Molecular mechanisms of the CdnG-Cap5 antiphage defense system employing 3',2'-cGAMP as the second messenger.

Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria. Here, we present the biochemical and structural characterization of two CBASS systems, composed of CdnG and Cap5, from Asticcacaulis sp. and Lactococcus lactis. We show that CdnG from Asticcacaulis sp. synthesizes 3',2'-cGAMP in vitro, and 3',2'-cGAMP is the biological signaling molecule that activates Cap5 for DNA degradation. Crystal structures of Cap5, together with the SAVED domain in complex with 3',2'-cGAMP, provide insight into the architecture of Cap5 as well as molecular recognition of 3',2'-cGAMP by the SAVED domain of Cap5. Amino acid conservation of the SAVED domain of Cap5, together with mutational studies, led us to propose a mechanism of Back-to-Front stacking of two SAVED domains, mediated by 3',2'-cGAMP, to activate HNH nuclease domain for DNA degradation. This study of the most abundant CBASS system provides insights into the mechanisms employed by bacteria in their conflicts against phage.

Genome-Based Taxonomy of Brevundimonas with Reporting Brevundimonas huaxiensis sp. nov.

Brevundimonas is a genus of Gram-negative bacteria widely distributed in nature and is also an opportunistic pathogen causing health care-associated infections. Brevundimonas strain 090558T was recovered from a blood culture of a cancer patient and was subjected to genome sequencing and analysis. The average nucleotide identity and in silico DNA-DNA hybridization values between 090558T and type strains of Brevundimonas species were 78.76% to 93.94% and 19.8% to 53.9%, respectively, below the cutoff to define bacterial species. Detailed phenotypic tests were performed, suggesting that 090558T can be differentiated from other Brevundimonas species by its ability to assimilate sodium acetate but not to utilize glucose, trypsin, or ß-glucosidase. Strain 090558T (GDMCC 1.1871T or KCTC 82165T) therefore represents a novel Brevundimonas species, for which the name Brevundimonas huaxiensis sp. nov. is proposed. All Brevundimonas genomes available in GenBank (accessed on 25 January 2021) were retrieved, discarding those labeled "excluded from RefSeq" by GenBank, and included 82 genomes for precise species curation. In addition to the 21 Brevundimonas species with genomes of type strains available, we identified 29 Brevundimonas taxa that either belong to the 12 Brevundimonas species without available genomes of type strains or represent novel species. We found that more than half (57.3%) of the 82 Brevundimonas genomes need to be corrected for species assignation, including species mislabeling of a type strain. Our analysis highlights the complexity of Brevundimonas taxonomy. We also found that only some Brevundimonas species are associated with human infections, and more studies are warranted to understand their pathogenicity and epidemiology. IMPORTANCEBrevundimonas is a genus of the family Caulobacteraceae and comprises 33 species. Brevundimonas can cause various infections but remains poorly studied. In this study, we reported a novel Brevundimonas species, Brevundimonas huaxiensis, based on genome and phenotype studies of strain 090558T recovered from human blood. We then examined the species assignations of all Brevundimonas genomes (n = 82) in GenBank and found that in addition to the known Brevundimonas species with genome sequences of type strains available, there are 29 Brevundimonas taxa based on genome analysis, which need to be further studied using phenotype-based methods to establish their species status. Our study significantly updates the taxonomy of Brevundimonas and enhances our understanding of this genus of clinical relevance. The findings also encourage future studies on the characterization of novel Brevundimonas species.

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.

Brevundimonas mongoliensis sp. nov., A Novel Psychrotolerant Bacterium Isolated from Oil-Contaminated Soil.

Two yellow-coloured, Gram-stain-negative, motile, and rod-shaped bacteria, designated strains R-10-10T and R-10-15 were isolated from oil-contaminated soil. Both strains were able to grow at 4-40 °C, pH 5.5-10.5, and 0-4% (w/v) NaCl concentration. These strains were taxonomically characterized by a polyphasic approach. Based on the 16S rRNA gene sequence analysis, both strains, R-10-10T and R-10-15, could be affiliated to the genus Brevundimonas and shared highest sequence similarity with Brevundimonas staleyi FWC43T (98.8%), Brevundimonas bullata TK0051T (98.6%), and Brevundimonas subvibrioides CB81T (98.3%). The pairwise sequence similarity between strains R-10-10T and R-10-15 was 99.9%. Both strains R-10-10T and R-10-15 contained phosphatidylglycerol, diphosphatidylglycerol, and four unidentified glycolipids as major polar lipids; ubiquinone-10 as sole respiratory quinone; and summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and C18:1ω9c as major fatty acids. The genomic DNA G+C content values of strains R-10-10T and R-10-15 were 67.1 and 66.9 mol%, respectively. The DNA-DNA relatedness between R-10-10T and R-10-15 was higher than 70% but the values were less than 55% with closely related reference type strains. The morphological, physiological, chemotaxonomic, and phylogenetic data clearly distinguished strain R-10-10T from its closest phylogenetic neighbors. Thus, strain R-10-10T is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas mongoliensis sp. nov. is proposed. The type strain is R-10-10T (=KEMB 9005-696T = KACC 19387T = JCM 32172T), and strain R-10-15 is considered as an additional strain of the novel species.

Isolation and Characterization of PHA-Producing Bacteria from Propylene Oxide Saponification Wastewater Residual Sludge.

A polyhydroxyalkanoate (PHA)-producing strain was isolated from propylene oxide (PO) saponification wastewater activated sludge and was identified as Brevundimonas vesicularis UJN1 through 16S rDNA sequencing and Biolog microbiological identification. Single-factor and response surface methodology experiments were used to optimize the culture medium and conditions. The optimal C/N ratio was 100/1.04, and the optimal carbon and nitrogen sources were sucrose (10 g/L) and NH4Cl (0.104 g/L) respectively. The optimal culture conditions consisted of initial pH of 6.7 and an incubation temperature of 33.4 °C for 48 h, with 15% inoculum and 100 mL medium at an agitation rate of 180 rpm. The PHA concentration reached 34.1% of the cell dry weight and increased three times compared with that before optimization. The only report of PHA-producing bacteria by Brevundimonas vesicularis showed that the conversion rate of PHAs using glucose as the optimal carbon source was 1.67%. In our research, the conversion rate of PHAs with sucrose as the optimal carbon source was 3.05%, and PHA production using sucrose as the carbon source was much cheaper than that using glucose as the carbon source.

Brevundimonas humi sp. nov., an alphaproteobacterium isolated from forest soil.

During a study of bacterial diversity of soil, a novel strain, CA-15T, was isolated from Kyonggi University forest soil. Cells were aerobic, Gram-stain-negative, motile, non-spore-forming, rod-shaped, oxidase-positive and catalase- negative. Tyrosine was not oxidized but produced red pigmentation on an agar palte. Strain CA-15T hydrolysed Tween 60 and DNA. It grew at 15-35 °C (optimum, 25-30 °C), pH 6.0-10.0 (optimum, 7.0-9.0) and at 1.5 % (w/v) NaCl concentration. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain CA-15T formed a lineage within the family Caulobacteraceae of the class Alphaproteobacteria that was distinct from various species of the genus Brevundimonas. Brevundimonas bullata DSM 7126T was the closest member of strain CA-15T on the basis of 16S rRNA gene sequence similarity (98.48 %). Q-10 was only an isoprenoid quinone detected for strain CA-15T. The major polar lipids were 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-αd-glucopyranuronosyl]glycerol, 1,2-di-O-acyl-3-O-[αd-glucopyranosyl]-sn-glycerol, 1,2-di-O-acyl-3-O-αd-glucopyranuronosylglycerol, 1,2-diacyl-3-O-[6'-phosphatidyl-αd-glucopyranosyl]glycerol and phosphatidylglycerol. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, C18 : 1ω7c 11-methyl and C17 : 1ω8c. The DNA G+C content of strain CA-15T was 63.6 mol%. The polyphasic characterization indicated that strain CA-15T represents a novel species in the genus Brevundimonas, for which the name Brevundimonas humi sp. nov. is proposed. The type strain of Brevundimonas humi is CA-15T (=KEMB 9005-528T=KACC 19106T=NBRC 112677T).

Phenylobacterium hankyongense sp. nov., isolated from ginseng field soil.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterial strain, designated HKS-05T, was isolated from ginseng field soil. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. HKS-05T grew at 10-37 °C and at pH 6.0-8.0 on R2A agar. On the basis of 16S rRNA gene sequence similarity, HKS-05T was shown to represent a member of the family Caulobacteraceaeand to be related to Phenylobacterium lituiforme FaiI3T (98.1 % sequence similarity), 'Phenylobacterium zucineum' HLK1 (97.9 %), Phenylobacterium muchangponense A8T (97.7 %), Phenylobacteriumcomposti 4T-6T (97.2 %) and Phenylobacterium immobile ET (97.1 %). The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids were phosphatidylglycerol, unidentified glycolipids and unidentified polar lipids. The G+C content of the genomic DNA was 70.4 mol%. DNA-DNA relatedness values between HKS-05T and its closest phylogenetically neighbours were low. HKS-05T could be differentiated genotypically and phenotypically from the species of the genus Phenylobacterium with validly published names. The isolate therefore represents a novel species, for which the name Phenylobacteriumhankyongense sp. nov. is proposed, with the type strain HKS-05T (=KACC 18628T=LMG 30081T).

Phenylobacterium deserti sp. nov., isolated from desert soil.

A novel bacterial strain, designated YIM 73061T, was isolated from the Cholistan desert in Punjab, Pakistan, and characterized by using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis revealed the highest levels of sequence similarity with respect to Phenylobacterium conjunctum FWC21T (97.6 %), Phenylobacterium lituiforme FaiI3T (97.4 %), Phenylobacteriumcomposti 4T-6T (97.0 %) and Phenylobacterium aquaticum W2-3-4T (96.8 %). Cells were Gram-stain-negative, aerobic and motile rods that formed orange colonies. The strain was oxidase- and catalase-positive. Growth occurred at 20-40 °C (optimum, 30-37 °C) at pH 5.0-8.0 (optimum, pH 7.0) and with 0-1 % (w/v) NaCl (optimum, 0-0.5 %). The major cellular fatty acids (>10 %) were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The polar lipid profile consisted of phosphatidylglycerol and four unidentified glycolipids. The major isoprenoid quinone was ubiquinone-10 (Q-10). The G+C content of the genomic DNA was 66.8 mol%. Strain YIM 73061T showed low levels of DNA-DNA relatedness to P. conjunctum FWC21T (27.2±2.6 %), P. lituiforme FaiI3T (24.6±1.1 %) and P.composti 4T-6T (18.4±3.1 %). On the basis of phylogenetic inference, chemotaxonomic characteristics and phenotypic data, strain YIM 73061T should be classified as representing a novel species, for which the name Phenylobacterium deserti sp. nov. is proposed. The type strain is YIM 73061T (=DSM 103871T=CCTCC AB 2016297T).

The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca.

Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' ß-oxygenase (crtW) and 3,3' ß-hydroxylase (crtZ) genes from Brevundimonas sp. SD-212 were expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility of Brevundimonas sp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.

Phenylobacterium aquaticum sp. nov., isolated from the reservoir of a water purifier.

A Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, rod-shaped bacterial strain designated W2-3-4T was isolated from the reservoir of a water purifier. This bacterium was characterized to determine its taxonomic position by using a polyphasic approach. Strain W2-3-4T grew well at 25-30 °C on nutrient and R2A agars. On the basis of 16S rRNA gene sequence similarity, strain W2-3-4T was shown to belong to the family Caulobacteraceaeand to be related to Phenylobacterium conjunctumFWC21T (98.0 % sequence similarity) and Phenylobacterium haematophilum CCUG 26751T (97.2 %). Lower sequence similarities were found with the type strains of all other recognized members of the genus Phenylobacterium (95.7-97.1 %). The G+C content of the genomic DNA was 68.7 mol%. The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, C18 : 1ω7c 11-methyl and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids were phosphatidylglycerol, an unknown phospholipid, four unknown glycolipids and three unidentified polar lipids. DNA-DNA relatedness values between strain W2-3-4Tand its closest phylogenetically neighbours were below 7 %. Strain W2-3-4T could be differentiated genotypically and phenotypically from recognized species of the genus Phenylobacterium. The isolate therefore represents a novel species, for which the name Phenylobacterium aquaticum sp. nov. is proposed, with the type strain W2-3-4T (=KACC 18306T=LMG 28593T).

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate.

Organophosphate hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane ofBrevundimonas diminutain a pre-folded conformation by thetwinargininetransport (Tat) pathway. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Interaction of OPH with PstS appears to facilitate transport of Pigenerated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model,opdnull mutants ofB. diminutafailed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate.

Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection.

BACKGROUND: While 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung's bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients. RESULTS: Targeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-L-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria. CONCLUSIONS: In bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.

Brevundimonas diminuta bacteremia in a man with myelodysplastic syndromes.

Brevundimonas diminuta are ubiquitous in the environment, but are infrequently isolated from clinical samples. Here we report a case of B. diminuta bacteremia in a man with myelodysplastic syndromes (MDS) at a teaching hospital in China and review the previously reported cases. The organism was confirmed by culture and 16s rRNA sequence analysis with highly sensitivity to broad-spectrum antibiotics. Our report and other cases demonstrated that the optimal therapeutic duration for B. diminuta infections in various situations remains to be established.

Salix purpurea Stimulates the Expression of Specific Bacterial Xenobiotic Degradation Genes in a Soil Contaminated with Hydrocarbons.

The objectives of this study were to uncover Salix purpurea-microbe xenobiotic degradation systems that could be harnessed in rhizoremediation, and to identify microorganisms that are likely involved in these partnerships. To do so, we tested S. purpurea's ability to stimulate the expression of 10 marker microbial oxygenase genes in a soil contaminated with hydrocarbons. In what appeared to be a detoxification rhizosphere effect, transcripts encoding for alkane 1-monooxygenases, cytochrome P450 monooxygenases, laccase/polyphenol oxidases, and biphenyl 2,3-dioxygenase small subunits were significantly more abundant in the vicinity of the plant's roots than in bulk soil. This gene expression induction is consistent with willows' known rhizoremediation capabilities, and suggests the existence of S. purpurea-microbe systems that target many organic contaminants of interest (i.e. C4-C16 alkanes, fluoranthene, anthracene, benzo(a)pyrene, biphenyl, polychlorinated biphenyls). An enhanced expression of the 4 genes was also observed within the bacterial orders Actinomycetales, Rhodospirillales, Burkholderiales, Alteromonadales, Solirubrobacterales, Caulobacterales, and Rhizobiales, which suggest that members of these taxa are active participants in the exposed partnerships. Although the expression of the other 6 marker genes did not appear to be stimulated by the plant at the community level, signs of additional systems that rest on their expression by members of the orders Solirubrobacterales, Sphingomonadales, Actinomycetales, and Sphingobacteriales were observed. Our study presents the first transcriptomics-based identification of microbes whose xenobiotic degradation activity in soil appears stimulated by a plant. It paints a portrait that contrasts with the current views on these consortia's composition, and opens the door for the development of laboratory test models geared towards the identification of root exudate characteristics that limit the efficiency of current willow-based rhizoremediation applications.

Biomineralization processes of calcite induced by bacteria isolated from marine sediments

Biomineralization is a known natural phenomenon associated with a wide range of bacterial species. Bacterial-induced calcium carbonate precipitation by marine isolates was investigated in this study. Three genera of ureolytic bacteria, Sporosarcina sp., Bacillus sp. and Brevundimonas sp. were observed to precipitate calcium carbonate minerals. Of these species, Sporosarcina sp. dominated the cultured isolates. B. lentus CP28 generated higher urease activity and facilitated more efficient precipitation of calcium carbonate at 3.24 ± 0.25 × 10−4 mg/cell. X-ray diffraction indicated that the dominant calcium carbonate phase was calcite. Scanning electron microscopy showed that morphologies of the minerals were dominated by cubic, rhombic and polygonal plate-like crystals. The dynamic process of microbial calcium carbonate precipitation revealed that B. lentus CP28 precipitated calcite crystals through the enzymatic hydrolysis of urea, and that when ammonium ion concentrations reached 746 mM and the pH reached 9.6, that favored calcite precipitation at a higher level of 96 mg/L. The results of this research provide evidence that a variety of marine bacteria can induce calcium carbonate precipitation, and may influence the marine carbonate cycle in natural environments.

Asticcacaulis endophyticus sp. nov., a prosthecate bacterium isolated from the root of Geum aleppicum.

A strictly aerobic, light-yellow-coloured, stalked bacterium, designated strain ZFGT-14(T), was isolated from the root of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi province, north-west China, and was subjected to a taxonomic study using a polyphasic approach. This novel isolate grew at 7-33 °C (optimum 25-28 °C) and pH 6.0-10.0 (optimum pH 7.0-8.0). Flexirubin-type pigments were not produced. Cells were Gram-stain-negative, rod-shaped and motile with a single polar flagellum. The predominant respiratory quinone was Q-10. The major cellular fatty acids were summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C19 : 0 cyclo ω8c and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and the major polar lipids were phosphatidylglycerol and glycolipids. The DNA G+C content was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFGT-14(T) was most closely related to the genus Asticcacaulis and had low sequence similarity (95.0-95.9 %) with all species with validly published names within the genus Asticcacaulis. Based on the phenotypic, phylogenetic and genotypic data, strain ZFGT-14(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis endophyticus sp. nov. is proposed. The type strain is ZFGT-14(T) ( = CCTCC AB 2013012(T) = KCTC 32296(T) = LMG 27605(T)).

Discovery and characterization of an isopeptidase that linearizes lasso peptides.

Lasso peptides are a class of ribosomally derived natural products with diverse bioactivities. The characteristic threaded lasso structure in these peptides derives from an isopeptide bond attaching the N-terminus of the peptide to an acidic side chain. Here we describe the heterologous expression of a lasso peptide gene cluster encoding two lasso peptides, astexin-2 and astexin-3, and solve the solution structure of astexin-3. This cluster also encodes an enzyme annotated as a protease. We show that this enzyme, AtxE2, is a lasso peptide isopeptidase that specifically hydrolyzes astexins-2 and -3, converting them to linear peptides. Astexin-3 is highly thermostable and resists unthreading after extensive heat treatment. In contrast, astexin-2 unthreads upon heat treatment. AtxE2 has no activity toward unthreaded astexin-2, demonstrating that this isopeptidase must recognize a knotted structure in order to function. We also use this isopeptidase as a tool to study evolutionary relationships between lasso peptide gene clusters.

Asticcacaulis solisilvae sp. nov., isolated from forest soil.

An obligately aerobic, chemoheterotrophic, mesophilic prosthecate bacterium, designated strain CGM1-3EN(T), was isolated from the enrichment cultures of forest soil from Cheonggyesan Mountain, Republic of Korea. Cells were Gram-reaction-negative, motile rods (1.3-2.4 µm long by 0.30-0.75 µm wide) with single flagella. The strain grew at 10-37 °C (optimum 25-30 °C) and at pH 4.5-9.5 (optimum 5.0-7.0). The major cellular fatty acids were C16 : 0, C18 : 1ω7c 11-methyl, C12 : 1 3-OH and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c). The genomic DNA G+C content of strain CGM1-3EN(T) was 63.7 mol%. The closest phylogenetic neighbour to strain CGM1-3EN(T) was identified as Asticcacaulis biprosthecium DSM 4723(T) (97.2 % 16S rRNA gene sequence similarity) and the DNA-DNA hybridization value between strain CGM1-3EN(T) and A. biprosthecium DSM 4723(T) was less than 24.5 %. Strain CGM1-3EN(T) used d-glucose, d-fructose, sucrose, maltose, trehalose, d-mannose, d-mannitol, d-sorbitol, d-galactose, cellobiose, lactose, raffinose, fumarate, pyruvate, dl-alanine and glycerol as carbon sources. Based on data from the present polyphasic study, the forest soil isolate CGM1-3EN(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis solisilvae sp. nov. is proposed. The type strain is CGM1-3EN(T) ( = AIM0088(T) = KCTC 32102(T) = JCM 18544(T)).

The astexin-1 lasso peptides: biosynthesis, stability, and structural studies.

Lasso peptides are a large family of natural products that owe their name to a unique structure formed by a side chain to backbone macrocyclization, resembling a knotted lasso. The unique structure has significant impact on their biological and physical properties, as lasso peptides are usually more stable than linear ones. Current work examines stability, structure, and biosynthesis of recently discovered lasso peptide astexin-1, a heat-sensitive lasso peptide. The obtained results revealed a new lasso structure with a tight loop and long tail as well as narrow specificity of the maturation machinery for some essential residues associated with the protease processing site, involved in macrolactam ring formation and entrapment of the tail. Using the astexin-1 structure, it was possible to rationally construct a thermostable variant of this lasso peptide.

Brevundimonas abyssalis sp. nov., a dimorphic prosthecate bacterium isolated from deep-subsea floor sediment.

A novel Gram-negative, aerobic, psychrotolerant, alkali-tolerant, heterotrophic and dimorphic prosthecate bacterium, designated strain TAR-001(T), was isolated from deep-sea floor sediment in Japan. Cells of this strain had a dimorphic life cycle and developed an adhesive stalk at a site not coincident with the centre of the cell pole, and the other type of cell, a swarm cell, had a polar flagellum. Colonies were glossy, viscous and yellowish-white in colour. The temperature, pH and salt concentration range for growth were 2-41 °C, pH 6.5-10.0 and 1-4% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-001(T) belongs to the family Caulobacteraceae of the class Alphaproteobacteria, and lies between the genus Brevundimonas and the genus Caulobacter. Levels of similarity between the 16S rRNA gene sequence of strain TAR-001(T) and those of the type strains of Brevundimonas species were 93.3-95.7%; highest sequence similarity was with the type strain of Brevundimonas diminuta. Levels of sequence similarity between those of the type strains of Caulobacter species were 94.9-96.0%; highest sequence similarity was with the type strain of Caulobacter mirabilis. The G+C content of strain TAR-001(T) was 67.6 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C18:1ω7c and C16:0, and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1→4)-α-D-glucopyranuronosyl]glycerol suggests strain TAR-001(T) is more closely to the genus Brevundimonas than to the genus Caulobacter. The mean DNA-DNA hybridization levels between strain TAR-001(T) and the type strains of two species of the genus Brevundimonas were higher than that of the genus Caulobacter. On the basis of polyphasic biological features and the 16S rRNA gene sequence comparison presented here, strain TAR-001(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas abyssalis sp. nov. is proposed; the type strain is TAR-001(T) (=JCM 18150(T)=CECT 8073(T)).