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.

Erwinia chrysanthemi causes soft‐rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall‐degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe–S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription–polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB::uidA fusion was found to be induced in iron‐deficient growth conditions and to be repressed by the iron‐sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non‐polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron‐activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR‐dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe–S cluster‐assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe–S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.

The phytopathogenic bacterium Erwinia chrysanthemi, unlike other members of the family Enterobacteriaceae, is able to metabolize the beta-glucosides, arbutin, and salicin. A previous genetic analysis of the E. chrysanthemi arb genes, which mediate beta-glucoside metabolism, suggested that they were homologous to the Escherichia coli K-12 bgl genes. We have now determined the nucleotide sequence of a 5,065-bp DNA fragment containing three genes, arbG, arbF, and arbB. Deletion analysis, expression in minicell systems, and comparison with sequences of other proteins suggest that arbF and arbB encode a beta-glucoside-specific phosphotransferase system-dependent permease and a phospho-beta-glucosidase, respectively. The ArbF amino acid sequence shares 55% identity with that of the E. coli BglF permease and contains most residues thought to be important for a phosphotransferase. One change, however, was noted, since BglF Arg-625, presumably involved in phosphoryl transfer, was replaced by a Cys residue in ArbF. An analysis of the ArbB sequence led to the definition of a protein family which contained enzymes classified as phospho-beta-glucosidases, phospho-beta-galactosidases, beta-glucosidases, and beta-galactosidases and originating from gram-positive and gram-negative bacteria, archebacteria, and mammals, including humans. An analysis of this family allowed us (i) to speculate on the ways that these enzymes evolved, (ii) to identify a glutamate residue likely to be a key amino acid in the catalytic activity of each protein, and (iii) to predict that domain II of the human lactate-phlorizin hydrolase, which is involved in lactose intolerance, is catalytically nonactive. A comparison between the untranslated regions of the E. chrysanthemi arb cluster and the E. coli bgl operon revealed the conservation of two regions which, in the latter, are known to terminate transcription under noninducing conditions and be the target of the BglG transcriptional antiterminator under inducing conditions. ArbG was found to share a high level of similarity with the BglG antiterminator as well as with Bacillus subtilis SacT and SacY antiterminators, suggesting that ArbG functions as an antiterminator in regulating the expression of the E. chrysanthemi arb genes.

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine‐rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA‐binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh‐dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as‐yet undescribed link between protein targeting and oxidation.

ObjectiveTo investigate antioxidant activity, total phenolic and flavonoid content of ethanol (E) and ethyl acetate (A) extracts of Inula viscosa aerial parts selected from three regions of Morocco (Imouzzer, Sefrou and Taounate).MethodsAntioxidants properties were measured by three different test systems of assay namely free radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl, total antioxidant capacity and iron-reducing capacity. Total phenolic content was measured by Folin-Ciocalteu reagent.ResultsAll the extracts showed significant antioxidant activities and contained important levels of phenols. The ethanol extract (0.3 mg/mL) from Sefrou showed the greatest antioxidant capacity in the three systems of assay, which was probably due to its high content of polyphenols (274.39±6.94) mg gallic acid equivalent/g dry extract. Total flavonoid content was found equal for all extracts.ConclusionsOur results of antioxidant assays were justified and partially supported the popular usage of the tested plants. The high antioxidant activity found in the plant from Sefrou and its great biomass in this region suggested that Inula viscosa is a good source of natural antioxidants compounds which might have benefits for health