Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1

<p>Abstract</p> <p>Background</p> <p><it>Lactobacillus plantarum </it>is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for <it>in situ </it>delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.</p> <p>Results</p> <p>We have constructed a library of 76 Sec-type signal peptides from <it>L. plantarum </it>WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.</p> <p>Conclusion</p> <p>This is the first comprehensive experimental study showing that predicted SPs in the <it>L. plantarum </it>genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in <it>L. plantarum</it>. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.</p

Identification of proteins related to the stress response in Enterococcus faecalis V583 caused by bovine bile

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecalis </it>is an opportunistic pathogen and one of the most important causes of hospital infections. Bile acids are a major stress factor bacteria have to cope with in order to colonize and survive in the gastro-intestinal tract. The aim of this study was to investigate the effects of bile acids on the intracellular proteome of <it>E. faecalis </it>V583.</p> <p>Results</p> <p>The proteomes of cells challenged with 1% bile were analyzed after 20 - 120 minutes exposure, using 2D gel electrophoresis and mass spectrometry. Among the approximately 500 observed proteins, 53 unique proteins were found to be regulated in response to bile and were identified with mass spectrometry. The identified proteins belonged to nine different functional classes, including fatty acid- and phospholipid-biosynthesis, energy metabolism, and transport and binding. Proteins involved in fatty acid and phospholipid biosynthesis pathways were clearly overrepresented among the identified proteins and all were down-regulated upon exposure to bile. The proteome data correlated reasonably well with data from previous transcriptome experiments done under the same conditions, but several differences were observed.</p> <p>Conclusion</p> <p>The results provide an overview of potentially important proteins that <it>E. faecalis </it>V583 needs to regulate in order to survive and adapt to a bile-rich environment, among which are several proteins involved in fatty acid and phospholipid biosynthesis pathways. In addition, this study reveals several hypothetical proteins, which are both abundant and clearly regulated and thus stand out as targets for future studies on bile stress.</p

Quantitative transcript analysis of the inducible expression system pSIP: comparison of the overexpression of Lactobacillus spp. β-galactosidases in Lactobacillus plantarum

<p>Abstract</p> <p>Background</p> <p>Two sets of overlapping genes, <it>lacLMReu </it>and <it>lacLMAci</it>, encoding heterodimeric β-galactosidases from <it>Lactobacillus reuteri </it>and <it>Lactobacillus acidophilus</it>, respectively, have previously been cloned and expressed using the pSIP vector system and <it>Lactobacillus plantarum </it>WCSF1 as host. Despite the high similarity between these <it>lacLM </it>genes and the use of identical cloning and expression strategies, strains harboring <it>lacLMReu </it>produced about twenty-fold more β-galactosidase than strains containing <it>lacLMAci</it>.</p> <p>Results</p> <p>In this study, the plasmid copy numbers (PCN) of expression vectors pEH9R (<it>lacLMReu</it>) and pEH9A (<it>lacLMAci</it>) as well as the transcription levels of both <it>lacLM </it>genes were compared using quantitative PCR methods. Analyses of parallel fermentations of <it>L. plantarum </it>harboring either pEH9R or pEH9A showed that the expression plasmids were present in similar copy numbers. However, transcript levels of <it>lacLM </it>from <it>L. reuteri </it>(pEH9R) were up to 18 times higher than those of <it>lacLM </it>from <it>L. acidophilus </it>(pEH9A). As a control, it was shown that the expression levels of regulatory genes involved in pheromone-induced promoter activation were similar in both strains.</p> <p>Conclusion</p> <p>The use of identical expression strategies for highly similar genes led to very different mRNA levels. The data indicate that this difference is primarily caused by translational effects that are likely to affect both mRNA synthesis rates and mRNA stability. These translational effects thus seem to be a dominant determinant for the success of gene expression efforts in lactobacilli.</p

Identification of surface proteins in Enterococcus faecalis V583

<p>Abstract</p> <p>Background</p> <p>Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design.</p> <p>Results</p> <p>Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in <it>Enterococcus faecalis </it>V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31) or to be secreted (5). Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species.</p> <p>Conclusions</p> <p>The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium <it>E. faecalis</it>. The 36 identified secreted (5) and surface (31) proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between <it>E. faecalis </it>and its environment.</p

Novel enzymes for the degradation of cellulose

Abstract The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.</p

Neutron and high‐resolution room‐temperature X‐ray data collection from crystallized lytic polysaccharide monooxygenase

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1-3 mm(3)) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes

Brazil builds in Campinas: a symbolic tool of the strategy to legitimate the implementation of the urban-improvement plan

Este artículo trae una reflexión sobre la exposición Brazil Builds que se presentó en Campinas en febrero de 1945. La ciudad era parte del circuito de la exposición que comenzó el 13 de enero de 1943 en el Museum of Modern Art de Nueva York (MoMA). El evento nos lleva a reflexionar sobre cómo se han concertado los distintos intereses involucrados en su realización en esa ciudad. Los esfuerzos del poder público y de sectores de la elite local evidencian una tela de intereses relacionados a la exposición de arquitectura. El objetivo principal es discutir las estructuras de poder locales que se han beneficiado de la dimensión simbólica del arte, usando la exposición como instrumento para legitimar la implantación de un plan de urbanismo para la ciudad (Plan de Mejorías Urbanas), que fue desarrollado por el urbanista Prestes Maia, contratado por la alcadía de la ciudad desde 1934. Ese plan era una parte de un gran proyecto para modernizar la ciudad