Draft genome sequences of two thermophilic, spore-forming Aeribacillus pallidus strains isolated from dairy products.

The presence of thermophilic spore-forming bacteria is challenging in industrial food processing. The presented genome sequences of Aeribacillus pallidus, isolated from raw milk and cocoa powder, provide insights into how to prevent damage to minimally processed foods and products with extended shelf life, such as milk products.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR.

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85-97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103-107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. KEY POINTS: • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential.

Genetic Organization of the aprX-lipA2 Operon Affects the Proteolytic Potential of Pseudomonas Species in Milk.

Psychrotolerant Pseudomonas species are a main cause of proteolytic spoilage of ultra-high temperature (UHT) milk products due to the secretion of the heat-resistant metallopeptidase AprX, which is encoded by the first gene of the aprX-lipA2 operon. While the proteolytic property has been characterized for many different Pseudomonas isolates, the underlying aprX-lipA2 gene organization was only described for a few strains so far. In this study, the phylogenomic analysis of 185 Pseudomonas type strains revealed that the presence of aprX is strongly associated to a monophylum composed of 81 species, of which 83% carried the aprX locus. Furthermore, almost all type strains of known milk-relevant species were shown to be members of the three monophyletic groups P. fluorescens, P. gessardii, and P. fragi. In total, 22 different types of aprX-lipA2 genetic organizations were identified in the genus, whereby 31% of the species tested carried the type 1 operon structure consisting of eight genes (aprXIDEF prtAB lipA2). Other genetic structures differed from type 1 mainly in the presence and location of genes coding for two lipases (lipA1 and lipA2) and putative autotransporters (prtA and prtB). The peptidase activity of 129 strains, as determined on skim milk agar and in UHT-milk, correlated largely with different aprX-lipA2 gene compositions. Particularly, isolates harboring the type 1 operon were highly proteolytic, while strains with other operon types, especially ones lacking prtA and prtB, exhibited significantly lower peptidase activities. In conclusion, the phylogenomic position and the aprX-lipA2 gene organization specify the proteolytic potential of Pseudomonas isolates. In addition, however, an interplay of several environmental factors and intrinsic traits influences production and activity of AprX, leading to strain-specific proteolytic phenotypes.

Reprogramming Human Siderocalin To Neutralize Petrobactin, the Essential Iron Scavenger of Anthrax Bacillus.

Bacillus anthracis owes its pronounced virulence-apart from specific toxins-to a twofold import mechanism for FeIII ions. This pathogenic bacterium secretes the siderophores bacillibactin (BB) and petrobactin (PB), of which only BB is neutralized by human siderocalin, an abundant lipocalin in plasma. We describe its reshaping via combinatorial protein design to bind PB⋅FeIII instead of BB⋅FeIII , and with even higher affinity (KD ≈20 pm). X-ray crystallographic analysis of the resulting "petrocalin" in complex with PB⋅GaIII reveals a positively charged ligand pocket while the extended butterfly-like conformation of the bound PB provides a rationale for the missing recognition by the natural siderocalin. In microbiological studies, a combination of petrocalin and siderocalin effectively suppressed the growth of a BB+ /PB+ strain of Bacillus cereus under iron-limiting culture conditions. Thus, our reprogrammed lipocalin may offer novel treatment options for devastating infections caused by B. anthracis.

Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels.

The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

Multiparametric Quantitation of the Bacillus cereus Toxins Cereulide and Isocereulides A-G in Foods.

Consumption of food products contaminated with cereulide (1), a toxin produced by Bacillus cereus, might cause intoxications with symptoms reported to range from indigestion pain and emesis to death. Recently, a series of structural variants, coined isocereulides A-G (2-8), were identified for the first time to be produced along with cereulide (1). The observation that isocereulide A (2) shows an ∼ 8-fold increased cytotoxicity when compared to 1 urges the development of analytical tools enabling an accurate quantitation of these toxins. Therefore, a rapid, sensitive, and robust stable isotope dilution assay (SIDA) was developed for the combined quantitation of 1-8 by means of UPLC-MS/MS. On average, trueness and precision of the method were 112.5 ± 1.8% RSD, repeatability and reproducibility were 2 and 4% for cereulide and isocereulides A-G, and the LOD and LOQ of 0.1 and 0.5 ng/g, respectively, demonstrated a high sensitivity for the developed SIDA method. Application of this method to food samples revealed elevated levels of 1-8 in two suspicious noodle samples, for example, ranging from 0.59 (7) to 189.08 ng/g (1) in sample 1 and from 5.77 (7) to 6198.17 ng/g (1) in sample 2, whereas the analysis of 25 randomly selected food samples, which have not been the subject to any complaints, did not contain detectable amounts of any of these toxins. As a consequence, this SIDA method could add an important contribution to the knowledge-based risk assessment of B. cereus toxins in foods.

Depsipeptide Intermediates Interrogate Proposed Biosynthesis of Cereulide, the Emetic Toxin of Bacillus cereus.

Cereulide and isocereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a non-ribosomal peptide synthetase (NRPS) called Ces. Although a thiotemplate mechanisms involving cyclo-trimerization of ready-made D-O-Leu-D-Ala-L-O-Val-L-Val via a thioesterase (TE) domain is proposed for cereulide biosynthesis, the exact mechanism is far from being understood. UPLC-TOF MS analysis of B. cereus strains in combination with (13)C-labeling experiments now revealed tetra-, octa-, and dodecapeptides of a different sequence, namely (L-O-Val-L-Val-D-O-Leu-D-Ala)1-3, as intermediates of cereulide biosynthesis. Surprisingly, also di-, hexa-, and decadepsipeptides were identified which, together with the structures of the previously reported isocereulides E, F, and G, do not correlate to the currently proposed mechanism for cereulide biosynthesis and violate the canonical NRPS biosynthetic logic. UPLC-TOF MS metabolite analysis and bioinformatic gene cluster analysis highlighted dipeptides rather than single amino or hydroxy acids as the basic modules in tetradepsipeptide assembly and proposed the CesA C-terminal C* domain and the CesB C-terminal TE domain to function as a cooperative esterification and depsipeptide elongation center repeatedly recruiting the action of the C* domain to oligomerize tetradepsipeptides prior to the release of cereulide from the TE domain by macrocyclization.

Chemodiversity of cereulide, the emetic toxin of Bacillus cereus.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage.

Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n=467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100°C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125°C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay.

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC-TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC-electrospray ionization (ESI)-TOF MS and the HPLC-ESI-MS/MS-SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits.

Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

Transcriptional kinetic analyses of cereulide synthetase genes with respect to growth, sporulation and emetic toxin production in Bacillus cereus.

In light of the increasing number of serious food borne outbreaks caused by emetic Bacillus cereus, a better understanding of the cereulide synthetase (ces) gene expression and toxin synthesis is required. Here, the relative expression levels of three ces genes (cesP, cesA and cesB) were investigated using quantitative real-time reverse transcription PCR in relation to growth, degree of sporulation and toxin production of the emetic reference strain B. cereus F4810/72 and the weakly emetic strain IH41385. The strict co-transcription of all three genes confirmed the operon structure of the ces gene cluster responsible for cereulide formation. ces transcription turned out to be highly temporal and tightly regulated; ces mRNA was only detectable during mid to late exponential growth in both strains. The low toxigenic potential of the weakly emetic strain IH41385 correlated well with its respective ces transcripts, showing reduced activity at a transcriptional level. Two non-sporulating mutants (F4810/72Δspo0A and F4810/72INsigH) demonstrated that cereulide synthesis is part of the Spo0A regulon but independent of later sporulation processes. Besides strain specific intrinsic factors, ces transcription was found to be significantly influenced by the cellular growth state as well as by extrinsic abiotic factors, like salt. An increase of sodium chloride in the media resulted in lower ces transcription and coincided with lower cereulide toxin levels. Interestingly, at 25 gl(-1) NaCl, toxin levels were already reduced without strongly affecting the growth of B. cereus, indicating an inhibitory effect of NaCl on cereulide biosynthesis independent of growth. This illustrates that ces gene expression and toxicity cannot be predicted solely from growth rates or cell numbers, but is influenced by complex interactions of various intrinsic as well as extrinsic factors, which remain to be clarified in detail.

Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR.

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The sigma(A)-promoted transcription of spo0A was found to be crucial for cereulide production, while the sigma(H)-driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Proteome approaches combined with Fourier transform infrared spectroscopy revealed a distinctive biofilm physiology in Bordetella pertussis.

Proteome analysis was combined with whole-cell metabolic fingerprinting to gain insight into the physiology of mature biofilm in Bordetella pertussis, the agent responsible for whooping cough. Recent reports indicate that B. pertussis adopts a sessile biofilm as a strategy to persistently colonize the human host. However, since research in the past mainly focused on the planktonic lifestyle of B. pertussis, knowledge on biofilm formation of this important human pathogen is still limited. Comparative studies were carried out by combining 2-DE and Fourier transform infrared (FT-IR) spectroscopy with multivariate statistical methods. These complementary approaches demonstrated that biofilm development has a distinctive impact on B. pertussis physiology. Results from MALDI-TOF/MS identification of proteins together with results from FT-IR spectroscopy revealed the biosynthesis of a putative acidic-type polysaccharide polymer as the most distinctive trait of B. pertussis life in a biofilm. Additionally, expression of proteins known to be involved in cellular regulatory circuits, cell attachment and virulence was altered in sessile cells, which strongly suggests a significant impact of biofilm development on B. pertussis pathogenesis. In summary, our work showed that the combination of proteomics and FT-IR spectroscopy with multivariate statistical analysis provides a powerful tool to gain further insight into bacterial lifestyles.

her7 and hey1, but not lunatic fringe show dynamic expression during somitogenesis in medaka (Oryzias latipes).

Epithelialized somites form repeatedly from the unsegmented presomitic mesoderm (PSM) in the tailbud of vertebrate embryos. Mutant analysis has shown that the Delta-Notch pathway is essential for the temporal and spatial control of somite formation. Several components of this pathway show cyclic transcription, which is driven by a molecular oscillator. This oscillator is thought to act similarly in different vertebrates. In this study, we used the Japanese Medaka (Oryzias latipes) to examine the expression of three factors of the Delta-Notch cascade that are known to show cyclic expression in the PSM of higher vertebrates. We report that in contrast to the situation in mice, lunatic fringe (lfng) in medaka is expressed in a non-dynamic fashion in the rostral halves of the formed somites and the anteriormost PSM. On the other hand, her7, a member of the hairy/Enhancer-of-split related (Her) gene family, shows cyclic expression in the medaka PSM. Although this is similar in zebrafish, there are important differences in the distribution of transcripts in the PSM indicating different modes of regulation in both fish species. Finally, we show that hey1, another Delta-Notch regulated bHLH gene, is dynamically expressed in the PSM of medaka, similar to hey1 in zebrafish and the hey2 orthologs in mice and chicken. Interestingly, medaka hey1 is also expressed in the dorsal aorta and the heart, two tissues where hey2, but not hey1, is expressed in zebrafish. This shows that several components of the Delta-Notch pathway are differently regulated during somitogenesis in different species.