ABSTRACT: BACKGROUND: Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism. In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses. RESULTS: Two hours after the shift of cells from glucose to methanol nearly 20 %(1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal beta-oxidation. A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11. Approximately 20 %(1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation. CONCLUSION: Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal beta-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1h vs td methanol 4,5h).

Sigma factors direct specific binding of the bacterial RNA polymerase to the promoter. Here we present the elucidation of the sigma54-regulon in Lactobacillus plantarum. A sequence-based regulon-prediction of sigma54 dependent promoters revealed an operon encoding a mannose phosphotransferase system (PTS) as best candidate for sigma54 mediated control. A sigma54 (rpoN) mutant derivative does not grow on mannose, confirming this prediction. Additional mutation analyses established the presence of one functional mannose PTS in L. plantarum, of which expression is controlled by sigma54 in concert with the sigma54-activator ManR. Genome-wide transcription comparison of the wild type and the rpoN-deletion strain revealed 9 up-regulated genes in the wild type, including the genes of the mannose PTS, and 21 up-regulated genes in the rpoN mutant. In addition, the sigma54-controlled mannose PTS was shown to also transport glucose in L. plantarum wild type cells, and its presence causes a lag phase when cultures are transferred from glucose to galactose containing media. The mannose PTS appeared to drain phosphoenolpyruvate (PEP)-pools in resting cells, since no PEP could be detected in resting wild type cells, while mannose PTS mutant derivatives contained 1-3 muM PEP (mg protein)-1. Our data provide new insight in the role of sigma54 in L. plantarum and possibly other Gram-positive bacteria in the control of expression of an important glucose transporter that contributes to glucose mediated catabolite control via modulation of the PEP pool.

Despite significant efforts by all parties involved, there is still a considerable burden of foodborne illness, in which micro-organisms play a prominent role. Microbes can enter the food chain at different steps, are highly versatile and can adapt to the environment allowing survival, growth and production of toxic compounds. This sets them apart from chemical agents and thus their study from food toxicology. We summarize the discussions of a conference organized by the Dutch Food and Consumer Products Safety Authority and the European Food Safety Authority. The goal of the conference was to discuss new challenges to food safety that are caused by micro-organisms as well as strategies and methodologies to counter these. Management of food safety is based on generally accepted principles of Hazard Analysis Critical Control Points and of Good Manufacturing Practices. However, a more pro-active, science-based approach is required, starting with the ability to predict where problems might arise by applying the risk analysis framework. Developments that may influence food safety in the future occur on different scales (from global to molecular) and in different time frames (from decades to less than a minute). This necessitates development of new risk assessment approaches, taking the impact of different drivers of change into account. We provide an overview of drivers that may affect food safety and their potential impact on foodborne pathogens and human disease risks. We conclude that many drivers may result in increased food safety risks, requiring active governmental policy setting and anticipation by food industries whereas other drivers may decrease food safety risks. Monitoring of contamination in the food chain, combined with surveillance of human illness and epidemiological investigations of outbreaks and sporadic cases continue to be important sources of information. New approaches in human illness surveillance include the use of molecular markers for improved outbreak detection and source attribution, sero-epidemiology and disease burden estimation. Current developments in molecular techniques make it possible to rapidly assemble information on the genome of various isolates of microbial species of concern. Such information can be used to develop new tracking and tracing methods, and to investigate the behavior of micro-organisms under environmentally relevant stress conditions. These novel tools and insight need to be applied to objectives for food safety strategies, as well as to models that predict microbial behavior. In addition, the increasing complexity of the global food systems necessitates improved communication between all parties involved: scientists, risk assessors and risk managers, as well as consumers.

Abstract In spring 2008, 15 Salmonella Panama laboratory-confirmed cases were reported within 2 weeks, twice the average annual number of reported cases of this infrequent serotype in The Netherlands. To identify the source responsible for this national outbreak, we carried out an epidemiological, microbiological, and trace-back investigation. In total, 33 cases were reported, and a matched case-control study (23 cases/24 controls) identified consumption of fresh (unpasteurized) fruit juice purchased from a large retailer (X) as the only significant risk factor for illness (matched odds ratio: 7.4, 95% confidence interval: 1.5-37.2). Though the bacterium could not be isolated from fruit juice, the minimal pH value for growth of the causative strain of the outbreak (3.4) was compatible with survival in fruit juice from X. The outbreak strain showed acid resistance and adaptive properties that may explain how it could have caused infection through fresh orange juice. To our knowledge, this is the first documented outbreak related to fresh fruit juice consumption in western Europe since 1922. A growing number of consumers who are seeking healthy food practices are exposed to the infectious risks related to unpasteurized fresh fruit juice. Labeling regulations should be adapted to properly indicate to the consumers that unpasteurized fresh fruit juices remain vulnerable to microbial contamination. Frequent microbiological screening and strict compliance with food safety procedures should reduce the infectious hazards of fresh fruit juices.

The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways.

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Reverse proteolysis and transpeptidation lead to the generation of polypeptide sequences that cannot be inferred directly from genome sequences as they are post-translational phenomena. These phenomena have so far received little attention although the physiological consequences may reach far. The protease-mediated synthesis of several immunodominant MHC class I antigens was recently reported, underscoring its importance to immunity. Reverse proteolytic and transpeptidation mechanisms as well as conditions that favor successful protease-catalyzed synthetic events are discussed here.

Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3 days and was characterised by syncytia. None of the samples produced cytopathic effect (CPE) on African green monkey kidney (Vero) cells or hamster kidney (BSR) cells. Four of the samples, which were positive in the EHV-5 PCR, produced CPE on rabbit kidney (RK13) cells and equine dermis (ED) cells. EHV-5 CPE on both cell lines was slow and was apparent after four 7-day passages. On RK13 cells, the CPE was characteristic of equid herpesvirus, with the formation of syncytia. However, in ED cells, the CPE was characterised by ring-shaped syncytia. For the first time, a case of equine respiratory disease involving dual infection with EHV-4 and EHV-5 has been reported in Queensland (Australia). This was shown by simultaneously isolating EHV-4 and EHV-5 from clinical samples. EHV5 was recovered from all samples except one, suggesting that EHV5 was more prevalent in young horses than EHV2.

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure which allows protein quantitation using differential isotopic dimethyl labeling of peptide N-termini and lysine residues. We show that the method allows reliable quantitation over several orders of magnitude and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20S core proteasome complexes. We find that, although all catalytically active immunoproteasome subunits were upregulated in spleen (compared to liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue specific (immuno) is more diverse than previously assumed.

The efficacy of enrofloxacin against Riemerella anatipestifer (formerly Pasteurella anatipestifer) septicaemia of Muscovy and Pekin ducklings was assessed in an artificial challenge model which reproduced typical duck riemerellosis with very high mortality. Mortality, clinical signs, gross lesions, microbiological clearance, feed intake and body weight gain were the efficacy criteria studied. A pulsing medication in drinking water for 4 h at 25, 50 or 100 ppm enrofloxacin on the first day, followed by 12.5, 25 or 50 ppm, respectively, on the following 4 days, provided clinical cure of R. anatipestifer septicaemia at all dosages tested. An optimum dosage of 50 ppm enrofloxacin, followed by 25 ppm was established.