Zbyněk Zdráhal

Sample preparation methods for MALDI-MS profiling of bacteria

Direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) bacterial cell or lysate analysis appears to meet all the criteria required for a rapid and reliable analytical microorganism identification and taxonomical classification tool. Few-minute analytical procedure providing information extending up to sub-species level underlines the potential of the MALDI-MS profiling in comparison with other methods employed in the field. However, the quality of MALDI-MS profiles and consequently the performance of the method are influenced by numerous factors, which involve particular steps of the sample preparation procedure. This review is aimed at advances in development and optimization of the MALDI-MS profiling methodology. Approaches improving the quality of the MALDI-MS profiles and universal feasibility of the method are discussed.

Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to accommodate Acinetobacter genomic species 10 and 11, respectively

Acinetobacter genospecies (genomic species) 10 and 11 were described by Bouvet and Grimont in 1986 on the basis of DNA-DNA reassociation studies and comprehensive phenotypic analysis. In the present study, the names Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., respectively, are proposed for these genomic species based on the congruence of results of polyphasic analysis of 33 strains (16 and 17 strains of genomic species 10 and 11, respectively). All strains were investigated by selective restriction fragment amplification (i.e. AFLP) analysis rpoB sequence analysis, amplified rDNA restriction analysis and tDNA intergenic length polymorphism analysis, and their nutritional and physiological properties were determined. Subsets of the strains were studied by 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or had been classified previously by DNA-DNA reassociation. Results indicate that A. bereziniae and A. guillouiae represent two phenetically and phylogenetically distinct groups within the genus Acinetobacter. Based on the comparative analysis of housekeeping genes (16S rRNA and rpoB genes), these species together represent a monophyletic branch within the genus. Despite their overall phenotypic similarity, the ability to oxidize d-glucose and to grow at 38 degrees C can be used in the presumptive differentiation of these two species from each other: with the exception of three strains that were positive for only one test, A. bereziniae strains were positive for both tests, whereas A. guillouiae strains were negative in these tests. The strains of A. bereziniae originated mainly from human clinical specimens, whereas A. guillouiae strains were isolated from different environmental sources in addition to human specimens. The type strain of A. bereziniae sp. nov. is LMG 1003(T) (=CIP 70.12(T) =ATCC 17924(T)) and that of A. guillouiae sp. nov. is LMG 988(T) (=CIP 63.46( T) =ATCC 11171(T) =CCUG 2491(T)).

Identification of lactobacilli isolated from food by genotypic methods and MALDI-TOF MS

Lactobacilli are bacteria with important implications in food and feed fermentation. Detailed knowledge of the lactobacilli composition is of high relevance to food and health control, various industrial or biotechnological applications, etc., but accurate identification of the Lactobacillus species is not an easy task. In this study, three methods, i.e. polymerase chain reaction (PCR), amplified 16S rDNA restriction analysis with restrictase MseI (16S-ARDRA), and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were evaluated for their capabilities to identify Lactobacillus species. After comparison of the three methods on a set of 19 type/reference Lactobacillus strains, 148 strains isolated from dairy and meat products were characterized by PCR and MALDI-TOF MS. The strains were differentiated into nine and ten lactobacilli species by PCR and MALDI-TOF MS, respectively, with nine of these species being congruent. The success rates of species level assignment were 77% for PCR and 93% for MALDI-TOF MS that proved superior in the species identification. However, to differentiate between closely related Lactobacillus species, MALDI-TOF MS needs to be used in combination with genotypic techniques to achieve a more reliable identification.

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Huwe1-Mediated Ubiquitylation of Dishevelled Defines a Negative Feedback Loop in the Wnt Signaling Pathway

By ubiquitylating a protein interaction domain in Dishevelled, Huwe1 acts as a negative feedback regulator of Wnt signaling. UbiqWNTin Feedback The Wnt/β-catenin pathway directs the migration of the Q neuroblasts in Caenorhabditis elegans. In response to Wnt pathway activation, Dishevelled (Dvl) is activated and the degradation of β-catenin is prevented. Dvl activity is regulated by phosphorylation and ubiquitylation. Using an RNA interference screen targeting ubiquitin ligases and deubiquitylating enzymes, de Groot et al. identified the E3 ubiquitin ligase EEL-1 as a Wnt signaling inhibitor that affected Q neuroblast migration. Knockdown of the EEL-1 homolog Huwe1 in human embryonic kidney 293T cells enhanced the activity of the Wnt/β-catenin transcriptional pathway. Huwe1 interacted and colocalized with Dvl. Huwe1 promoted the ubiquitylation of the DIX domain in Dvl and inhibited Dvl multimerization. Thus, Huwe1 functions as an evolutionarily conserved feedback inhibitor of Wnt signaling. Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/β-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/β-catenin pathway, we performed an RNA interference screen in Caenorhabditis elegans and identified the HECT domain–containing ubiquitin ligase EEL-1 as an inhibitor of Wnt signaling. In human embryonic kidney 293T cells, knockdown of the EEL-1 homolog Huwe1 enhanced the activity of a Wnt reporter in cells stimulated with Wnt3a or in cells that overexpressed casein kinase 1 (CK1) or a constitutively active mutant of the Wnt co-receptor low-density lipoprotein receptor–related protein 6 (LRP6). However, knockdown of Huwe1 had no effect on reporter gene expression in cells expressing constitutively active β-catenin, suggesting that Huwe1 inhibited Wnt signaling upstream of β-catenin and downstream of CK1 and LRP6. Huwe1 bound to and ubiquitylated the cytoplasmic Wnt pathway component Dishevelled (Dvl) in a Wnt3a- and CK1ε-dependent manner. Mass spectrometric analysis showed that Huwe1 promoted K63-linked, but not K48-linked, polyubiquitination of Dvl. Instead of targeting Dvl for degradation, ubiquitylation of the DIX domain of Dvl by Huwe1 inhibited Dvl multimerization, which is necessary for its function. Our findings indicate that Huwe1 is part of an evolutionarily conserved negative feedback loop in the Wnt/β-catenin pathway.

New perspectives of valproic acid in clinical practice.

Introduction: Valproic acid (VPA) has been used in clinical practice as an anticonvulsant for more than four decades. Its pharmacokinetics and toxicity are thus well documented. VPA is also a potent class-selective histone deacetylase (HDAC) inhibitor at nontoxic therapeutic concentrations. New areas of application for VPA are currently opening up in clinical practice. Areas covered: The authors discuss VPA and how it may serve as an effective drug for cancer therapy. This is due to its ability to induce differentiation of a number of cancer cells in vitro and also to decrease tumor growth and metastases in animal models. The authors highlight how the utilization of VPA as an HDAC inhibitor is not limited to a single-agent therapy. Early clinical studies have also revealed promising potency of VPA in combination treatment with classic anticancer drugs. The authors do this by summarizing the published results and providing insight into the potential future developments for this field. Expert opinion: VPA was shown to restore or improve responsiveness of tumors to conventional therapeutic agents, to enhance the efficacy of adenoviral gene therapy, to sensitize TRAIL-resistant tumor cells to apoptosis, and to enhance radiosensitivity of tumor cells. Drawbacks in VPA medical applications include its teratogenicity and complexity of its effects.

Achromobacter marplatensis sp. nov., isolated from a pentachlorophenol-contaminated soil

A polyphasic taxonomic approach was applied to the study of a Gram-negative bacterium (B2(T)) isolated from soil by selective enrichment with pentachlorophenol. 16S rRNA gene sequence analysis of strain B2(T) showed that the strain belongs to the genus Achromobacter within the Betaproteobacteria. The 16S rRNA gene sequence displayed more than 99 % similarity to the sequences of the type strains of all species of Achromobacter, with the highest sequence similarity to those of Achromobacter spanius CCM 7183(T) and A. piechaudii CCM 2986(T) (99.8 %). On the basis of phylogenetic analysis, genomic DNA-DNA relatedness and phenotypic characteristics, including chemotaxonomic (cellular fatty acid profile) analysis, a novel species is proposed, Achromobacter marplatensis sp. nov., with the type strain B2(T) ( = CCM 7608(T)  = CCUG 56371(T)  = CECT 7342(T)).

Nanosecond Time-Dependent Stokes Shift at the Tunnel Mouth of Haloalkane Dehalogenases

The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide-hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.

Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus—Acinetobacter baumannii complex

MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.

Proteome Analysis in Arabidopsis Reveals Shoot- and Root-Specific Targets of Cytokinin Action and Differential Regulation of Hormonal Homeostasis

Summary: The plant hormone cytokinin regulates the Arabidopsis proteome in a shoot- and root-specific way, and the cytokinin-mediated tissue-specific modulation of hormonal metabolism is an intrinsic component of the Arabidopsis response to cytokinins. 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.