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The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.

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.

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.

Abstract Background Two sets of overlapping genes, lacLMReu and lacLMAci, encoding heterodimeric β-galactosidases from Lactobacillus reuteri and Lactobacillus acidophilus, respectively, have previously been cloned and expressed using the pSIP vector system and Lactobacillus plantarum WCSF1 as host. Despite the high similarity between these lacLM genes and the use of identical cloning and expression strategies, strains harboring lacLMReu produced about twenty-fold more β-galactosidase than strains containing lacLMAci. Results In this study, the plasmid copy numbers (PCN) of expression vectors pEH9R (lacLMReu) and pEH9A (lacLMAci) as well as the transcription levels of both lacLM genes were compared using quantitative PCR methods. Analyses of parallel fermentations of L. plantarum harboring either pEH9R or pEH9A showed that the expression plasmids were present in similar copy numbers. However, transcript levels of lacLM from L. reuteri (pEH9R) were up to 18 times higher than those of lacLM from L. acidophilus (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. Conclusion 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.

Abstract Background Enterococcus faecalis 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 E. faecalis V583. Results 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. Conclusion The results provide an overview of potentially important proteins that E. faecalis 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.

Abstract Background Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ 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. Results We have constructed a library of 76 Sec-type signal peptides from L. plantarum 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. Conclusion This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum 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 L. plantarum. 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.