Discovery of Cyclic Lipopeptides Ralstopeptins A and B from Ralstonia solanacearum Species Complex and Analysis of Biosynthetic Gene Evolution.

Ralstonia solanacearum species complex (RSSC) strains are plant pathogens that produce lipopeptides (ralstonins and ralstoamides) by the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) enzyme hybrid. Recently, ralstonins were found to be key molecules in the parasitism of RSSC to other hosts, Aspergillus and Fusarium fungi. The PKS-NRPS genes of RSSC strains in the GenBank database suggest the production of additional lipopeptides, although it has not been confirmed to date. Here, we report the genome-driven and mass-spectrometry-guided discovery, isolation, and structural elucidation of ralstopeptins A and B from strain MAFF 211519. Ralstopeptins were found to be cyclic lipopeptides with two amino acid residues less than ralstonins. The partial deletion of the gene encoding PKS-NRPS obliterated the production of ralstopeptins in MAFF 211519. Bioinformatic analyses suggested possible evolutionary events of the biosynthetic genes of RSSC lipopeptides, where intragenomic recombination may have occurred within the PKS-NRPS genes, reducing the gene size. The chlamydospore-inducing activities of ralstopeptins A and B, ralstonins A and B, and ralstoamide A in the fungus Fusarium oxysporum indicated a structural preference for ralstonins. Altogether, we propose a model for the evolutionary processes that contribute to the chemical diversity of RSSC lipopeptides and its relation to the endoparasitism of RSSC in fungi.

A Unique Combination of Two Different Quorum Sensing Systems in the ß-Rhizobium Cupriavidus taiwanensis.

Cupriavidus taiwanensis LMG19424, a ß-rhizobial symbiont of Mimosa pudica, harbors phc and tqs quorum sensing (QS), which are the homologous cell-cell communication systems previously identified from the plant pathogen Ralstonia solanacearum and the human pathogen Vibrio cholerae, respectively. However, there has been no experimental evidence reported that these QS systems function in C. taiwanensis LMG19424. We identified (R)-methyl 3-hydroxymyristate (3-OH MAME) and (S)-3-hydroxypentadecan-4-one (C15-AHK) as phc and tqs QS signals, respectively, and characterized these QS systems. The expression of the signal synthase gene phcB and tqsA in E. coli BL21(DE3) resulted in the high production of 3-OH MAME and C15-AHK, respectively. Their structures were elucidated by comparison of EI-MS data and GC/chiral LC retention times with synthetic standards. The deletion of phcB reduced cell motility and increased biofilm formation, and the double deletion of phcB/tqsA caused the accumulation of the metal chelator coproporphyrin III in its mutant culture. Although the deletion of phcB and tqsA slightly reduced its ability to nodulate on aseptically grown seedlings of M. pudica, there was no significant difference in nodule formation between LMG19424 and its QS mutants when commercial soils were used. Taken together, this is the first example of the simultaneous production of 3-OH MAME/C15-AHK as QS signals in a bacterial species, and the importance of the phc/tqs QS systems in the saprophytic stage of C. taiwanensis LMG19424 is suggested.

Functional interaction between cyclooxygenase-2 and p53 in response to an endogenous electrophile.

Cyclooxygenase-2 (Cox-2) is rapidly expressed by various stimuli and plays a key role in conversion of free arachidonic acid to prostaglandins. We have previously identified 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived electrophile, as the potent Cox-2 inducer in rat epithelial RL34 cells and revealed that the HNE-induced Cox-2 expression resulted from the stabilization of Cox-2 mRNA that is mediated by the p38 mitogen-activated protein kinase signaling pathway. In the present study, we investigated an alternative regulatory mechanism of Cox-2 expression mediated by a transcription factor p53. In addition, to characterize the causal role for Cox-2, we examined the effects of Cox-2 overexpression in RL34 cells. To examine whether the HNE-induced Cox-2 expression was mechanistically linked to the p53 expression, we analyzed changes in Cox-2 and p53 expression levels in response to HNE and observed that the Cox-2 levels were inversely correlated with the p53 levels. Down-regulation of p53 followed by the activation of a transcription factor Sp1 was suggested to be involved in the HNE-induced Cox-2 gene expression. To characterize the effect of Cox-2 expression in the cells, we established the Cox-2-overexpressing derivatives of RL34 cells by stable transfection with Cox-2 cDNA. An oligonucleotide microarray analysis revealed a dramatic down-regulation of the proteasome subunit RC1 in the Cox-2 overexpressed cells compared to the empty-vector transfected control cells. Consistent with the Cox-2-mediated down-regulation of proteasome, a moderate reduction of the proteasome activities was observed. This proteasome dysfunction mediated by the Cox-2 overproduction was associated with the enhanced accumulation of p53 and ubiquitinated proteins, leading to the enhanced sensitivity toward electrophiles. These results suggest the existence of a causal link between Cox-2 and p53, which may represent a toxic mechanism of electrophilic lipid peroxidation products.

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages.

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.

Higher reactivity of apolipoprotein B-100 and alpha-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction.

Radical reaction of low-density lipoprotein (LDL) is a key step in atherogenesis and causes both a decrease in the sialic acid moiety and modification of apolipoprotein B-100 (apoB). Although apoB modification (cross-link and fragmentation) increases in atherosclerosis, the change in apoB-bound sialic acid in atherosclerosis is controversial. To elucidate the physiological implications of desialylation of LDL by radical reaction, the reactivity of sialic acid of LDL was compared with that of apoB, which underwent facile fragmentation in radical reactions. ApoB was determined by immunoblot analysis with anti-apoB antiserum, and the sialic acid moiety was measured by blot analysis with a biotin-bound lectin [biotin-SSA from Japanese elderberry (Sambucus sieboldiana)] specific to sialic acid. When human LDL was oxidized with Cu(2+) at 37 degrees C, apoB and apoB-attached sialic acid decreased simultaneously. Comparison of the staining bands with anti-apoB and with biotin-SSA shows that sialic acid moieties still remain on fragmented apoB proteins, indicating that the decrease in sialic acid is much slower than that of apoB fragmentation. In addition, human plasma was oxidized with 400 microM of Cu(2+) at 37 degrees C. Similar analysis indicates that the decrease in sialic acid attached to apoB also results from the fragmentation of apoB. This study indicates that the fragmentation of apoB proceeds at a much faster rate than the decrease in sialic acid content when a free radical reaction is induced in isolated LDL as well as in plasma LDL exposed to Cu(2+)-induced oxidative stress. On the basis of these results, the modification of apoB is much more sensitive than the decrease in sialic acid as an indicator of oxidative stress.

Evaluation of apolipoprotein B-100 fragmentation and cross-linkage in serum as an index of atherosclerosis.

It is well established that radical reaction of low density lipoprotein (LDL) causes fragmentation and cross-linkage of apolipoprotein B-100 (apoB). Our previous studies demonstrated that fragmented and cross-linked apoB proteins are present in normal human serum and tended to increase with age based on immunoblot analysis. These observations suggest that the fragmentation and cross-linkage pattern of apoB reflects the oxidative stress in an individual and that this pattern is a good atherosclerotic index. In this study, a method was developed to evaluate the fragmentation and conjugation pattern of apoB. A parameter named B-ox was introduced for each serum sample to quantitate the staining bands of the immunoblotting analysis. B-ox represents the relative abundance of radical reaction products (a sum of fragmented and conjugated apoB proteins) based on one control subject. If this value increases, it indicates that radical reaction products have increased, i.e., the oxidative stress has increased in the subject. Based on measurements of subjects in a rural area of Japan, B-ox showed significant positive correlation with intima-media thickness (IMT) of the carotid artery, LDL cholesterol, and age, while it showed significant negative correlation with high density lipoprotein (HDL) cholesterol and vitamin C. These results suggest that B-ox is a reliable indicator of atherosclerosis.