Identification of Apolipoprotein N-Acyltransferase (Lnt) in Mycobacteria

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the α-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.Proteins of various organisms are modified in numerous ways, one of them is lipidation. Lipid modification of proteins is common in eucaryal and bacterial organisms and can involve myristoyl, palmitoyl, and isoprenyl polymers of various lengths or aminoglycan-linked phospholipids (1, 2). Lipoprotein modifications investigated here are restricted to bacteria.The lipoprotein biosynthesis pathway is a major virulence factor in Mycobacterium tuberculosis, the causative agent of human tuberculosis. Every year 1.6 million people fall prey to tuberculosis and one-third of the population of the world are infected. Thus, tuberculosis is responsible for 2.5% of deaths in the world, which is the highest rate claimed by a single infectious agent. An M. tuberculosis knock-out mutant deficient in lipoprotein signal peptidase lspA showed reduced multiplication in bone marrow-derived macrophages, complete absence of lung pathology and a 1000-fold reduced number of colony forming units in a mouse model of infection (3, 4). Likewise, lipoprotein synthesis contributes to virulence of other Gram-positive pathogens, Listeria, Staphylococci, and Streptococci (5).Bacterial lipoproteins are a functionally diverse class of lipidated proteins involved in cell wall synthesis, nutrient uptake, adhesion, and transmembrane signaling (6) and about 2% of open reading frames encode this kind of proteins (7). Lipidation allows anchoring of these proteins to the cell surface. Lipoproteins are characterized by the presence of a consensus sequence, the “lipobox,” located in the C-terminal part of the leader sequence and consisting of four amino acids (LVI/ASTVI/GAS/C) (7). Precursor lipoproteins are mainly translocated in a Sec-dependent manner across the plasma membrane and modified subsequently on the universally conserved, essential cysteine residue located in the lipobox motif. The modifications taking place after translocation are consecutively mediated by three enzymes: 1) formation of a thioether linkage between the conserved cysteine residue and a diacylglycerol catalyzed by phosphatidylglycerol:pre-prolipoprotein diacylglycerol transferase (Lgt), 2) cleavage of the N-terminal signal peptide by the prolipoprotein signal peptidase/signal peptidase II (LspA), and 3) in the case of Gram-negative bacteria, aminoacylation of the N-terminal cysteine residue by phospholipid:apolipoprotein N-acyltransferase (Lnt) (6–8). In Escherichia coli, most of the mature triacylated lipoproteins are finally transported across the periplasm by the LolABCDE transport system (9). Homologues of the Lol-transport system are absent in Mycobacteria. Although lipoprotein modifying enzymes act sequentially, Lgt-independent LspA-mediated signal sequence cleavage has recently been demonstrated in Listeria monocytogenes (10). Although Lgt and LspA are universally present in both Gram-positive and Gram-negative bacteria, Lnt has been reported to be restricted to Gram-negative bacteria (11), although some indications for N-acylation in Bacillus subtilis and Staphylococcus aureus were reported (12–15).Mycobacterial lipoproteins are immunodominant antigens (16) and several manipulate innate immune mechanisms and antigen presenting cells (17). It is known that mycobacterial lipoproteins, e.g. the 19-kDa lipoprotein, activate Toll-like receptor 2 (TLR2) and co-receptors TLR1, which recognize triacylated peptides, but also TLR6, which recognize diacylated peptides (18, 19). However, the lipid linkage of mycobacterial lipoproteins has not been determined.In this study, we show that Lnt activity is more widely distributed than previously assumed. We demonstrate apolipoprotein N-acyltransferase activity in a Gram-positive Mycobacterium and give complete structural information about the lipid modification of mycobacterial lipoproteins. Hereby, the functionality of Lnt homologues in actinomycetes is revealed (5). We show that mycobacterial lipoproteins are triacylated and carry mycobacteria-specific fatty acids.     

A proteomics approach to study synergistic and antagonistic interactions of the fungal–bacterial consortium Fusarium oxysporum wild‐type MSA 35

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil‐borne plant pathogen, including non‐pathogenic F. oxysporum strains. In this study, F. oxysporum wild‐type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to γ‐proteobacteria, was analyzed by two complementary metaproteomic approaches (2‐DE combined with MALDI‐Tof/Tof MS and 1‐D PAGE combined with LC‐ESI‐MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter‐part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.     

Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose

The wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss. Both polar and non-polar mutations in the sixth gene of the wss operon (wssF ) or adjacent downstream genes (wssGHIJ ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa, which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.     

Responses in the start of Betula (birch) pollen seasons to recent changes in spring temperatures across Europe

A shift in the timing of birch pollen seasons is important because it is well known to be a significant aeroallergen, especially in NW Europe where it is a notable cause of hay fever and pollen-related asthma. The research reported in this paper aims to investigate temporal patterns in the start dates of Betula (birch) pollen seasons at selected sites across Europe. In particular it investigates relationships between the changes in start dates and changes in spring temperatures over approximately the last 20 years. Daily birch pollen counts were used from Kevo, Turku, London, Brussels, Zurich and Vienna, for the core period from 1982 to 1999 and, in some cases, from 1970 to 2000. The sites represent a range of biogeographical situations from just within the Arctic Circle through to North West Maritime and Continental Europe. Pollen samples were taken with Hirst-type volumetric spore traps. Weather data were obtained from the sites nearest to the pollen traps. The timing of birch pollen seasons is known to depend mostly on a non-linear balance between the winter chilling required to break dormancy, and spring temperatures. Pollen start dates and monthly mean temperatures for January through to May were compiled to 5-year running means to examine trends. The start dates for the next 10 years were calculated from regression equations for each site, on the speculative basis that the current trends would continue. The analyses show regional contrasts. Kevo shows a marked trend towards cooler springs and later starts. If this continues the mean start date will become about 6 days later over the next 10 years. Turku exhibits cyclic patterns in start dates. A current trend towards earlier starts is expected to continue until 2007, followed by another fluctuation. London, Brussels, Zurich and Vienna show very similar patterns in the trends towards earlier start dates. If the trend continues the mean start dates at these sites will advance by about 6 days over the next 10 years. Following this work, amendments will be needed to pollen calendars and local predictive models. It will also be important to assess the implications of earlier seasons for allergy sufferers.     

Fatty Acid Oxidation by Spores of Penicillium roqueforti

When 1 μm sodium octanoate was the substrate for spores, most of the molecule was recovered as CO₂ and no ketone was produced. However, when larger concentrations (20 μm) were used as substrate, part of the molecule was converted to methyl ketone and part was completely oxidized. Optimal conditions for the production of 2-heptanone were determined because of the importance of this compound in giving aroma and flavor to mold-ripened cheeses. Optimal ketone formation was not dependent upon the temperature and length of time at which the spores were stored. The spore suspensions were stored for over 36 months at 4 C without losing their ability to convert octanoic acid to 2-heptanone. The oxidation of octanoic acid was inhibited by cyanide, carbon monoxide, mercury, 2,3-dimercapto-1-propanol, and α, α-dipyridyl. No ketone was produced under anaerobic conditions. Although no intermediates of fatty acid oxidation were isolated, since an active cell-free preparation could not be obtained, this investigation has yielded some evidence for the beta oxidation of the fatty acids by spores of Penicillium roqueforti.     

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.     

Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.     

A bacterial-fungal metaproteomic analysis enlightens an intriguing multicomponent interaction in the rhizosphere of Lactuca sativa

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic isolate that protects plants against pathogenic Fusaria. This strain lives in association with ectosymbiotic bacteria. When cured of the prokaryotic symbionts [cured (CU) form], the fungus is pathogenic, causing wilt symptoms similar to those of F. oxysporum f.sp. lactucae. The aim of this study was to understand if and how the host plant Lactuca sativa contributes to the expression of the antagonistic/pathogenic behaviors of MSA 35 strains. A time-course comparative analysis of the proteomic profiles of WT and CU strains was performed. Fungal proteins expressed during the early stages of plant-fungus interaction were involved in stress defense, energy metabolism, and virulence and were equally induced in both strains. In the late phase of the interkingdom interaction, only CU strain continued the production of virulence- and energy-related proteins. The expression analysis of lettuce genes coding for proteins involved in resistance-related processes corroborated proteomic data by showing that, at the beginning of the interaction, both fungi are perceived by the plant as pathogen. On the contrary, after 8 days, only the CU strain is able to induce plant gene expression. For the first time, it was demonstrated that an antagonistic F. oxysporum behaves initially as pathogen, showing an interesting similarity with other beneficial organisms such as mychorrizae.