Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

In Vitro Formation of Dickeya zeae MS1 Biofilm.

Bacterial soft rot caused by Dickeya zeae MS1 (Erwinia chrysanthemi) is one of the most devastating banana diseases worldwide. However, knowledge of the development and ecological interactions of D. zeae MS1 biofilm is limited. Here, we visualized the development and architecture of D. zeae MS1 biofilm using confocal laser scanning microscopy, and we evaluated the ability of D. zeae MS1 to form biofilms under different environmental conditions (carbon sources, temperatures, pH levels and mineral elements) using a microtiter plate assay. We found that the development of D. zeae MS1 biofilm could be categorized into four phases and that mature biofilm consisted of a highly organized architecture of both bacterial cells and a self-produced matrix of extracellular polysaccharides. Furthermore, sucrose was the most suitable carbon source for supporting the growth of biofilm cells and that 32 °C and pH 7.0 were the most favorable of the temperatures and pH levels examined. Meanwhile, the addition of Ca2+, Fe2+, K+ and Na+ enhanced the formation of biofilm in minimal medium cultures, whereas 2.5 mM Cu2+ and Mn2+ was inhibitory. A better understanding of biofilm formation under different environmental parameters will improve our knowledge of the growth kinetics of D. zeae MS1 biofilm.

Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.

l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations-open and closed-corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.

Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation.

L-asparaginase is an integral component of therapy for acute lymphoblastic leukemia. However, asparaginase-related complications, including the development of hypersensitivity reactions, can limit its use in individual patients. Of considerable concern in the setting of clinical allergy is the development of neutralizing antibodies and associated asparaginase inactivity. Also problematic in the use of asparaginase is the potential for the development of silent inactivation, with the formation of neutralizing antibodies and reduced asparaginase activity in the absence of a clinically evident allergic reaction. Here we present guidelines for the identification and management of clinical hypersensitivity and silent inactivation with Escherichia coli- and Erwinia chrysanthemi- derived asparaginase preparations. These guidelines were developed by a consensus panel of experts following a review of the available published data. We provide a consensus of expert opinions on the role of serum asparaginase level assessment, indications for switching asparaginase preparation, and monitoring after change in asparaginase preparation.

Trigalacturonate-producing pectate lyase PelQ1 from Saccharobesus litoralis with unique exolytic activity.

PelQ1 from Saccharobesus litoralis is a Ca2+-dependent pectate lyase belonging to the polysaccharide lyase family 1 (PL1). Although being an endolytic enzyme, it degraded polygalacturonate into predominantly unsaturated trimer in an exolytic manner with delayed production of dimer, tetramer and pentamer. The enzyme harbours a C-terminal domain from the carbohydrate-binding module family 13 (CBM13), whose presence facilitated the production of dimer. PelQ1's homology model showed that it possessed a well-conserved catalytic cleft, with R232 acting as the general base and R203 as the general acid. Structural comparison with DcPelC, a similar trimer-generating pectate lyase from Dickeya chrysanthemi EC16, implied that both enzymes' catalytic clefts encompassed at least eight subsites, i.e. -5 to +3. The unequal distribution of the subsites between the reducing and non-reducing ends of the cleavage site might be responsible for the exolytic generation of the trimer. As all but the -1, +1 and + 2 subsites could accommodate methylated galacturonate, this subclass of PL1 pectate lyases may function to help break up methylated pectin.

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer.

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass.

Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.

AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis.

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy.

The Right-Handed Parallel ß-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure.

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-ß pectinolytic enzyme with a right-handed parallel ß-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol-1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner.

Regulatory mechanisms of exoribonuclease PNPase and regulatory small RNA on T3SS of Dickeya dadantii.

The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.

ClpXP protease regulates the type III secretion system of Dickeya dadantii 3937 and is essential for the bacterial virulence.

The type III secretion system (T3SS) is considered one of the major virulence factors in many bacterial pathogens. This report demonstrates that RssB, ClpXP, and RpoS play a role in T3SS regulation of Dickeya dadantii 3937. ClpP is a serine-type protease which associates with the ClpX chaperone to form a functional Clp proteolytic complex for degradation of proteins. With the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor. RpoS positively regulates the expression of the rsmA gene encoding an RNA-binding regulatory protein. By interacting with the hrpL mRNA, RsmA reduces HrpL production and downregulates the T3SS genes in the HrpL regulon. In addition, ClpXP, RssB, and RpoS affect pectinolytic enzyme production in D. dadantii 3937, probably through RsmA. The ClpXP and RssB proteins are essential for bacterial virulence.

Determination of glycation levels in Erwinia chrysanthemi asparaginase drug product by liquid chromatography - mass spectrometry.

Erwinase (Erwinia chrysanthemi L-asparaginase) Drug Product (DP) is a freeze-dried formulation with a three-year shelf life at 2-8 °C, and an established safety, stability and efficacy profile over the more than three decades of clinical use. Seven Erwinase® DP batches, released over a 7-year period, were screened by reversed-phase liquid chromatography coupled to time-of-flight mass spectrometry for glycation levels. This modification is a known and natural consequence of exposure of Erwinase Drug Product to glucose excipients in stabilizing formulations. Although glycation is detected in current release and stability methods, glycation, including the conditions under which this reaction occurs, has not been previously characterised in detail. We have found that glycation levels of different DP lots generally correlated with age, when they were stored at low temperature. This suggests that the glycation reaction continues over time within the Drug Product formulation in the lyophilised state, even under low temperature (+2-8 °C) conditions. We were also able to examine glycation levels of one DP lot, Lot D, held under long term stability at 3 different temperatures over a 5-year period. The 2 samples held at -20 °C and -80 °C, were glycated to levels of 12% and 17%, respectively. However, the DP Lot D sample held at +2-8 oC in this time period was found to be glycated to a level of 35.6%, with multiple glycations of individual subunits observed. For analytical reference materials, it is important to keep parameters such as glycation levels as constant as possible, to avoid a 'moving target' with respect to comparisons with release and stability testing. These data suggest that storage of DP as reference standards at a lower temperature (e.g., -20 °C) can significantly reduce levels of glycation over the longer time periods required for analytical reference standards.

Catabolism of raffinose, sucrose, and melibiose in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi (Dickeya dadantii) is a plant pathogenic bacterium that has a large capacity to degrade the plant cell wall polysaccharides. The present study reports the metabolic pathways used by E. chrysanthemi to assimilate the oligosaccharides sucrose and raffinose, which are particularly abundant plant sugars. E. chrysanthemi is able to use sucrose, raffinose, or melibiose as a sole carbon source for growth. The two gene clusters scrKYABR and rafRBA are necessary for their catabolism. The phenotypic analysis of scr and raf mutants revealed cross-links between the assimilation pathways of these oligosaccharides. Sucrose catabolism is mediated by the genes scrKYAB. While the raf cluster is sufficient to catabolize melibiose, it is incomplete for raffinose catabolism, which needs two additional steps that are provided by scrY and scrB. The scr and raf clusters are controlled by specific repressors, ScrR and RafR, respectively. Both clusters are controlled by the global activator of carbohydrate catabolism, the cyclic AMP receptor protein (CRP). E. chrysanthemi growth with lactose is possible only for mutants with a derepressed nonspecific lactose transport system, which was identified as RafB. RafR inactivation allows the bacteria to the assimilate the novel substrates lactose, lactulose, stachyose, and melibionic acid. The raf genes also are involved in the assimilation of alpha- and beta-methyl-D-galactosides. Mutations in the raf or scr genes did not significantly affect E. chrysanthemi virulence. This could be explained by the large variety of carbon sources available in the plant tissue macerated by E. chrysanthemi.

Erwinia chrysanthemi iron metabolism: the unexpected implication of the inner membrane platform within the type II secretion system.

The type II secretion (T2S) system is an essential device for Erwinia chrysanthemi virulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important for E. chrysanthemi virulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores of E. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly, outE and outL mutants also exhibited similar phenotypes, but, outD and outJ mutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni(2+) affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.

High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray-based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.

Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence. IscR was able to suppress all of the phenotypes listed above in a sufC-dependent manner while depletion of the Isc system led to IscR-dependent activation of the suf operon. No virulence defects were found associated with csdA or nifS mutations. Surprisingly, we found that the sufC mutant was virulent against A. thaliana, whereas its virulence had been found altered in Saintpaulia. Collectively, these results lead us to propose that E. chrysanthemi possess the Fe/S biogenesis strategy suited to the physico-chemical conditions encountered in its host upon infection. In this view, the IscR regulator, which controls both Isc and Suf, is predicted to play a major role in the ability of E. chrysanthemi to colonize a wide array of different plants.

The acyl-homoserine lactone-type quorum-sensing system modulates cell motility and virulence of Erwinia chrysanthemi pv. zeae.

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI(Ecz)) and a cognate response regulator (expR(Ecz)). Chromatography analysis showed that expI(Ecz) encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI(Ecz) in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI(Ecz). These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.

Differential role of ferritins in iron metabolism and virulence of the plant-pathogenic bacterium Erwinia chrysanthemi 3937.

During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.