Repurposing drugs with specific activity against L-form bacteria.

Cell wall deficient "L- form" bacteria are of growing medical interest as a possible source of recurrent or persistent infection, largely because of their complete resistance to cell wall active antibiotics such as ß-lactams. Antibiotics that specifically kill L-forms would be of potential interest as therapeutics, but also as reagents with which to explore the role of L-forms in models of recurrent infection. To look for specific anti-L-form antibiotics, we screened a library of several hundred FDA-approved drugs and identified compounds highly selective for L-form killing. Among the compounds identified were representatives of two different classes of calcium channel blockers: dihydropyridines, e.g., manidipine; and diphenylmethylpiperazine, e.g., flunarizine. Mode of action studies suggested that both classes of compound work by decreasing membrane fluidity. This leads to a previously recognized phenotype of L-forms in which the cells can continue to enlarge but fail to divide. We identified a considerable degree of variation in the activity of different representatives of the two classes of compounds, suggesting that it may be possible to modify them for use as drugs for L-form-dependent infections.

A Small Molecule Inhibitor of CTP Synthetase Identified by Differential Activity on a Bacillus subtilis Mutant Deficient in Class A Penicillin-Binding Proteins.

In the course of screening for compounds with differential growth inhibition activity on a mutant of Bacillus subtilis lacking all four class A penicillin-binding proteins (Δ4), we came across an isoquinoline derivative, IQ-1 carboxylic acid (IQC) with relatively high activity on the mutant compared to the wild type strain. Treated cells were slightly elongated and had altered chromosome morphology. Mutants of Δ4 resistant to IQC were isolated and subjected to whole genome sequencing. Most of the mutants were affected in the gene, pyrG, encoding CTP synthetase (CTPS). Purified wild type CTPS was inhibited in vitro by IQC. Two of the three mutant proteins purified showed decreased sensitivity to IQC in vitro. Finally, inhibition by IQC was rescued by addition of cytidine but not uridine to the growth medium, consistent with the notion that IQC acts by reducing the synthesis of CTP or a related compound. IQC provides a promising new starting point for antibiotic inhibitors of CTPS.

Chitin is a functional component of the larval adhesive of barnacles.

Barnacles are the only sessile crustaceans, and their larva, the cyprid, is supremely adapted for attachment to surfaces. Barnacles have a universal requirement for strong adhesion at the point of larval attachment. Selective pressure on the cyprid adhesive has been intense and led to evolution of a tenacious and versatile natural glue. Here we provide evidence that carbohydrate polymers in the form of chitin provide stability to the cyprid adhesive of Balanus amphitrite. Chitin was identified surrounding lipid-rich vesicles in the cyprid cement glands. The functional role of chitin was demonstrated via removal of freshly attached cyprids from surfaces using a chitinase. Proteomic analysis identified a single cement gland-specific protein via its association with chitin and localized this protein to the same vesicles. The role of chitin in cyprid adhesion raises intriguing questions about the evolution of barnacle adhesion, as well as providing a new target for antifouling technologies.

Cold-modulated small proteins abundance in winter triticale (x Triticosecale, Wittm.) seedlings tolerant to the pink snow mould (Microdochium nivale, Samuels and Hallett) infection.

Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4-50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo's leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- ß-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.

RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway.

The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides. For many years, the penicillin-binding proteins were thought to be the key enzymes required for wall synthesis. RodA and possibly other proteins in the wider SEDS (shape, elongation, division and sporulation) family have now emerged as a previously unknown class of essential glycosyltranferase enzymes, which play key morphogenetic roles in bacterial cell wall synthesis. We provide evidence in support of this role and the discovery of small natural product molecules that probably target these enzymes. The SEDS proteins have exceptional potential as targets for new antibacterial therapeutic agents.

MALDI-TOF Mass Spectrometry Discriminates Known Species and Marine Environmental Isolates of Pseudoalteromonas.

The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies.

Proteomic-based biotyping reveals hidden diversity within a microalgae culture collection: An example using Dunaliella.

Accurate and defendable taxonomic identification of microalgae strains is vital for culture collections, industry and academia; particularly when addressing issues of intellectual property. We demonstrate the remarkable effectiveness of Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) biotyping to deliver rapid and accurate strain separation, even in situations where standard molecular tools prove ineffective. Highly distinctive MALDI spectra were obtained for thirty two biotechnologically interesting Dunaliella strains plus strains of Arthrospira, Chlorella, Isochrysis, Tetraselmis and a range of culturable co-occurring bacteria. Spectra were directly compared with genomic DNA sequences (internal transcribed spacer, ITS). Within individual Dunaliella isolates MALDI discriminated between strains with identical ITS sequences, thereby emphasising and enhancing knowledge of the diversity within microalgae culture collections. Further, MALDI spectra did not vary with culture age or growth stage during the course of the experiment; therefore MALDI presents stable and accurate strain-specific signature spectra. Bacterial contamination did not affect MALDI's discriminating power. Biotyping by MALDI-TOF-MS will prove effective in situations wherein precise strain identification is vital, for example in cases involving intellectual property disputes and in monitoring and safeguarding biosecurity. MALDI should be accepted as a biotyping tool to complement and enhance standard molecular taxonomy for microalgae.

Chemistry-specific surface adsorption of the barnacle settlement-inducing protein complex.

Gregarious settlement in barnacle larvae (cyprids) is induced by a contact pheromone, the settlement-inducing protein complex (SIPC). The SIPC has been identified both in the cuticle of adult barnacles and in the temporary adhesive secretion (footprint) of cyprids. Besides acting as a settlement inducer, the presence of the SIPC in footprints points to its additional involvement in the adhesion process. SIPC adsorption behaviour was therefore investigated on a series of self-assembled monolayers (SAMs) by surface plasmon resonance at the pH of seawater (8.3). Fibrinogen and α2-macroglobulin (A2M) (blood complement protease inhibitors with which the SIPC shares 29% sequence homology) were used in the adsorption experiments as positive and negative standards, respectively. The mass uptake of the SIPC was comparable to that of fibrinogen, with adsorption observed even on the protein-resistant oligo(ethylene glycol) surface. Notably, on the positively charged SAM the SIPC showed a kinetic overshoot, indicating a metastable configuration causing the amount of adsorbed protein to temporarily exceed its equilibrium value. A2M adsorption was low or negligible on all SAMs tested, except for the positively charged surface, indicating that A2M adsorption is mainly driven by electrostatics. Evaluation of SIPC non-specific adsorption kinetics revealed that it adsorbed irreversibly and non-cooperatively on all surfaces tested.

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.

Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.

The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.

Acclimation to high CO2 in maize is related to water status and dependent on leaf rank.

The responses of C(3) plants to rising atmospheric CO(2) levels are considered to be largely dependent on effects exerted through altered photosynthesis. In contrast, the nature of the responses of C(4) plants to high CO(2) remains controversial because of the absence of CO(2) -dependent effects on photosynthesis. In this study, the effects of atmospheric CO(2) availability on the transcriptome, proteome and metabolome profiles of two ranks of source leaves in maize (Zea mays L.) were studied in plants grown under ambient CO(2) conditions (350 +/- 20 µL L(-1) CO(2) ) or with CO(2) enrichment (700 +/- 20 µL L(-1) CO(2) ). Growth at high CO(2) had no effect on photosynthesis, photorespiration, leaf C/N ratios or anthocyanin contents. However, leaf transpiration rates, carbohydrate metabolism and protein carbonyl accumulation were altered at high CO(2) in a leaf-rank specific manner. Although no significant CO(2) -dependent changes in the leaf transcriptome were observed, qPCR analysis revealed that the abundance of transcripts encoding a Bowman-Birk protease inhibitor and a serpin were changed by the growth CO(2) level in a leaf rank specific manner. Moreover, CO(2) -dependent changes in the leaf proteome were most evident in the oldest source leaves. Small changes in water status may be responsible for the observed responses to high CO(2,) particularly in the older leaf ranks.

Changes in protein expression profiles between a low phytic acid rice ( Oryza sativa L. Ssp. japonica) line and its parental line: a proteomic and bioinformatic approach.

The seed proteome of a low phytic acid (lpa) rice line (Os-lpa-XS110-1), developed as a novel food source, was compared to that of its parental line, Xiushui 110 (XS-110). Analysis by surfaced enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) and two-dimensional gel electrophoresis (2-DE) allowed the detection of a potential low molecular weight biomarker and identification of 23 differentially expressed proteins that include stress-related proteins, storage proteins, and potential allergens. Bioinformatic analyses revealed that triose phosphate isomerase (TPI) and fructose bisphosphatealdolase (FBA), two major differentially expressed proteins, are involved in myo-inositol metabolism. Accumulation of globulin was also significantly decreased in the lpa line. This study demonstrates the potential of proteomic and bioinformatic profiling techniques for safety assessment of novel foods. Furthermore, these techniques provide powerful tools for studying functional genomics due to the possibility of identifying genes related to the mutated traits.

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies.

BACKGROUND: Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids. RESULTS: We used proteomics to search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. The interaction between Sam68 and hnRNP L proteins was observed in a cell line which exhibits low frequency of SNBs suggesting that this association also takes place outside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. CONCLUSION: Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system.

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.

A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA).

Genetically modified plants expressing insecticidal traits offer a new strategy for crop protection, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of a rice variety expressing the snowdrop Galanthus nivalis lectin (GNA lectin), and forms part of a EU-funded project where the objective has been to develop and validate sensitive and specific methods to assess the safety of genetically modified foods. Male and female Wistar rats were given a purified diet containing either 60% genetically modified or parental rice for 90 days. This corresponds to a mean daily GNA lectin intake of approximately 58 and 67mg/kg body weight for males and females, respectively. Prior to the animal study comprehensive analytical characterization of both rice materials was performed. The chemical analyses showed a number of statistically significant differences, with the majority being within the ranges reported in the literature. In the animal study a range of clinical, biological, immunological, microbiological and pathological parameters were examined. A number of significant differences were seen between groups fed the two diets, but none of them were considered to be adverse. In conclusion, the design of the present animal study did not enable us to conclude on the safety of the GM food. Additional group(s) where the expressed gene products have been spiked to the diet should be included in order to be able to distinguish whether the observed effects were due to the GNA lectin per se or to secondary changes in the GM rice.

Safety testing of GM-rice expressing PHA-E lectin using a new animal test design.

The 90-day animal study is the core study for the safety assessment of genetically modified foods in the SAFOTEST project. The model compound tested in the 90-day study was a rice variety expressing the kidney bean Phaseolus vulgaris lectin agglutinin E-form (PHA-E lectin). Female Wistar rats were given a nutritionally balanced purified diet with 60% parental rice, 60% PHA-E rice or 60% PHA-E rice spiked with 0.1% recombinant PHA-E lectin for 90 days. This corresponded to a mean daily PHA-E lectin intake of approximately 0, 30 and 100mg/kg body weight for each group, respectively. The spiking was used to increase the specificity and to demonstrate the sensitivity of the study. A range of biological, biochemical, microbiological and pathological parameters were examined and significant differences in weight of small intestine, stomach and pancreas and plasma biochemistry were seen between groups. Included in this paper are also data from the molecular characterisation and chemical analysis of the PHA-E rice, from the construction and production of the PHA-E lectin, and from the preceding 28-day in vivo study where the toxicity of the pure PHA-E lectin was determined. In conclusion, the combined use of information from the compositional analysis, the 28-day study and the characterisation of the PHA-E rice and the PHA-E lectin has improved the design of the 90-day study. The spiking procedure has facilitated the interpretation of the results of the study and transferred it into a valuable tool for the future safety testing of genetically modified foods.

Conservation of XYN11A and XYN11B xylanase genes in Bipolaris sorghicola, Cochliobolus sativus, Cochliobolus heterostrophus, and Cochliobolus spicifer.

Two types of xylanase gene, XYN11A ( XYL1) and XYN11B ( XYL2), were amplified by PCR and partially sequenced in four phytopathogenic species of the ascomycete fungal genus Cochliobolus (anamorph genus Bipolaris). Three of the species, C. heterostrophus ( B. maydis), C. sativus ( B. sorokiniana), and Bipolaris sorghicola (no teleomorph known), are interrelated; the fourth, C. spicifer ( B. spicifera), was found, through analysis of the 5.8S RNA and internal transcribed spacer (ITS) sequences of its ribosomal DNA, to be more distantly related to the other three. Isolates from all four species contain orthologous XYN11A and XYN11B genes, but a set of laboratory strains of C. heterostrophus gave no product corresponding to the XYN11B gene. The patterns of evolution of the two xylanase genes and ribosomal DNA sequences are mutually consistent; the results indicate that the two genes were present in the common ancestor of all Cochliobolus species and are evolving independently of each other.

The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan.

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.