Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum

Many species of Proteobacteria communicate by using LuxI-LuxR-type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p-coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI, and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI-linked bjaR(1) gene is involved in the response to IV-HSL, and BjaR(1) is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.     

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.     

Combination of interferon-beta and angiotensin-converting enzyme inhibitor, perindopril, attenuates the murine liver fibrosis development.

Recent studies have revealed that both interferon (IFN) and angiotensin-converting enzyme inhibitor (ACE-I) exert an anti-fibrotic effect. The aim of this study was to examine the combined effect of the ACE-I and IFN on the murine hepatic fibrosis development. A model of CCl(4)-induced hepatic fibrosis was used to assess the effect of the clinically used ACE-I, perindopril (PE), and IFN-beta. The PE and IFN were administered after 2-week treatment with CCl(4), and the hepatic indices of fibrosis were assessed at 8 weeks. Single treatment with either PE or IFN at the clinically available comparable doses significantly attenuated liver fibrogenesis associated with suppression of the hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells, and the hepatic alpha1(I)-procollagen mRNA were also markedly inhibited. The inhibitory effect of PE was more potent than IFN, and the combination treatment with PE and IFN almost completely attenuated liver fibrosis development. In vitro, the angiotensin-II (AT-II) type 1 receptor blocker and IFN suppressed the AT-II-induced proliferation and alpha1(I)-procollagen mRNA expression of the activated hepatic stellate cells. The combination treatment of the clinically used PE and IFN may provide a new strategy for anti-liver fibrosis therapy.     

The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi

Peptide methionine sulfoxide reductase (MsrA), which repairs oxidized proteins, is present in most living organisms, and the cognate structural gene belongs to the so-called minimum gene set [Mushegian, A. R. & Koonin, E. V., (1996) Proc. Natl. Acad. Sci. USA 93, 10268–10273]. In this work, we report that MsrA is required for full virulence of the plant pathogen Erwinia chrysanthemi. The following differences were observed between the wild-type and a MsrA⁻ mutant: (i) the MsrA⁻ mutant was more sensitive to oxidative stress; (ii) the MsrA⁻ mutant was less motile on solid surface; (iii) the MsrA⁻ mutant exhibited reduced virulence on chicory leaves; and (iv) no systemic invasion was observed when the MsrA⁻ mutant was inoculated into whole Saintpaulia ionantha plants. These results suggest that plants respond to virulent pathogens by producing active oxygen species, and that enzymes repairing oxidative damage allow virulent pathogens to survive the host environment, thereby supporting the theory that active oxygen species play a key role in plant defense.     

IS6110 in oriC affects the morphology and growth of Mycobacterium tuberculosis and attenuates virulence in mice

The IS6110 element is widely used in studies of molecular epidemiology of tuberculosis and it is considered the gold standard for genotyping Mycobacterium tuberculosis strains. Because of its high frequency of transposition, IS6110 is probably a major contributor to the evolution of M. tuberculosis. Nevertheless, very few studies of the effect of IS6110 insertions on the virulence of M. tuberculosis have been reported. We analysed two isogenic groups of M. tuberculosis strains isolated from the sputa of two patients. Strains belonging to the same isogenic group differed from one another by one IS6110-oriC hybridising band, but they showed identical spoligo and MIRU-VNTR profiles. Isogenic strains containing the IS6110 element in oriC exhibited a diminished growth rate and average dimensions of the bacilli were modified; moreover, they were less virulent in a mouse model.     

Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants

Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus, C. palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.     

Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression

BACKGROUND/AIMS: Chemoprevention should be a promising approach to improve the prognosis of the patients with hepatocellular carcinoma (HCC). Angiogenesis is now recognized as a crucial step not only in tumor growth, but also in early carcinogenesis. The aim of this study was to elucidate the combination effect of the clinically used vitamin K(2) (VK) and the angiotensin-converting enzyme inhibitor, perindopril (PE), on hepatocarcinogenesis, especially in conjunction with angiogenesis. METHODS: In a diethylnitrosamine-induced rat hepatocarcinogenesis model, the effects of VK and PE on the development of liver enzyme-altered preneoplastic lesions and angiogenesis were examined. RESULTS: Treatment with both VK and PE markedly inhibited the development of preneoplastic lesions in association with suppression of neovascularization in the liver. The combination treatment with VK and PE exerted a more potent inhibitory effect as compared with the single agent treatments. The in vitro study demonstrated that VK and PE inhibited the endothelial cell (EC) tubular formation. VK also suppressed the EC proliferation in a dose-dependent manner. CONCLUSIONS: The combination of VK and PE exerted a chemopreventive effect against rat liver carcinogenesis via suppression of angiogenesis. Since both agents are widely used in the clinical practice, this combination therapy may represent a potential new strategy for chemoprevention against HCC in the future.     

NAD+ and NADH regulate an ATP-dependent kinase that phosphorylates enzyme I of the Escherichia coli phosphotransferase system.

Crude extracts of Escherichia coli contain a protein kinase, EI-K, that phosphorylates enzyme I (EI) of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Phosphorylation occurs at the active site histidine residue. The activity of EI-K was lost during purification. However, kinase activity was restored by adding NAD+ or NADP+.NADH reversed NAD+ activation of the kinase, and the level of EI-K activity was dependent on the NAD+/NADH ratio. Although crude preparations of EI-K showed no NAD+ requirement, they were completely inhibited by NADH, either in the assay mixture or when the enzyme was pretreated and the NADH was removed prior to the assay. NAD+ restored full activity to the NADH-pretreated inactive fractions. The results suggest that EI-K contains a bound cofactor that is lost during purification and that may be analogous to NAD+. EI-K activity may serve to link some of the diverse functions of the PTS, such as sugar transport, to the metabolic state of the cell.     

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg(2+)-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg(2+) and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg(2+) and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189 approximately P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an alpha-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189 approximately P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.     

The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD have on virulence of Streptococcus pneumoniae

Four genes, cpsA-cpsD, at the 5' end of the capsular polysaccharide (CPS) biosynthesis locus are conserved in nearly all of the 90 known serotypes of Streptococcus pneumoniae. In the present study, the impact that mutations in cpsA, cpsB, and cpsD have on CPS production and on virulence in mice infected via systemic and intranasal routes was investigated. Strains exhibiting rough colony morphologies (in which either the cpsB or cpsD gene had been deleted) were avirulent, but a smooth, partially encapsulated strain (in which the cpsA gene had been deleted) was as virulent as the wild-type strain. Interestingly, mucoid strains containing mutations affecting the [YGX](3)-repeat domain of CpsD were unable to cause bacteremia after intranasal challenge of CD1 mice, even though such strains were capable of killing BALB/c mice after intraperitoneal challenge. In our model, the ability of S. pneumoniae to regulate, via CpsD phosphorylation, CPS production was required for its transition from the lung to the bloodstream.     

HecA,a member of a class of adhesins produced by diverse pathogenic bacteria,contributes to the attachment,aggregation, epidermal cell killing,and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings

Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.     

Imidapril, an angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and improving vascular resistance in the Zucker fatty rat.

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive agents, that inhibit the conversion of angiotensin I to angiotensin II, resulting in smooth-muscle relaxation and a reduction of vascular resistance. Recently, it has been suggested that ACE inhibitors improve insulin resistance in diabetic patients. To investigate the effect of an ACE inhibitor on insulin sensitivity, insulin signaling, and circulation, imidapril was administered orally or intraduodenally to Zucker fatty rats. Oral administration of imidapril improved insulin sensitivity based on the results of an oral glucose tolerance test (OGTT) and a decrease in urinary glucose secretion. Phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with hepatic insulin receptor substrate-1 (IRS-1) in the insulin-stimulated condition was significantly enhanced 110% without a significant alteration in tyrosine phosphorylation of IRS-1 in the imidapril-treated group. In muscle, IRS-1 tyrosine phosphorylation and PI 3-kinase activity associated with IRS-1 in the insulin-stimulated condition were enhanced 70% and 20%, respectively, in the imidapril-treated group. In contrast, an alteration of the IRS-2 pathway was observed only in liver; a significant insulin-induced increase in the IRS-2-associated PI 3-kinase over the basal level was observed in the imidapril-treated group but not in the control. In addition, treatment with imidapril was shown to significantly reduce blood pressure and increase blood flow in the liver and muscle. These results suggest that the ACE inhibitor imidapril may improve insulin sensitivity not only by acting directly on the insulin signaling pathway but also by increasing blood flow in tissues via normalization of vascular resistance, a major cause of hypertension.