Hydrogen peroxide inactivates the Escherichia coli Isc iron-sulphur assembly system, and OxyR induces the Suf system to compensate

Environmental H(2) O(2) creates several injuries in Escherichia coli, including the oxidative conversion of dehydratase [4Fe-4S] clusters to an inactive [3Fe-4S] form. To protect itself, H(2) O(2) -stressed E. coli activates the OxyR regulon. This regulon includes the suf operon, which encodes an alternative to the housekeeping Isc iron-sulphur cluster assembly system. Previously studied [3Fe-4S] clusters are repaired by an Isc/Suf-independent pathway, so the rationale for Suf induction was not obvious. Using strains that cannot scavenge H(2) O(2) , we imposed chronic low-grade stress and found that suf mutants could not maintain the activity of isopropylmalate isomerase, a key iron-sulphur dehydratase. Experiments showed that its damaged cluster was degraded in vivo beyond the [3Fe-4S] state, presumably to an apoprotein form, and thus required a de novo assembly system for reactivation. Surprisingly, submicromolar H(2) O(2) poisoned the Isc machinery, thereby creating a requirement for Suf both to repair the isomerase and to activate nascent Fe-S enzymes in general. The IscS and IscA components of the Isc system are H(2) O(2) -resistant, suggesting that oxidants disrupt Isc by oxidizing clusters as they are assembled on or transferred from the IscU scaffold. Consistent with these results, organisms that are routinely exposed to oxidants rely upon Suf rather than Isc for cluster assembly.     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

The GacA global regulator is required for the appropriate expression of Erwinia chrysanthemi 3937 pathogenicity genes during plant infection

Pathogenicity of the phytopathogenic enterobacterium Erwinia chrysanthemi , the causal agent of soft rot disease on many plants, is a complex process involving several factors whose production is regulated by a complex, intertwined regulatory network. In this work we characterized the GacA regulator, member of the GacS–GacA two-component system, as a global regulator which is required for disease expression but not for bacterial multiplication in planta during the first stages of the plant infection. GacA was shown to control the expression of plant cell wall-degrading enzymes and hrp genes in vitro . Analysis of virulence gene expression during infection of Arabidopsis thaliana revealed a coordinated expression of these virulence genes at 12 h post infection and showed that GacA is required for the appropriate production of virulence factors in planta . GacA might partly act by negatively controlling the expression of the pecT gene encoding the global repressor PecT, indicating a hierarchy in the pathways involved in the E. chrysanthemi regulatory network.     

Delivery of iron-sulfur clusters to the hydrogen-oxidizing [NiFe]-hydrogenases in Escherichia coli requires the A-type carrier proteins ErpA and IscA

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.     

Siderophore-controlled iron assimilation in the enterobacterium Erwinia chrysanthemi: evidence for the involvement of bacterioferritin and the Suf iron-sulfur cluster assembly machinery

The intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins.     

SufC: an unorthodox cytoplasmic ABC/ATPase required for [Fe-S] biogenesis under oxidative stress

Proteins containing [Fe-S] clusters perform essential functions in all domains of life. Previously, we identified the sufABCDSE operon as being necessary for virulence of the plant pathogen Erwinia chrysanthemi. In addition, we collected preliminary evidence that the sufABCDSE operon might be involved in the assembly of [Fe-S] clusters. Of particular interest are the sufB, sufC and sufD genes, which are conserved among Eubacteria, Archaea, plants and parasites. The present study establishes SufC as an unorthodox ATPase of the ABC superfamily that is located in the cytosol, wherein it interacts with both SufB and SufD. Moreover, under oxidative stress conditions, SufC was found to be necessary for the activity of enzymes containing oxygen-labile [Fe-S] clusters, but dispensable for glutamate synthase, which contains an oxidatively stable [Fe-S] cluster. Lastly, we have shown SufBCD to be essential for iron acquisition via chrysobactin, a siderophore of major importance in virulence. We discuss a model wherein the SufBCD proteins contribute to bacterial pathogenicity via their role in the assembly of [Fe-S] clusters under oxidative stress and iron limitation.     

Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid Hh, and regulation of pectolytic enzymes

Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad number of crops. The PhoP-PhoQ system is a key factor in pathogenicity of several bacteria and is involved in the bacterial resistance to different factors, including acid stress. Since E. chrysanthemi is confronted by acid pH during pathogenesis, we have studied the role of this system in the virulence of this bacterium. In this work, we have isolated and characterized the phoP and phoQ mutants of E. chrysanthemi strain 3937. It was found that: (i) they were not altered in their growth at acid pH; (ii) the phoQ mutant showed diminished ability to survive at acid pH; (iii) susceptibility to the antimicrobial peptide thionin was increased; (iv) the virulence of the phoQ mutant was diminished at low and high magnesium concentrations, whereas the virulence of the phoP was diminished only at low magnesium concentrations; (v) in planta Pel activity of both mutant strains was drastically reduced; and (vi) both mutants lagged behind the wild type in their capacity to change the apoplastic pH. These results suggest that the PhoP-PhoQ system plays a role in the virulence of this bacterium in plant tissues, although it does not contribute to bacterial growth at acid pH.     

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.     

Abscisic acid deficiency leads to rapid activation of tomato defence responses upon infection with Erwinia chrysanthemi

In addition to the important role of abscisic acid (ABA) in abiotic stress signalling, basal and high ABA levels appear to have a negative effect on disease resistance. Using the ABA-deficient sitiens tomato ( Solanum lycopersicum ) mutant and different application methods of exogenous ABA, we demonstrated the influence of this plant hormone on disease progression of Erwinia chrysanthemi . This necrotrophic plant pathogenic bacterium is responsible for soft rot disease on many plant species, causing maceration symptoms mainly due to the production and secretion of pectinolytic enzymes. On wild-type (WT) tomato cv. Moneymaker E. chrysanthemi leaf inoculation resulted in maceration both within and beyond the infiltrated zone of the leaf, but sitiens showed a very low occurrence of tissue maceration, which never extended the infiltrated zone. A single ABA treatment prior to infection eliminated the effect of pathogen restriction in sitiens , while repeated ABA spraying during plant development rendered both WT and sitiens very susceptible. Quantification of E. chrysanthemi populations inside the leaf did not reveal differences in bacterial growth between sitiens and WT. Sitiens was not more resistant to pectinolytic cell-wall degradation, but upon infection it showed a faster and stronger activation of defence responses than WT, such as hydrogen peroxide accumulation, peroxidase activation and cell-wall fortifications. Moreover, the rapid activation of sitiens peroxidases was also observed after application of bacteria-free culture filtrate containing E. chrysanthemi cell-wall-degrading enzymes and was absent during infection with an out E. chrysanthemi mutant impaired in secretion of these extracellular enzymes.