Carboxy-terminal polyglutamylation regulates signaling and phase separation of the Dishevelled protein.

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating.

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Pedobacter fastidiosus sp. nov., isolated from glacial habitats of maritime Antarctica.

Strains P8930T and 478 were isolated from Antarctic glaciers located on James Ross Island and King George Island, respectively. They comprised Gram-stain-negative short rod-shaped cells forming pink pigmented colonies and exhibited identical 16S rRNA gene sequences and highly similar MALDI TOF mass spectra, and hence were assigned as representatives of the same species. Phylogenetic analysis based on 16S rRNA gene sequences assigned both isolates to the genus Pedobacter and showed Pedobacter frigidisoli and Pedobacter terrae to be their closest phylogenetic neighbours, with 97.4 and 97.2 % 16S rRNA gene sequence similarities, respectively. These low similarity values were below the threshold similarity value of 98.7%, confirming the delineation of a new bacterial species. Further genomic characterization included whole-genome sequencing accompanied by average nucleotide identity (ANI) and digital DNA-DNA hybridization calculations, and characterization of the genome features. The ANI values between P8930T and P. frigidisoli RP-3-11T and P. terrae DSM 17933T were 79.7 and 77.6 %, respectively, and the value between P. frigidisoli RP-3-11T and P. terrae DSM 17933T was 77.7 %, clearly demonstrating the phylogenetic distance and the novelty of strain P8930T. Further characterization included analysis of cellular fatty acids, quinones and polar lipids, and comprehensive biotyping. All the obtained results proved the separation of strains P8930T and 478 from the other validly named Pedobacter species, and confirmed that they represent a new species for which the name Pedobacter fastidiosus sp. nov. is proposed. The type strain is P8930T (=CCM 8938T=LMG 32098T).

Ecological specialization and reproductive isolation among closely related sympatric ant-eating spiders.

Biological divergence results from several mechanisms. Defensive mechanisms, such as Batesian mimicry, can cause reproductive isolation via temporal segregation in foraging activity, particularly, in species that closely associate with their model. This seems to be the case of ant-eating spiders, which can be inaccurate Batesian mimics of their prey. Here, we focused on Zodarion nitidum, which has two forms occurring in sympatry, black and yellow. Given the expected noticeable impact of their colour differences on the spiders' interactions with their potential predators and prey, we investigated whether these morphotypes have diverged in other aspects of their biology. We measured the two morphotypes' phenotypic resemblance to a mimetic model, tested whether they were protected from predators, investigated their circadian activity, surveyed the prey they hunted, modelled their distributions, performed crossing experiments and estimated their degree of genetic differentiation. We found that the black morphotype is ant-like, resembling Messor ants, and it was not distinguishable from their ant models by four potential predators. In contrast, the yellow morphotype seems to use predator avoidance as a defensive strategy. Additionally, the two morphotypes differ in their circadian activity, the yellow morphotype being nocturnal and the black one being diurnal. The two morphotypes hunt and associate with different ant prey and possess marked differences in venom composition. Finally, crossing trials showed complete pre-mating isolation between the two morphotypes, but there was no evidence of genetic (mitochondrial data) or environmental niche differentiation. We conclude that the two morphotypes show evidence of a deep differentiation in morphological, behavioural, physiological and ecological traits that evolved together as part of the spider's diverging lifestyles.

Hymenobacter terrestris sp. nov. and Hymenobacter lapidiphilus sp. nov., isolated from regoliths in Antarctica.

A group of four psychrotrophic bacterial strains was isolated on James Ross Island (Antarctica) in 2013. All isolates, originating from different soil samples, were collected from the ice-free northern part of the island. They were rod-shaped, Gram-stain-negative, and produced moderately slimy red-pink pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, MALDI-TOF MS, rep-PCR analyses, chemotaxonomic methods and extensive biotyping was used to clarify the taxonomic position of these isolates. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to the genus Hymenobacter. The closest relative was Hymenobacter humicola CCM 8763T, exhibiting 98.3 and 98.9% 16S rRNA pairwise similarity with the reference isolates P5342T and P5252T, respectively. Average nucleotide identity, digital DNA-DNA hybridization and core gene distances calculated from the whole-genome sequencing data confirmed that P5252T and P5342T represent two distinct Hymenobacter species. The menaquinone systems of both strains contained MK-7 as the major respiratory quinone. The predominant polar lipids for both strains were phosphatidylethanolamine and one unidentified glycolipid. The major components in the cellular fatty acid composition were summed feature 3 (C16:1 ω7c/C16:1ω6c), C16:1ω5c, summed feature 4 (anteiso-C17:1 B/iso-C17:1 I), anteiso-C15:0 and iso-C15 : 0 for all isolates. Based on the obtained results, two novel species are proposed, for which the names Hymenobacter terrestris sp. nov. (type strain P5252T=CCM 8765T=LMG 31495T) and Hymenobacter lapidiphilus sp. nov. (type strain P5342T=CCM 8764T=LMG 30613T) are suggested.

Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements.

Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase gene, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

Hymenobacter humicola sp. nov., isolated from soils in Antarctica.

A set of three psychrotrophic bacterial strains was isolated from different soil samples collected at the deglaciated northern part of James Ross Island (Antarctica) in 2014. All isolates were rod-shaped, Gram-stain-negative, non-motile, catalase-positive and oxidase-negative, and produced moderately slimy red-pink pigmented colonies on Reasoner's 2A (R2A) agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, automated ribotyping, MALDI-TOF MS, chemotaxonomy methods and extensive biotyping using conventional tests and commercial identification kits was applied to the isolates in order to clarify their taxonomic position. Phylogenetic analysis based on the 16S rRNA gene showed that all isolates belonged to the genus Hymenobacter with the closest relative being Hymenobacter aerophilus DSM 13606T, exhibiting 98.5 % 16S rRNA gene pairwise similarity to the reference isolate P6312T. Average nucleotide identity values calculated from the whole-genome sequencing data proved that P6312T represents a distinct Hymenobacter species. The major components of the cellular fatty acid composition were summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 1 ω5c, summed feature 4 (C17 : 1 anteiso B/iso I), C15 : 0 anteiso and C15 : 0 iso. The menaquinone system of strain P6312T contained MK-7 as the major respiratory quinone. The predominant polar lipids were phosphatidylethanolamine and an unidentified phospholipid. Moderate to minor amounts of three unidentified polar lipids, four unidentified aminophospholipids, one unidentified glycolipid and one unidentified phospholipid were also present. Based on the obtained results, we propose a novel species for which the name Hymenobacterhumicola sp. nov. is suggested, with the type strain P6312T (=CCM 8763T=LMG 30612T).

Trophic niche, capture efficiency and venom profiles of six sympatric ant-eating spider species (Araneae: Zodariidae).

The arms race between specialist predators and their prey has resulted in the evolution of a variety of specific adaptations. In venomous predators, this can include venom composition, particularly if predators are specialized on dangerous prey. Here, we performed an integrative study using six species of highly specialized ant-eating spiders of the genus Zodarion to investigate their phylogeny, realized trophic niche, efficacy in the capture of various ant species and venom composition. Data on natural diet obtained by next-generation sequencing and field observations showed that the six Zodarion species exploit different ant species. Their phylogeny, based on mitochondrial and nuclear genes, correlated with the composition of their natural prey, indicating that closely related Zodarion species specialize on similar ant species. Prey-capture parameters differed among Zodarion species suggesting prey-specific efficacy. Similarly, the venom profiles of both low and high molecular compounds differed among species. Only the profiles of low molecular compounds were correlated with capture efficacy parameters, suggesting that the venom of Zodarion spiders contains prey-specific components. Our study suggests that Iberian Zodarion spiders are specialized on particular ant species.

Venom gland size and venom complexity-essential trophic adaptations of venomous predators: A case study using spiders.

Specialized predators possess a variety of adaptations. In venomous predators, this may include the size of the venom gland and venom composition. It is expected that due to different foraging strategies, predators with a wide trophic niche (generalists) should possess larger venom glands that contain more diversified components than predators with a narrow niche (specialists). We focused on spiders, as the most diversified group of venomous predators, in which a wide variety of trophic strategies have evolved. We conducted a comparative analysis using 40 spider species, in which we measured the size of their venom gland and venom complexity using proteome profiling methods. The species were classified into three trophic groups: generalists, facultative specialists and obligatory specialists. We found that the venom glands of generalists are larger than those of obligatory specialists, which is presumably due to more frequent prey capture by the former. The complexity of venom of peptides (2-15 kDa) and proteins (15-250 kDa) was more diverse in generalists than in specialists. Multivariate analysis of venom revealed significant differences among the three trophic categories only in the complexity of peptides. Our study thus shows that venom gland size and its content have taken different pathways during the evolution of different trophic strategies in spiders. Generalists evolved larger venom glands with more complex composition, whereas obligatory specialists possess smaller glands with less diverse chemical structures.

Proteomic characterization and antifungal activity of potato tuber proteins isolated from starch production waste under different temperature regimes.

Proteins were obtained from effluent of a starch manufacture by using different isolation temperatures (40, 60, 80, and 100 °C). The proteins, remaining in effluent after treatment of potato juice at 80 and 100 °C differed significantly in composition and in structural stability as well as in trypsin inhibitory and antifungal activities in comparison with the variants of 40 and 60 °C. The protein samples of 80 °C exhibited the highest antifungal activity and its average value of IC50 against five strains of two Fusarium species was determined in average at 0.18 mg ml-1. The 80 °C protein samples consisted predominantly of low-molecular proteins (7-17 kDa) identified as potato tuber protease inhibitors I and II. Predominantly, protease inhibitors II were identified for the protein samples obtained by 100 °C and here we identified 7 spots in comparison with 12 identified for the 80 °C samples. Samples of 40 and 60 °C with low antifungal activities represent high variability of detected and identified proteins. We identified various representatives of aspartic, cysteine, and serine protease inhibitors in both types of samples. These samples also contained Kunitz-type protease inhibitors that were not found in the 80 and 100 °C samples which documented thermal unstableness of Kunitz-type protease inhibitors. Functional stability at high temperatures and antifungal activity of isolated potato protease inhibitors I and II support the potential of this fraction usage in food, feed, pharmaceutical, or agricultural industry and offer new products for starch manufactures. At the same time, utilization of the stable protein fraction of waste deproteinized potato water promotes exploitation of potato starch production resources.

Acinetobacter colistiniresistens sp. nov. (formerly genomic species 13 sensu Bouvet and Jeanjean and genomic species 14 sensu Tjernberg and Ursing), isolated from human infections and characterized by intrinsic resistance to polymyxins.

Strains of the genusAcinetobacter, classified as genomic species 13BJ/14TU have been previously associated with human infections and resistance to colistin. To clarify the taxonomy of this provisional group, we investigated 24 strains that have been isolated from humans since the 1960s in 10 countries. The genus-wide analysis of the rpoB and gyrB sequences of all strains and whole-genome sequences of strains representing different rpoB/gyrB genotypes showed that the 24 strains formed a distinct monophyletic group within the so-called haemolytic clade of the genus Acinetobacter. The distinctness of the group at the species level was supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by matrix-assisted laser desorption ionization-time-of-flight MS. The 24 strains had very similar metabolic features and could be distinguished from other members of the genus by the combination of strong haemolytic and proteolytic activities and the ability to oxidize d-glucose and grow on phenylacetate and/or l-phenylalanine. The minimum inhibitory concentrations of the 24 strains to colistin and polymyxin B ranged from 16 to 64 mgl-1 and from 4 to 32 mgl-1, respectively, so uniformly reaching the current clinical resistance breakpoint (4 mg l-1) for these drugs. Genus-wide comparison revealed that such a consistently high level of resistance to polymyxins is a unique feature among species of the genus Acinetobacter,which occur in humans. We conclude that genomic species 13BJ/14TU represents a biologically meaningful and medically relevant species, for which the name Acinetobacter colistiniresistens sp. nov. is proposed. The type strain is NIPH 2036T (=CCM 8641T=CIP 110478T=CCUG 67966T=CNCTC 7573T).

Emergence of the First Catalytic Oligonucleotides in a Formamide-Based Origin Scenario.

50 years after the historical Miller-Urey experiment, the formamide-based scenario is perhaps the most powerful concurrent hypothesis for the origin of life on our planet besides the traditional HCN-based concept. The information accumulated during the last 15 years in this topic is astonishingly growing and nowadays the formamide-based model represents one of the most complete and coherent pathways leading from simple prebiotic precursors up to the first catalytically active RNA molecules. In this work, we overview the major events of this long pathway that have emerged from recent experimental and theoretical studies, mainly concentrating on the mechanistic, methodological, and structural aspects of this research.

Taxonomy of haemolytic and/or proteolytic strains of the genus Acinetobacter with the proposal of Acinetobacter courvalinii sp. nov. (genomic species 14 sensu Bouvet & Jeanjean), Acinetobacter dispersus sp. nov. (genomic species 17), Acinetobacter modestus sp. nov., Acinetobacter proteolyticus sp. nov. and Acinetobacter vivianii sp. nov.

We aimed to define the taxonomic status of 40 haemolytic and/or proteolytic strains of the genus Acinetobacter which were previously classified into five putative species termed as genomic species 14BJ (n=9), genomic species 17 (n=9), taxon 18 (n=7), taxon 19 (n=6) and taxon 20 (n=9). The strains were recovered mostly from human clinical specimens or soil and water ecosystems and were highly diverse in geographical origin and time of isolation. Comparative analysis of the rpoB and gyrB gene sequences of all strains, and the whole-genome sequences of selected strains, showed that these putative species formed five respective, well-supported clusters within a distinct clade of the genus Acinetobacter which typically, although not exclusively, encompasses strains with strong haemolytic activity. The whole-genome-based average nucleotide identity (ANIb) values supported the species status of each of these clusters. Moreover, the distinctness and coherence of the clusters were supported by whole-cell profiling based on MALDI-TOF MS. Congruent with these findings were the results of metabolic and physiological testing. We conclude that the five putative taxa represent respective novel species, for which the names Acinetobacter courvalinii sp. nov. (type strain ANC 3623T=CCUG 67960T=CIP 110480T=CCM 8635T), Acinetobacter dispersus sp. nov. (type strain ANC 4105T=CCUG 67961T=CIP 110500T=CCM 8636T), Acinetobacter modestus sp. nov. (type strain NIPH 236T=CCUG 67964T=CIP 110444T=CCM 8639T), Acinetobacter proteolyticus sp. nov. (type strain NIPH 809T=CCUG 67965T=CIP 110482T = CCM 8640T) and Acinetobacter vivianii sp. nov. (type strain NIPH 2168T=CCUG 67967T=CIP 110483T=CCM 8642T) are proposed.

Acinetobacter albensis sp. nov., isolated from natural soil and water ecosystems.

We have studied the taxonomic position of a phenetically unique group of eight strains of the genus Acinetobacter which were isolated from soil and water samples collected in protected landscape areas in the Czech Republic. Each of the comparative sequence analyses of the 16S rRNA, gyrB and rpoB genes showed that the eight strains formed a cohesive and tight cluster (intracluster sequence identities of ≥ 99.9 %, ≥ 98.5 % and ≥ 97.7 %, respectively), which was clearly separated from all hitherto known species of the genus Acinetobacter ( ≤ 98.6 %, ≤ 84.5 % and ≤ 89.3 %, respectively). Congruent with these findings were the results of comparative sequence analysis of three additional housekeeping genes (gltA, pyrG and recA). This genotypic distinctness was mirrored by the uniqueness of the combination of a number of independent phenotypic markers including the whole-cell spectra produced by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) MS and physiological and metabolic features. The most useful phenotypic features to differentiate the eight strains from all known species of the genus Acinetobacter were the ability to assimilate tricarballylate and the inability to grow at 35 °C or to assimilate ethanol or l-histidine. We conclude that the eight strains represent a novel environmental species for which the name Acinetobacter albensis sp. nov. is proposed. The type strain is ANC 4874T ( = CCUG 67281T = CCM 8611T).

Acinetobacter seifertii sp. nov., a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex isolated from human clinical specimens.

This study aimed to define the taxonomic status of a phenetically distinct group of 16 strains that corresponds to Acinetobacter genomic species 'close to 13TU', a provisional genomic species of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex recognized by Gerner-Smidt and Tjernberg in 1993. These strains have been isolated in different countries since the early 1990s and were mostly recovered from human clinical specimens. They were compared with 45 reference strains representing the known taxa of the ACB complex using taxonomic methods relevant to the genus Acinetobacter. Based on sequence analysis of the concatenated partial sequences (2976 bp) of seven housekeeping genes, the 16 strains formed a tight and well-supported cluster (intracluster sequence identity of ≥98.4 %) that was clearly separated from the other members of the ACB complex (≤94.7 %). The species status of the group was supported by average nucleotide identity values of ≤91.7 % between the whole genome sequence of representative strain NIPH 973(T) (NCBI accession no. APOO00000000) and those of the other species. In addition, whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS analyses indicated the distinctness of the group at the protein level. Metabolic and physiological tests revealed several typical features of the group, although they did not allow its reliable differentiation from the other members of the ACB complex. We conclude that the 16 strains represent a distinct novel species, for which we propose the name Acinetobacter seifertii sp. nov. The type strain is NIPH 973(T) ( = CIP 110471(T) = CCUG 34785(T) = CCM 8535(T)).

Acinetobacter variabilis sp. nov. (formerly DNA group 15 sensu Tjernberg & Ursing), isolated from humans and animals.

We aimed to define the taxonomic status of 16 strains which were phenetically congruent with Acinetobacter DNA group 15 described by Tjernberg & Ursing in 1989. The strains were isolated from a variety of human and animal specimens in geographically distant places over the last three decades. Taxonomic analysis was based on an Acinetobacter-targeted, genus-wide approach that included the comparative sequence analysis of housekeeping, protein-coding genes, whole-cell profiling based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), an array of in-house physiological and metabolic tests, and whole-genome comparative analysis. Based on analyses of the rpoB and gyrB genes, the 16 strains formed respective, strongly supported clusters clearly separated from the other species of the genus Acinetobacter. The distinctness of the group at the species level was indicated by average nucleotide identity values of ≤82 % between the whole genome sequences of two of the 16 strains (NIPH 2171(T) and NIPH 899) and those of the known species. In addition, the coherence of the group was also supported by MALDI-TOF MS. All 16 strains were non-haemolytic and non-gelatinase-producing, grown at 41 °C and utilized a rather limited number of carbon sources. Virtually every strain displayed a unique combination of metabolic and physiological features. We conclude that the 16 strains represent a distinct species of the genus Acinetobacter, for which the name Acinetobacter variabilis sp. nov. is proposed to reflect its marked phenotypic heterogeneity. The type strain is NIPH 2171(T) ( = CIP 110486(T) = CCUG 26390(T) = CCM 8555(T)).

Proteome analysis in Arabidopsis reveals shoot- and root-specific targets of cytokinin action and differential regulation of hormonal homeostasis.

The plant hormones cytokinins (CKs) regulate multiple developmental and physiological processes in Arabidopsis (Arabidopsis thaliana). Responses to CKs vary in different organs and tissues (e.g. the response to CKs has been shown to be opposite in shoot and root samples). However, the tissue-specific targets of CKs and the mechanisms underlying such specificity remain largely unclear. Here, we show that the Arabidopsis proteome responds with strong tissue and time specificity to the aromatic CK 6-benzylaminopurine (BAP) and that fast posttranscriptional and/or posttranslational regulation of protein abundance is involved in the contrasting shoot and root proteome responses to BAP. We demonstrate that BAP predominantly regulates proteins involved in carbohydrate and energy metabolism in the shoot as well as protein synthesis and destination in the root. Furthermore, we found that BAP treatment affects endogenous hormonal homeostasis, again with strong tissue specificity. In the shoot, BAP up-regulates the abundance of proteins involved in abscisic acid (ABA) biosynthesis and the ABA response, whereas in the root, BAP rapidly and strongly up-regulates the majority of proteins in the ethylene biosynthetic pathway. This was further corroborated by direct measurements of hormone metabolites, showing that BAP increases ABA levels in the shoot and 1-aminocyclopropane-1-carboxylic acid, the rate-limiting precursor of ethylene biosynthesis, in the root. In support of the physiological importance of these findings, we identified the role of proteins mediating BAP-induced ethylene production, METHIONINE SYNTHASE1 and ACC OXIDASE2, in the early root growth response to BAP.

Acinetobacter gandensis sp. nov. isolated from horse and cattle.

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467(T) showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467(T) showed the highest similarities with 'Acinetobacter bohemicus' ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467(T) were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467(T) and UG 60716 showed a DNA-DNA relatedness of 84 % with each other and a DNA-DNA relatedness with A. schindleri LMG 19576(T) of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467(T) was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467(T) ( = ANC 4275(T) = LMG 27960(T) = DSM 28097(T)).

Improvement in Staphylococcus and Bacillus strain differentiation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling by using microwave-assisted enzymatic digestion.

RATIONALE: Distinguishing between individual bacterial strains below the species level is a challenge to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial profiling. We propose a quick method for improving strain differentiation of two Staphylococcus and one Bacillus species. METHODS: An alternative procedure to the extraction protocol recommended by Bruker Daltonics was developed. Ethanol-sterilized cells of six S. aureus and six S. haemolyticus strains were digested by trypsin using 2-min microwave irradiation and were then analyzed. Twenty-eight strains belonging to two ecotypes of B. subtilis were subjected to the same procedure to extend the scope of the method. RESULTS: S. aureus and S. haemolyticus strains, only partially distinguishable by the standard sample preparation procedure, were subjected to microwave-assisted tryptic digestion. The repeatability of the procedure was checked in three experiments accomplished at weekly intervals. Clear distinction of the strains was achieved by cluster analysis. The differentiation of B. subtilis ecotypes was also improved significantly by the digestion method. The discriminatory power of the novel method was supported by an increase in the number of strain-specific peaks, as compared to the standard method. CONCLUSIONS: The method modulates the discriminatory power of MALDI-TOF MS profiling. The differentiation of a set of S. aureus, S. haemolyticus and B. subtilis strains was improved significantly after microwave-accelerated tryptic digestion of the cellular material.

A simplified method for peptide de novo sequencing using (18)O labeling.

Incorporation of an (18)O atom into a peptide C-terminus by proteolytic cleavage in the presence of H2(18)O is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a H2(16)O/H2(18)O mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for (18)O incorporation into tryptic peptides by adding H2(18)O and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)- MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.