Isolation and characterization of persisters of the pathogenic microorganism Staphylococcus aureus.

The presence of antibiotic persisters is one of the leading causes of recurrent and chronic diseases. One challenge in mechanistic research on persisters is the enrichment of pure persisters. In this work, we validated a proposed method to isolate persisters with notorious Staphylococcus aureus cultures. With this, we analyzed the proteome profile of pure persisters and revealed the distinct mechanisms associated with vancomycin and enrofloxacin induced persisters. Furthermore, morphological and metabolic characterizations were performed, indicating further differences between these two persister populations. Finally, we assessed the effect of ATP repression, protein synthesis inhibition, and reactive oxygen species (ROS) level on persister formation. In conclusion, this work provides a comprehensive understanding of S. aureus vancomycin and enrofloxacin induced persisters, facilitating a better mechanistic understanding of persisters and the development of effective strategies to combat them.

Tomato R-gene-mediated resistance against Fusarium wilt originates in roots and extends to shoots via xylem to limit pathogen colonization.

Vascular wilt disease, caused by the soil-borne fungus Fusarium oxysporum (Fo), poses a threat to many crop species. Four different tomato resistance (R) genes (I-1, I-2, I-3, and I-7) have been identified to confer protection against Fo f.sp. lycopersici (Fol). These I genes are root-expressed and mount an immune response upon perception of the invading fungus. Despite immune activation, Fol is still able to colonize the xylem vessels of resistant tomato lines. Yet, the fungus remains localized in the vessels and does not colonize adjacent tissues or cause disease. The molecular mechanism constraining Fol in the vascular system of the stem remains unclear. We here demonstrate that an I-2-resistant rootstock protects a susceptible scion from Fusarium wilt, notwithstanding fungal colonization of the susceptible scion. Proteomic analyses revealed the presence of fungal effectors in the xylem sap of infected plants, showing that the lack of fungal pathogenicity is not due to its inability to express its virulence genes. To identify mobile root-derived proteins, potentially involved in controlling fungal proliferation, comparative xylem sap proteomics was performed. We identified distinct pathogenesis-related (PR) protein profiles in xylem sap from Fol-inoculated I-1, I-2, I-3, and I-7 resistant lines. Despite structural diversity, all four immune receptors trigger the accumulation of a common set of four PR proteins: PR-5x, PR-P2, and two glucan endo-1,3-ß-D-glucosidases. This research provides insights into Fusarium resistance mechanisms and identifies a core set of proteins whose abundance correlates with defense against Fusarium wilt.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Integrative Metabolomics and Proteomics Allow the Global Intracellular Characterization of Bacillus subtilis Cells and Spores.

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores.

Molecular physiological characterization of the dynamics of persister formation in Staphylococcus aureus.

Bacteria possess the ability to enter a growth-arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely, vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences, depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy.

Interaction of Whitefly Effector G4 with Tomato Proteins Impacts Whitefly Performance.

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci. Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Discovery of Three Bemisia tabaci Effectors and Their Effect on Gene Expression in Planta.

Bemisia tabaci (whitefly) is a polyphagous agroeconomic pest species complex. Two members of this species complex, Mediterranean (MED) and Middle-East-Asia Minor 1 (MEAM1), have a worldwide distribution and have been shown to manipulate plant defenses through effectors. In this study, we used three different strategies to identify three MEAM1 proteins that can act as effectors. Effector B1 was identified using a bioinformatics-driven effector-mining strategy, whereas effectors S1 and P1 were identified in the saliva of whiteflies collected from artificial diet and in phloem exudate of tomato on which nymphs were feeding, respectively. These three effectors were B. tabaci specific and able to increase whitefly fecundity when transiently expressed in tobacco plants (Nicotiana tabacum). Moreover, they reduced growth of Pseudomonas syringae pv. tabaci in Nicotiana benthamiana. All three effectors changed gene expression in planta, and B1 and S1 also changed phytohormone levels. Gene ontology and KEGG pathway enrichment analysis pinpointed plant-pathogen interaction and photosynthesis as the main enriched pathways for all three effectors. Our data thus show the discovery and validation of three new B. tabaci MEAM1 effectors that increase whitefly fecundity and modulate plant immunity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Compatible solutes determine the heat resistance of conidia.

BACKGROUND: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. RESULTS: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. CONCLUSIONS: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Pichia kudriavzevii.

The pathogenic yeast Pichia kudriavzevii, previously known as Candida krusei, is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in P. kudriavzevii. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in P. kudriavzevii is similar to Saccharomyces cerevisiae and C. albicans and is comprised of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, P. kudriavzevii cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of P. kudriavzevii yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 33 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in P. kudriavzevii including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of P. kudriavzevii.

Identification and characterization of new proteins crucial for bacterial spore resistance and germination.

2Duf, named after the presence of a transmembrane (TM) Duf421 domain and a small Duf1657 domain in its sequence, is likely located in the inner membrane (IM) of spores in some Bacillus species carrying a transposon with an operon termed spoVA 2mob. These spores are known for their extreme resistance to wet heat, and 2Duf is believed to be the primary contributor to this trait. In this study, we found that the absence of YetF or YdfS, both Duf421 domain-containing proteins and found only in wild-type (wt) B. subtilis spores with YetF more abundant, leads to decreased resistance to wet heat and agents that can damage spore core components. The IM phospholipid compositions and core water and calcium-dipicolinic acid levels of YetF-deficient spores are similar to those of wt spores, but the deficiency could be restored by ectopic insertion of yetF, and overexpression of YetF increased wt spore resistance to wet heat. In addition, yetF and ydfS spores have decreased germination rates as individuals and populations with germinant receptor-dependent germinants and increased sensitivity to wet heat during germination, potentially due to damage to IM proteins. These data are consistent with a model in which YetF, YdfS and their homologs modify IM structure to reduce IM permeability and stabilize IM proteins against wet heat damage. Multiple yetF homologs are also present in other spore forming Bacilli and Clostridia, and even some asporogenous Firmicutes, but fewer in asporogenous species. The crystal structure of a YetF tetramer lacking the TM helices has been reported and features two distinct globular subdomains in each monomer. Sequence alignment and structure prediction suggest this fold is likely shared by other Duf421-containing proteins, including 2Duf. We have also identified naturally occurring 2duf homologs in some Bacilli and Clostridia species and in wt Bacillus cereus spores, but not in wt B. subtilis. Notably, the genomic organization around the 2duf gene in most of these species is similar to that in spoVA 2mob, suggesting that one of these species was the source of the genes on this operon in the extremely wet heat resistant spore formers.

Time-Resolved Proteomics of Germinating Spores of Bacillus cereus.

Bacillus cereus is a spore-forming human pathogen that is a burden to the food chain. Dormant spores are highly resistant to harsh environmental conditions, but lose resistance after germination. In this study, we investigate the B. cereus spore proteome upon spore germination and outgrowth so as to obtain new insights into the molecular mechanisms involved. We used mass spectrometry combined with co-expression network analysis and obtained a unique global proteome view of the germination and outgrowth processes of B. cereus spores by monitoring 2211 protein changeovers. We are the first to examine germination and outgrowth models of B. cereus spores experimentally by studying the dynamics of germinant receptors, other proteins involved in spore germination and resistance, and coat and exosporium proteins. Furthermore, through the co-expression analysis of 1175 proteins identified with high quality data, germination proteome data were clustered into eight modules (termed black, blue, brown, green, red, turquoise, grey, and yellow), whose associated functions and expression profiles were investigated. Germination related proteins were clustered into blue and brown modules, the abundances of which decreased after finishing germination. In the brown and blue we identified 124 proteins that could be vital during germination. These proteins will be very interesting to study in future genetic studies regarding their function in spore revival in B. cereus.

Changes in the Spore Proteome of Bacillus cereus in Response to Introduction of Plasmids.

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore proteome were investigated. Proteomic analysis showed that overexpression of the target proteins had negligible effects on the spore proteome. However, plasmid-bearing spores displayed dramatic abundance changes in spore proteins involved in signaling and metabolism. Our findings indicate that the introduction of a plasmid alone alters the spore protein composition dramatically, with 993 proteins significantly down-regulated and 415 proteins significantly up-regulated among 3323 identified proteins. This shows that empty vector controls are more appropriate to compare proteome changes due to plasmid-encoded genes than is the wild-type strain, when using plasmid-based genetic tools. Therefore, researchers should keep in mind that molecular cloning techniques can alter more than their intended targets in a biological system, and interpret results with this in mind.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

UPLC-MS/MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.

MAIN CONCLUSION: Solanoeclepin A is a hatching stimulant for potato cyst nematode in very low (pM) concentrations. We report a highly sensitive method for the analysis of SolA in plant root exudates using UHPLC-MS/MS and show that there is considerable natural variation in SolA production in Solanum spp. corresponding with their hatching inducing activity. Potato cyst nematode (PCN) is a plant root sedentary endoparasite, specialized in the infection of solanaceous species such as potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Earlier reports (Mulder et al. in Hatching agent for the potato cyst nematode, Patent application No. PCT/NL92/00126, 1996; Schenk et al. in Croat Chem Acta 72:593-606, 1999) showed that solanoeclepin A (SolA), a triterpenoid metabolite that was isolated from the root exudate of potato, induces the hatching of PCN. Its low concentration in potato root exudate has hindered progress in fully understanding its hatching inducing activity and exploitation in the control of PCN. To further investigate the role of SolA in hatching of PCN, the establishment of a highly sensitive analytical method is a prerequisite. Here we present the efficient single-step extraction and UHPLC-MS/MS based analysis for rapid determination of SolA in sub-nanomolar concentrations in tomato root exudate. This method was used to analyze SolA production in different tomato cultivars and related solanaceous species, including the trap crop Solanum sisymbriifolium. Hatching assays with PCN, Globodera pallida, with root exudates of tomato genotypes revealed a significant positive correlation between SolA concentration and hatching activity. Our results demonstrate that there is natural variation in SolA production within solanaceous species and that this has an effect on PCN hatching. The analytical method we have developed can potentially be used to support breeding for crop genotypes that induce less hatching and may therefore display reduced infection by PCN.

High Resolution Analysis of Proteome Dynamics during Bacillus subtilis Sporulation.

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation.

Towards low false discovery rate estimation for protein-protein interactions detected by chemical cross-linking.

Chemical cross-linking (CX) of proteins in vivo or in cell free extracts followed by mass spectrometric (MS) identification of linked peptide pairs (CXMS) can reveal protein-protein interactions (PPIs) both at a proteome wide scale and the level of cross-linked amino acid residues. However, error estimation at the level of PPI remains challenging in large scale datasets. Here we discuss recent advances in the recognition of spurious inter-protein peptide pairs and in diminishing the FDR for these PPI-signaling cross-links, such as the use of chromatographic retention time prediction, in order to come to a more reliable reporting of PPIs.

Molecular Physiological Characterization of a High Heat Resistant Spore Forming Bacillus subtilis Food Isolate.

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain's spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins.

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE-CotE and CotJA-CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.