Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer.

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3',5'-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3'-phosphate-5'-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium "Aromatoleum" sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances. Graphical abstract.

Genome mining- and synthetic biology-enabled production of hypermodified peptides.

The polytheonamides are among the most complex and biosynthetically distinctive natural products known to date. These potent peptide cytotoxins are derived from a ribosomal precursor processed by 49 mostly non-canonical posttranslational modifications. As the producer is a 'microbial dark matter' bacterium only distantly related to any cultivated organism, >70-step chemical syntheses have been developed to access these unique compounds. Here, we mined prokaryotic diversity to establish a synthetic platform based on the new host Microvirgula aerodenitrificans that produces hypermodified peptides within two days. Using this system, we generated the aeronamides, new polytheonamide-type compounds with near-picomolar cytotoxicity. Aeronamides, as well as the polygeonamides produced from deep-rock biosphere DNA, contain the highest numbers of D-amino acids in known biomolecules. With increasing bacterial genomes being sequenced, similar host mining strategies might become feasible to access further elusive natural products from uncultivated life.

Structural basis of the divergent oxygenation reactions catalyzed by the rieske nonheme iron oxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.

Electron acceptor-dependent identification of key anaerobic toluene degraders at a tar-oil-contaminated aquifer by Pyro-SIP.

Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil-contaminated aquifer in Germany, where a specialized community of contaminant degraders codominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase α-subunit, bssA) genes detected in situ appeared to be more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with (13)C(7)-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae in sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This and the absence of (13)C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy among anaerobic toluene degraders on site.

Paludibacterium yongneupense gen. nov., sp. nov., isolated from a wetland, Yongneup, in Korea.

A cream-coloured bacterial strain, 5YN8-15(T), was isolated from a wetland, Yongneup, in the Inje region of the Republic of Korea. The bacterium was facultatively anaerobic, Gram-negative, motile with a single polar flagellum and curved-rod-shaped. Based on 16S rRNA gene sequence analysis, strain 5YN8-15(T) is a member of the Betaproteobacteria. Closely related taxa were Gulbenkiania mobilis E4FC31(T) (94.9% sequence similarity), Chromobacterium species (94.1-94.4%), Aquitalea magnusonii TRO-001DR8(T) (93.2%) and Aquaspirillum serpens IAM 13944(T) (92.5%). All other species with validly published names analysed showed sequence similarities of below 92%. Strain 5YN8-15(T) had ubiquinone 8 as the major isoprenoid quinone. The major fatty acids were summed feature 3 (C(16:1)omega7c and/or iso-C(15:0) 2-OH), C(16:0) and C(18:1)omega7c. The DNA G+C content was 63.0 mol%. Based on the data from the polyphasic study, strain 5YN8-15(T) represents a novel genus and species of the family Neisseriaceae, for which the name Paludibacterium yongneupense gen. nov., sp. nov. is proposed. The type strain is 5YN8-15(T) (=KACC 11601(T)=DSM 18731(T)).

Chitinilyticum aquatile gen. nov., sp. nov., a chitinolytic bacterium isolated from a freshwater pond used for Pacific white shrimp culture.

Strain c14(T), originally isolated from surface water of a freshwater pond located in Pingtung (southern Taiwan) used for culture of Pacific white shrimp (Litopenaeus vannamei), was subjected to a polyphasic taxonomic approach. The strain exhibited strong chitinolytic activity and was able to grow under aerobic and anaerobic conditions by utilizing chitin exclusively as the carbon, nitrogen and energy source. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the proposed bacterium to the Betaproteobacteria, most closely related to Chitinibacter tainanensis S1(T), Deefgea rivuli WB 3.4-79(T) and Silvimonas terrae KM-45(T), with 94.6, 93.6 and 92.9 % 16S rRNA gene sequence similarity, respectively. The predominant fatty acids detected in cells of strain c14(T) were C(16 : 0), C(18 : 1)omega7c and summed feature 3 (C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH). The G+C content of the genomic DNA was 69.5 (+/-1.0) mol%. Biochemical, physiological, chemotaxonomic and phylogenetic analyses showed that strain c14(T) could not be assigned to any known genus of the Betaproteobacteria. Therefore, strain c14(T) is classified within a novel genus and species, for which the name Chitinilyticum aquatile gen. nov., sp. nov. is proposed. The type strain of Chitinilyticum aquatile is c14(T) (=LMG 23346(T) =BCRC 17533(T)).

Polaromonas aquatica sp. nov., isolated from tap water.

Two Gram-negative, rod-shaped, non-spore-forming bacteria (CCUG 39402T and CCUG 39797), isolated from different water sources, were investigated in a polyphasic study. The two isolates shared 100% 16S rRNA gene sequence similarity and it was shown that they belonged to the Betaproteobacteria, most closely related to Polaromonas vacuolata (97.8%) and Polaromonas naphthalenivorans (97.8%). A polyamine pattern with 2-hydroxyputrescine and putrescine, as well as ubiquinone Q-8, were in agreement with characteristics of Betaproteobacteria. The presence of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and major fatty acids C(16:1)omega7c, C(16:0) and C(17:0) cyclo supported the affiliation of the two strains to the genus Polaromonas. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the two isolates from the two Polaromonas species with validly published names. They therefore represent a novel species, for which the name Polaromonas aquatica sp. nov. is proposed, with the type strain CCUG 39402T (= CIP 108776T).

Inter-domain cross-talk controls the NifA protein activity of Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize. The activity of NifA, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through a mechanism involving its N-terminal domain. Here we show that this domain interacts specifically in vitro with the N-truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA-binding activities of the N-truncated NifA protein. We suggest that the N-terminal domain inhibits NifA-dependent transcriptional activation by an inter-domain cross-talk between the catalytic domain of the NifA protein and its regulatory N-terminal domain in response to fixed nitrogen.

Xenophilus azovorans gen. nov., sp. nov., a soil bacterium that is able to degrade azo dyes of the Orange II type.

The taxonomy of strain KF46FT, which was isolated previously after an aerobic enrichment with the azo compound 1-(4'-carboxyphenylazo)-2-naphthol as the sole source of energy and carbon, was investigated by a polyphasic approach. The organism contained a quinone system with ubiquinone Q-8 and 2-hydroxyputrescine and putrescine as the major polyamines, suggesting that strain KF46FT belonged to the beta-subclass of the Proteobacteria. The polar lipid profile consisted mainly of phosphatidylethanolamine and minor amounts of phosphatidylglycerol and diphosphatidylglycerol. Sequencing of the 16S rRNA gene supported its placement in the family Comamonadaceae, but the sequence similarities to the most closely related species of the genera Hydrogenophaga, Acidovorax, Comamonas and Xylophilus were only in the range 95.0 to 96.1%. Different methods for the construction of phylogenetic trees showed the separate position of strain KF46FT 'between' the genera Hydrogenophaga, Variovorax, Comamonas and Xylophilus. Analysis of the fatty acids revealed an unusual profile, with the presence of 8:0 3-OH, 10:0 3-OH, 16:1 2-OH, 16:0 2-OH and 18:1 2-OH in addition to 17:0 cyclo, which is unique among the previously described genera of the family Comamonadaceae. Thus, a new taxon is proposed for strain KF46FT, with the name Xenophilus azovorans gen. nov., sp. nov.

[Evaluation of effect of the peptide structure on energetics of reduction-oxidation reactions of proteins containing Fe4S4 clusters in computer experiments]. / Opredelenie vliianiia peptidnoi struktury na énergetiku reaktsii okisleniia-vosstanovleniia belkov, soderzhashchikh Fe4S4 klastery, s pomoshch'iu komp'iuternykh éksperimentov.

The methods of molecular dynamics were used to study the influence of peptide matrix and conformational relaxation on the thermal effects of redox reactions of two proteins containing Fe4S4 clusters with sharply differing redox potential values. It was shown that the depth of immersion of the Fe4S4 cluster in the protein globule affects the parameters of chemical equilibrium. It was found that the peptide matrix makes a determining contribution to the compensation of the Coulomb repulsion energy in Fe4S4 clusters.

Assignment of CDC weak oxidizer group 2 (WO-2) to the genus Pandoraea and characterization of three new Pandoraea genomospecies.

CDC weak oxidizer group 2 (WO-2) consists of nine phenotypically similar human clinical isolates received by the Centers for Disease Control and Prevention between 1989 and 1998. Four of the isolates were from blood, three were from sputum, and one each was from bronchial fluid and maxillary sinus. All are aerobic nonfermentative, motile gram-negative rods with one to eight polar flagella per cell. All grew at 25 and 35 degrees C and were positive for catalase, urease (usually delayed 3 to 7 days), citrate, alkalinization of litmus milk, oxidization of glycerol (weakly), and growth on MacConkey agar and in nutrient broth without NaCl. All except one strain were oxidase positive with the Kovács method, and all except one isolate weakly oxidized D-glucose. All were negative for oxidation of D-xylose, D-mannitol, lactose, sucrose, maltose, and 20 other carbohydrates, esculin hydrolysis, indole production, arginine dihydrolase, and lysine and ornithine decarboxylase. Only two of nine isolates reduced nitrate. Broth microdilution susceptibilities were determined for all strains against 13 antimicrobial agents. Most of the strains were resistant to ampicillin, extended-spectrum cephalosporins, and aminoglycosides, including gentamicin, tobramycin, and amikacin, but they varied in their susceptibility to fluoroquinolones. High-performance liquid chromatographic and mass spectrometric analyses of the WO-2 group identified ubiquinone-8 as the major quinone component. The percent G+C of the WO-2 strains ranged from 65.2 to 70.7% (thermal denaturation method). All shared a common cellular fatty acid (CFA) profile, which was characterized by relatively large amounts (7 to 22%) of 16:1omega7c, 16:0, 17:0cyc, 18:1omega7c, and 19:0cyc(11-12); small amounts (1 to 3%) of 12:0 and 14:0; and eight hydroxy acids, 2-OH-12:0 (4%), 2-OH-14:0 (trace), 3-OH-14:0 (12%), 2-OH-16:1 (1%), 2-OH-16:0 (3%), 3-OH-16:0 (4%), 2-OH-18:1 (2%), and 2-OH-19:0cyc (3%). This profile is similar to the CFA profile of Pandoraea, a recently described genus associated with respiratory infections in cystic fibrosis patients (T. Coenye et al., Int. J. Syst. Evol. Microbiol., 50:887-899, 2000). Sequencing of the 16S rRNA gene (1,300 bp) for all nine strains indicated a high level (> or =98.8%) of homogeneity with Pandoraea spp. type strains. DNA-DNA hybridization analysis (hydroxyapatite method; 70 degrees C) confirmed the identity of WO-2 with the genus Pandoraea and assigned three strains to Pandoraea apista and three to Pandoraea pnomenusa, and identified three additional new genomospecies containing one strain each (ATCC BAA-108, ATCC BAA-109, ATCC BAA-110). This study also shows that Pandoraea isolates may be encountered in blood cultures from patients without cystic fibrosis.

Description of Pandoraea gen. nov. with Pandoraea apista sp. nov., Pandoraea pulmonicola sp. nov., Pandoraea pnomenusa sp. nov., Pandoraea sputorum sp. nov. and Pandoraea norimbergensis comb. nov.

A polyphasic taxonomic study was performed on a group of isolates tentatively identified as Burkholderia cepacia, Ralstonia pickettii or Ralstonia paucula (formerly known as CDC group IVc-2). The isolates were mainly cultured from sputum of cystic fibrosis patients or from soil. SDS-PAGE of whole-cell proteins and AFLP fingerprinting distinguished at least five different species, and this was confirmed by DNA-DNA hybridizations. 16S rDNA sequence analysis of representative strains indicated that these organisms belong to the beta-subclass of the Proteobacteria, with the genera Burkholderia and Ralstonia as closest neighbours. Based on genotypic and phenotypic characteristics, the organisms were classified in a novel genus, Pandoraea. The DNA base composition of the members of the new genus is between 61.2 and 64.3 mol%. This novel genus includes four new species, Pandoraea apista (the type species) (type strain is LMG 16407T), Pandoraea pulmonicola (type strain is LMG 18106T), Pandoraea pnomenusa (type strain is LMG 18087T) and Pandoraea sputorum (type strain is LMG 18819T), and Pandoraea norimbergensis (Wittke et al. 1997) comb. nov. (type strain is LMG 18379T). The available clinical data indicate that at least some of these organisms may cause chronic infection in, and can be transmitted amongst, cystic fibrosis patients.