A salivary GMC oxidoreductase of Manduca sexta re-arranges the green leaf volatile profile of its host plant.

Green leaf volatiles (GLVs) are short-chain oxylipins that are emitted from plants in response to stress. Previous studies have shown that oral secretions (OS) of the tobacco hornworm Manduca sexta, introduced into plant wounds during feeding, catalyze the re-arrangement of GLVs from Z-3- to E-2-isomers. This change in the volatile signal however is bittersweet for the insect as it can be used by their natural enemies, as a prey location cue. Here we show that (3Z):(2E)-hexenal isomerase (Hi-1) in M. sexta's OS catalyzes the conversion of the GLV Z-3-hexenal to E-2-hexenal. Hi-1 mutants that were raised on a GLV-free diet showed developmental disorders, indicating that Hi-1 also metabolizes other substrates important for the insect's development. Phylogenetic analysis placed Hi-1 within the GMCß-subfamily and showed that Hi-1 homologs from other lepidopterans could catalyze similar reactions. Our results indicate that Hi-1 not only modulates the plant's GLV-bouquet but also functions in insect development.

Vegetative Cell and Spore Proteomes of Clostridioides difficile Show Finite Differences and Reveal Potential Protein Markers.

Clostridioides difficile-associated infection (CDI) is a health-care-associated infection caused, as the name suggests, by obligate anaerobic pathogen C. difficile and thus mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate into cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of C. difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic, and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative cell proteins CD1228, CD3301 and spore proteins CD2487, CD2434, and CD0684 are identified as potential protein markers for C. difficile infection.

Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis.

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ⁻ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament⁻ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

"One-Pot" Sample Processing Method for Proteome-Wide Analysis of Microbial Cells and Spores.

PURPOSE: Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against a variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. EXPERIMENTAL DESIGN: The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead beating in 6 m urea under reductive conditions. The heterogeneous mixture was then double digested with LysC and trypsin. Next, the peptide mixture was pre-fractionated with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) followed by reverse-phase LC-FT-MS analysis of the fractions. RESULTS: "One-pot" method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from B. subtilis, B. cereus, and P. difficile. CONCLUSIONS AND MEDICAL RELEVANCE: This method can be employed for proteome-wide analysis of non-spore-forming as well as spore-forming pathogens. Analysis of spore protein profile will help to understand the sporulation and germination processes and to distinguish immunogenic protein markers.

Stoichiometry, Absolute Abundance, and Localization of Proteins in the Bacillus cereus Spore Coat Insoluble Fraction Determined Using a QconCAT Approach.

Spores of Bacillus cereus pose a threat to food safety due to their high resistance to the heat or acid treatments commonly used to make food microbiologically safe. Spores may survive these treatments and later resume growth either on foodstuffs or, after ingestion, upon entering the gut they are capable of producing toxins, which cause either vomiting or diarrhea. The outer layers of the spore, the spore coat and exosporium, consist primarily of proteins that may serve as potential biomarkers for detection. The major morphogenetic protein CotE is important for correct assembly and attachment of the outermost layer, the exosporium, and by extension retention of many proteins. However, characterization of the proteins affected by deletion of CotE has been limited to electrophoretic patterns. Here we report the effect of CotE deletion on the insoluble fraction of the spore proteome through liquid chromatography-Fourier transform tandem mass spectrometry (LC-FTMS/MS) analysis. A total of 560 proteins have been identified in both mutant and wild-type spore coat isolates. A further 163 proteins were identified exclusively in wild-type spore isolates indicating that they are dependent on CotE for their association with the spore. Several of these are newly confirmed as associated with the exosporium, namely BC_2569 (BclF), BC_3345, BC_2427, BC_2878, BC_0666, BC_2984, BC_3481, and BC_2570. A total of 153 proteins were only identified in ΔCotE spore isolates. This was observed for proteins that are known or likely to be interacting with or are encased by CotE. Crucial spore proteins were quantified using a QconCAT reference standard, the first time this was used in a biochemically heterogeneous system. This allowed us to determine the absolute abundance of 21 proteins, which spanned across three orders of magnitude and together covered 5.66% ± 0.51 of the total spore weight. Applying the QconCAT methodology to the ΔCotE mutant allowed us to quantify 4.13% ± 0.14 of the spore total weight and revealed a reduction in abundance for most known exosporium associated proteins upon CotE deletion. In contrast, several proteins, either known or likely to be interacting with or encased by CotE (i.e., GerQ), were more abundant. The results obtained provide deeper insight into the layered spore structure such as which proteins are exposed on the outside of the spore. This information is important for developing detection methods for targeting spores in a food safety setting. Furthermore, protein stoichiometry and determination of the abundance of germination mediating enzymes provides useful information for germination and outgrowth model development.

Identification and Characterization of (3Z):(2E)-Hexenal Isomerases from Cucumber.

E-2-hexenal is a volatile compound that is commonly emitted by wounded or stressed plants. It belongs to the group of so-called green leaf volatiles (GLVs), which play an important role in transferring information to plants and insects. While most biosynthetic enzymes upstream of E-2-hexenal have been studied extensively, much less is known about the enzyme responsible for the conversion from Z-3- to E-2-hexenal. In this study we have identified two (3Z):(2E)-hexenal isomerases (HIs) from cucumber fruits by classical biochemical fractionation techniques and we were able to confirm their activity by heterologous expression. Recombinant protein of the HIs did not only convert the leaf aldehyde Z-3-hexenal to E-2-hexenal, but also (Z,Z)-3,6-nonadienal to (E,Z)-2,6-nonadienal, these last two representing major flavor volatiles of cucumber fruits. Transient expression of the cucumber HIs in Nicotiana benthamiana leaves drastically changed the GLV bouquet of damaged plants from a Z-3- to an E-2-enriched GLV profile. Furthermore, transcriptional analysis revealed that the two HIs showed distinct expression patterns. While HI-1 was specifically expressed in the flesh of cucumber fruits HI-2 was expressed in leaves as well. Interestingly, wounding of cucumber leaves caused only a slight increase in HI-2 transcript levels. These results demonstrate that cucumber HIs are responsible for the rearrangement of Z-3-aldehydes in both leaves and fruits. Future research will reveal the physiological importance of an increased conversion to E-2-aldehydes for plants and insects.

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein-Protein Interactions at the Peptide Level.

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.

'Omics' for microbial food stability: Proteomics for the development of predictive models for bacterial spore stress survival and outgrowth.

Bacterial spores are ubiquitous in nature. They are stress resistant entities that are a concern to microbiological food stability due to their environmental stress resistance. In addition germinating and outgrowing spores at undesired times and places pose a significant health burden. The challenge is amplified due to the heterogeneous germination and outgrowth behaviour of isogenic spore populations. We discuss the role of different 'omics' techniques, proteomics in particular, to study spore biology in detail. With examples, the use of label-based and label-free quantitative proteomics approaches in understanding the spore physiology is demonstrated. Also the need of genomics, single cell analyses and analysis of cellular physiology is discussed briefly. Certainly accurate comprehensive data obtained from omics methods and molecular physiology will underpin the development of robust molecular models of bacterial spore germination and outgrowth.

The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores.

Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Corroborating the proteomic analyses, electron microscopy analyses show a significantly thinner outer coat layer of the spores cultured on the solid agar medium.

Bacillus subtilis Spore Inner Membrane Proteome.

The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803.

BACKGROUND: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. RESULTS: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. CONCLUSION: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression.

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

Temperature Dependence of the Proteome Profile of the Psychrotolerant Pathogenic Food Spoiler Bacillus weihenstephanensis Type Strain WSBC 10204.

Bacillus weihenstephanensis is a subspecies of the Bacillus cereus sensu lato group of spore-forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7 °C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. Comparison of the proteome profile of B. weihenstephanensis upon its exposure to different culturing conditions can reveal clues to the mechanistic basis of its psychrotolerant phenotype as well as elucidate relevant aspects of its toxigenic profile. To this end, the genome of the type strain B. weihenstephanensis WSBC 10204 was sequenced and annotated. Subsequently, the proteome profiles of cells grown at either 6 or 30 °C were compared, which revealed considerable differences and indicated several hundred (uncharacterized) proteins as being subproteome- and/or temperature-specific. In this manner, several processes were newly indicated to be dependent on growth temperature, such as varying carbon flux routes and a different role for the urea cycle. Furthermore, a possible post-translational regulatory function for acetylation was suggested. Toxin production was determined to be largely independent of growth temperature.

Simulation of the rate of transfer of antibiotic resistance between Escherichia coli strains cultured under well controlled environmental conditions.

It was demonstrated that the tetracycline resistance plasmid in Escherichia coli resembling K-12 23:06 containing the E. coli plasmid DM0133 could be transferred to tetracycline sensitive E. coli K-12 MG1655 YFP. The sensitive recipient strain has a slight metabolic advantage in continuous fermentation in absence of tetracycline pressure and as a result the numbers of the resistant recipient strain increase during fermentation. In presence of tetracycline pressure the sensitive strain is eliminated, but when it acquires tetracycline resistance the strain has still the same metabolic advantage as its sensitive parent strain in absence of tetracycline. Here a model will be shown that could explain the rate of transformation of a sensitive into a resistant recipient strain and its subsequent growth during continuous fermentation. According to the model the probability of formation of mutants would be much higher at the dilution rate of 0.09 compared to 0.28, whereas the growth of mutants would be much faster at high dilution rate. The growth model shows how the recipient mutants and the donor cells behave in relation to the dilution rate and the number of mutants. Apart from a deterministic model describing the growth rate of both the donor strain and the resistant recipient strain a stochastic model was developed that is particularly useful when low numbers of mutants are formed.

Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation.

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.

Spore proteomics: the past, present and the future.

Endospores are metabolically dormant, multi-layered cellular structures formed by Gram-positive bacteria belonging to the genera Bacillus, Clostridium and related organisms. Their external layers are composed of proteins which in part play a role in the resistance behaviour of spores to varied chemical and environmental assaults. Thus, protein analysis is of major interest in spore biology. Spore proteomic studies have been carried out previously but these studies have focused on the soluble coat protein fraction. Using gel-based techniques, protein identification and analysis were performed. Mass spectrometry-driven proteomics has opened new avenues to resolve in particular the insoluble part of the spore layer proteomes. Mass spectrometry-based qualitative and quantitative proteomics methods expand the knowledge about both the actual composition and the amount of proteins in their various layers. The techniques can also be used to study the integrity of the layers as well as spore biology in general. This notion is explored concisely in this mini-review.

Isolation of cross-linked peptides by diagonal strong cation exchange chromatography for protein complex topology studies by peptide fragment fingerprinting from large sequence databases.

Knowledge of spatial proximity of amino acid residues obtained by chemical cross-linking and mass spectrometric analysis provides information about protein folding, protein-protein interactions and topology of macromolecular assemblies. We show that the use of bis(succinimidyl)-3-azidomethyl glutarate as a cross-linker provides a solution for two major analytical problems of cross-link mapping by peptide fragment fingerprinting (PFF) from complex sequence databases, i.e., low abundance of protease-generated target peptides and lack of knowledge of the masses of linked peptides. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in cross-linked peptides to an amine group in competition with cleavage of an amide bond formed in the cross-link reaction. TCEP-induced reaction products were separated by diagonal strong cation exchange (SCX) from unmodified peptides. The relation between the sum of the masses of the cleavage products and the mass of the parent cross-linked peptide enables determination of the masses of candidate linked peptides. By reversed phase LC-MS/MS analysis of secondary SCX fractions, we identified several intraprotein and interprotein cross-links in a HeLa cell nuclear extract, aided by software tools supporting PFF from the entire human sequence database. The data provide new information about interacting protein domains, among others from assemblies involved in splicing.

A gas phase cleavage reaction of cross-linked peptides for protein complex topology studies by peptide fragment fingerprinting from large sequence database.

A high molecular weight fraction of a HeLa cell nuclear extract containing nearly 1100 identified proteins was cross-linked with bis(succinimidyl)-3-azidomethyl glutarate (BAMG). The azido group in cross-linked peptides can be reduced to an amine group. Reduction enables isolation of cross-linked peptides by diagonal strong cation exchange chromatography. Collision-induced dissociation (CID) of reduced cross-linked peptides shows abundant cleavage of the cross-link amide bonds, along with the cleavage of peptide bonds of the composing peptide pair. A defined relationship exists between the sum of the masses of a pair of cleavage products and the mass of the parent compound. This relationship enables accurate mass determination of the two composing peptides. With this knowledge, the identity of the pair of peptides in a cross-link is revealed at an extremely low false discovery rate by peptide fragment fingerprinting with MS1MS2 data from the entire human sequence databases with a conventional search engine for peptide identification. Our approach resulted in identification of 229 intraprotein and 18 interprotein cross-links. BIOLOGICAL SIGNIFICANCE: Mapping protein-protein interactions in complex samples like digests of in vitro cross-linked extracts, by interrogation of entire species specific sequence database with tandem mass spectrometric data may yield repositories of cross-linked peptides. Results will reveal interactions between proteins, the identity of which may lead to new hypotheses about molecular mechanisms and regulations of biological function, or new targets for drug development. In this paper we describe a new analytical strategy that improves existing approaches of cross-link mapping in complex samples. The cross-linker that we have designed and synthesized for our approach is membrane permeable. This opens avenues for in vivo cross-linking for better understanding of dynamic protein complex topologies involved in many biological processes.