <98.4 %. The genome sequence of JJ-125T was 4 516 360 bp long and had a DNA G+C content of 37.3 %. A DNA-DNA hybridization with the type strain of S. zhanjiangensis DSM 23010T resulted in values of 42.3 and 43.9 % (reciprocal). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the JJ-125T genome assembly and those of the other type strains of species of the genus Sutcliffiella were <75%, <80 % and <21 %, respectively. Chemotaxonomic features supported the grouping of the strain with the genus Sutcliffiella, e.g. the major fatty acids included iso-C15 : 0, iso-C17 : 1 ω10c and iso-C17 : 0, the polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, the only quinone was menaquinone MK-7 and the characteristic diamino acid was meso-diaminopimelic acid. Physiological and biochemical test results were also different from those of the most closely related species. As a consequence, JJ-125T represents a novel species of the genus Sutcliffiella, for which we propose the name Sutcliffiella rhizosphaerae sp. nov., with JJ-125T (= CIP 111883T = LMG 32156T = CCM 9046T) as the type strain.