Effect of Iron Concentration on the Growth Rate of Pseudomonas syringae and the Expression of Virulence Factors in hrp-Inducing Minimal Medium

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp-inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe³⁺. The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.The type III secretion system (T3SS) is used by diverse plant and animal pathogens to invade and colonize their hosts (1). This secretion system translocates bacterial proteins (effectors) from the bacterial cytoplasm directly into the eukaryotic host cell cytosol, where the effectors subvert host cell processes to the advantage of the pathogen. In Pseudomonas syringae pv. tomato DC3000, the T3SS is responsible for the elicitation of hypersensitive reactions of nonhost plants and is essential for disease on host plants (14). Many T3SS genes in plant pathogens are denoted hrp, for hypersensitive response and pathogenicity. We know of several regulatory elements that control T3SS genes in P. syringae pv. tomato DC3000 (7, 27), including HrpL, an alternative sigma factor. However, the exact environmental signals that the bacteria respond to are unknown.The expression of avrB, a T3SS effector, varies depending on the carbon source in Pseudomonas syringae pv. glycinea race 0 (9). Other environmental factors affecting the expression of virulence-related genes have also been studied. Nitrogen and osmolarity are important for the expression of the Pseudomonas syringae pv. syringae 61 hrp genes (28). Osmotic strength, pH, and carbon source differentially affected the expression of T3SS genes in Pseudomonas syringae pv. phaseolicola (18). These results imply that catabolite repression by the tricarboxylic acid cycle intermediates may be involved in the induction process. With other pathogenic bacteria, nutritional conditions are reported to be an important factor for the induction of virulence. For example, the Xanthomonas hrp genes are induced by sucrose and sulfur-containing amino acids (21). The optimal condition for hrp gene expression may simulate leaf apoplast environmental factors, including hypo-osmotic pressure, low pH, and limited nutrient concentration (18).Iron is a micronutrient (required in concentrations less than 10⁻⁴ M) for in vitro cultures (22), and the typical concentration needed for optimal bacterial growth is 0.3 to 1.8 μM (24). Iron is an essential element for bacteria due to its participation in the tricarboxylic acid cycle, electron transport, amino acid and pyrimidine biosynthesis, DNA synthesis, and other critical functions (3). Iron uptake must also be regulated due to its lethal effect through the Fenton reaction (2). The effect of iron limitation on bacterial growth has been documented for Escherichia coli cultures (6, 19, 20). Two studies have shown that production of the phytotoxins, syringomycin, and syringotoxin from P. syringae responds in batch culture to iron supplementation (5, 15). Iron is known to alter the physiology of other pseudomonads in both batch and chemostat cultures (11, 16). Although iron is the fourth most abundant element in the earth''s crust, its availability is very low due to its low solubility in aqueous solution ([Fe³⁺] at pH 7, 10⁻¹⁸ μM) (24). Bacteria have evolved complex mechanisms to ensure that iron requirements are met but not exceeded. Siderophore-mediated transport of iron is one of the mechanisms used by bacteria to uptake iron from their environment (17).In this study, medium components in hrp-inducing minimal medium were evaluated systematically with a chemostat culture. Iron was found to be both a growth-limiting nutrient in hrp-inducing minimal medium and a mediator of virulence gene expression in the model plant pathogen P. syringae pv. tomato DC3000.     

Recombineering Using RecTE from Pseudomonas syringae

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.There are currently more than 1,500 completed or draft bacterial genome sequences available for public access. This data resource continues to grow rapidly and provides potential insights into the roles of individual genes and regulons. However, testing hypotheses based on sequence data requires direct experimental manipulation of each genome. While many established methods for modifying bacterial DNA can assist in genetic analysis of these organisms, they are often time-consuming and limited with respect to the types of changes that can be directed.New advances in recombineering (genetic engineering by recombination) offer powerful alternative strategies for site-directed mutagenesis of genomic loci and provide methods for rapid and precise functional genomic analysis in some organisms (9, 29, 36-38, 41, 43). In these cases, recombineering is very efficient when phage-encoded recombinases are supplied, such that in vivo expression of these proteins enables direct genetic engineering of chromosomal and episomal replicons. These proteins catalyze RecA-independent recombination (21) of linear DNA substrates with homologous genomic target loci. The phage recombination functions typically involve the coordinated action of a 5′-to-3′ exonuclease (i.e., RecE or lambda Exo) and a single-stranded DNA (ssDNA)-annealing and strand invasion protein (i.e., RecT or lambda Beta), which we shall refer to as recombinases for brevity. The recombinase binds to 3′ ssDNA ends that are exposed by the action of the exonuclease, forming a protein-DNA filament, which protects the substrate DNA and promotes annealing with the homologous genomic sequence (4, 17, 19, 24). The recombinases are sufficient to facilitate recombination of ssDNA oligonucleotides, presumably because the oligonucleotides resemble the 5′-end-resected double-stranded DNA (dsDNA) substrate (11). Most of the recombinase proteins that have been shown to facilitate recombination are located in operons and are adjacent to the exonuclease-encoding genes, although there are cases where functional recombinase proteins have been identified without an accompanying exonuclease (9).Recombineering technologies have great potential in functional genomic applications and have worked exceptionally well in a few species, but adapting current systems to different bacteria is often problematic. Evidence suggests that these recombination systems have narrow species specificity such that a given system may catalyze robust recombination in one species and be essentially nonfunctional when expressed in another (9, 37). The reasons for this are not known but may be due to a requirement for specific interactions between the recombinase and host-encoded factors (9). Although there is a need to apply recombineering techniques to Pseudomonas species, only marginal success using the characterized phage recombination systems has been reported (14, 23). Most notably, recombinant strains of Pseudomonas aeruginosa were generated using long-homology substrates in the presence of plasmids expressing the lambda Red genes, but the relative influence of the Red genes was not reported (23).Here, we describe the identification of new recombineering proteins that function in a pseudomonad. The genes that encode proteins with similarity to the RecE/RecT proteins of the Rac prophage and lambda Red Exo and Beta were identified in Pseudomonas syringae pv. syringae B728a. These proteins promote efficient homologous recombination between genomic loci and linear DNA substrates introduced directly into P. syringae pv. tomato DC3000 cells by electroporation. These findings provide a foundation for more efficient site-directed mutagenesis of chromosomal loci in P. syringae and serve as a strategy for identifying similar proteins for recombineering in other bacteria.     

Analysis of the Role of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola 1448a in Interaction with the Plant

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.Type III secretion systems (T3SS) are complex and specialized machineries that inject effector proteins directly into the host cell cytosol (2). In Pseudomonas syringae, T3SS-mediated secretion is essential for disease in compatible hosts and for eliciting the hypersensitive response (HR) in incompatible hosts (1). P. syringae pathovars secrete a variable number of type III effectors that form their so-called secretomes and are expressed within the plant under the control of the alternative sigma factor HrpL (47). Understanding how the T3SS determines pathogenicity requires the functional characterization of the complete type III effector inventory. However, this characterization has been partially hindered by the fact that mutation of individual effectors, usually the most straightforward approach, rarely causes virulence attenuation (14). Thus, reports showing the contribution of the type III effector to virulence in P. syringae pathovars have resorted to ectopic expression in homolog-lacking related strains (40), plasmid-cured derivatives (21), double mutants (6, 28), or polymutants (3, 26). In relation to this, we have previously established the use of the competitive index (CI) in mixed infections (13, 42) as a more sensitive virulence assay for P. syringae pathovars than traditional assays (31). Using CIs, we demonstrated for the first time the individual contribution of AvrPto, an otherwise thoroughly characterized type III effector from Pseudomonas syringae pv. tomato (9, 17, 18, 27, 36, 39, 40, 46), to pathogen growth within its natural host (31). Therefore, analysis of effector mutants by use of the CI may provide the means to establish the quantitative contribution of the members of P. syringae T3SS secretomes to virulence. In addition, genetic analysis of the effects of combinations of effector mutations on virulence has already proven a useful approach to establishing the contribution of the members of the P. syringae pv. tomato DC3000 secretome to virulence by revealing a functional overlap (6, 26, 28). Thus, generation of knockouts in all individual effector genes of a given secretome, achieved in such a manner as to allow for easy combination of these strains into double or multiple mutant strains, is a desirable task, albeit a cumbersome one, considering the size of most secretomes.The secretome of the fully sequenced wild-type (wt) representative of the Pseudomonas syringae pv. phaseolicola 1448a strain (Pph1448a) has previously been analyzed, using a differential fluorescence induction screen (7) and bioinformatics (44), to identify effector genes. Our laboratory contributed to establishing this secretome through the development and application of a very sensitive assay for T3SS-mediated translocation based on CI assays (30). This assay represents an improvement over the sensitivity of the commonly used AvrRpt2 reporter assay. When fused to a T3SS-secreted protein, AvrRpt2^81-255 is translocated inside the host cell, eliciting a hypersensitive response (HR), dependent on the resistance protein RPS2 (32). By using CIs to measure the bacterial growth reduction associated with the AvrRpt2-RPS-mediated defense response, we detected translocation for two out of four Pph1448a effector candidates previously discarded by other assays, HopAJ1 and HopAK1 (30), and demonstrated translocation for two out of five previously untested candidates, HopAH2 and A0129. However, although in planta expression has been shown to take place in an HrpL-dependent manner for HopAJ1 and HopAK1 (7), it has not been established for HopAH2 and A0129. Effector nomenclature guidelines recommend that the abbreviation for the pathovars as well as the name of the strain should be included within the effector name (29). For simplicity, we include this indication only when effectors from other pathovars are mentioned. In summary, to date, 22 effectors in Pph1448a have been experimentally validated (7, 30, 44), one HrpL-regulated candidate has given inconsistent translocation results (AvrE1) (7), two translocated candidates have not been analyzed for expression in planta (HopAH2 and A0129) (30), one bioinformatically identified candidate has not been experimentally tested (AvrB4-2) (23), and six additional candidates have been proposed but experimentally ruled out (PSPPH3757, HopAN1, HopAJ2, HopW1-2, HopV1, and HopJ1) (7, 30).In this work, we analyzed the translocation and/or expression of these and other candidate effectors to close the type III effector inventory of Pph1448a. Our results indicate that the Pph1448a complete type III secretome is formed by 27 validated effectors, including a new one, HopAY1, which does not belong to any of the previously described effector families. The work includes the development of a simplified process for quick generation of single and double knockout mutants and its application to constructing a collection of single mutants for almost all members of the Pph1448a type III secretome. Additionally, we generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the four single and two double mutant strains as well as the double mutants expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to the virulence of Pph1448a. The tools and approach used in this work set the groundwork for dissecting the contribution of the entire Pph1448a type III secretome to virulence.     

The Siderophore Pyoverdine of Pseudomonas syringae pv. tabaci 6605 Is an Intrinsic Virulence Factor in Host Tobacco Infection

To investigate the role of iron uptake mediated by the siderophore pyoverdine in the virulence of the plant pathogen Pseudomonas syringae pv. tabaci 6605, three predicted pyoverdine synthesis-related genes, pvdJ, pvdL, and fpvA, were mutated. The pvdJ, pvdL, and fpvA genes encode the pyoverdine side chain peptide synthetase III l-Thr-l-Ser component, the pyoverdine chromophore synthetase, and the TonB-dependent ferripyoverdine receptor, respectively. The ΔpvdJ and ΔpvdL mutants were unable to produce pyoverdine in mineral salts-glucose medium, which was used for the iron-depleted condition. Furthermore, the ΔpvdJ and ΔpvdL mutants showed lower abilities to produce tabtoxin, extracellular polysaccharide, and acyl homoserine lactones (AHLs), which are quorum-sensing molecules, and consequently had reduced virulence on host tobacco plants. In contrast, all of the mutants had accelerated swarming ability and increased biosurfactant production, suggesting that swarming motility and biosurfactant production might be negatively controlled by pyoverdine. Scanning electron micrographs of the surfaces of tobacco leaves inoculated with the mutant strains revealed only small amounts of extracellular polymeric matrix around these mutants, indicating disruption of the mature biofilm. Tolerance to antibiotics was drastically increased for the ΔpvdL mutant, as for the ΔpsyI mutant, which is defective in AHL production. These results demonstrated that pyoverdine synthesis and the quorum-sensing system of Pseudomonas syringae pv. tabaci 6605 are indispensable for virulence in host tobacco infection and that AHL may negatively regulate tolerance to antibiotics.Phytopathogenic bacteria employ a variety of virulence mechanisms to overcome the defense systems of plants. Pseudomonas syringae pv. tabaci 6605 is a gram-negative bacterium that causes wildfire disease on host tobacco plants. Previously, we demonstrated that flagellin, a component of the flagellar filament of this organism, is a major elicitor of the hypersensitive reaction and is posttranslationally modified by glycosylation (26, 28-30). A genetic region composed of three open reading frames (ORFs), namely, fgt1, fgt2, and orf3, was previously identified in a flagellum gene cluster. fgt1 and fgt2 encode flagellin glycosyltransferase, and orf3 shows significant homology to the 3-oxoacyl-(acyl carrier protein [ACP]) synthase III in the fatty acid elongation cycle, required for the synthesis of acyl homoserine lactones (AHLs) (11, 30, 31). Analysis of an orf3 deletion (Δorf3) mutant revealed that orf3 played no role in the glycosylation of flagellin, although the virulence of the Δorf3 mutant on tobacco plants was remarkably reduced.Many virulence factors of bacteria have been reported to be under the regulation of a cell density-dependent system called quorum sensing. AHLs are synthesized by the coupling of the homoserine lactone ring from S-adenosylmethionine and acyl chains from the acyl-ACP by PsyI in P. syringae (9, 11). P. syringae pv. tabaci 6605 secretes three types of AHLs as signal molecules: N-hexanoyl-l-homoserine lactone, N-(3-oxohexanoyl)-l-homoserine lactone, and N-octanoyl-l-homoserine lactone (31). Our previous study indicated that the Δorf3 mutant and the quorum-sensing molecule-defective ΔpsyI mutant had significantly reduced abilities to produce AHLs and to take up iron (31). Furthermore, a scanning electron micrograph revealed little extracellular polymeric substance matrix surrounding the inoculated Δorf3 and ΔpsyI mutants on the tobacco leaf surface, indicating a lack of biofilm development (31). Iron acquisition has been reported to affect biofilm formation (2), and iron uptake is also involved in biofilm development under the regulation of quorum sensing in P. syringae pv. tabaci 6605 (2, 3, 11, 31).Iron is indispensable for the growth of almost all organisms, and the ability to acquire iron is thought to be an important factor in virulence (27). Because the concentration of Fe(III) in the environment is quite low, owing to its insolubility under environmental conditions, the fluorescent Pseudomonas group produces a yellow-green Fe(III)-chelating siderophore called pyoverdine in order to acquire iron effectively (24). The genes required for pyoverdine synthesis are well characterized in the Pseudomonas aeruginosa strain PAO1 (33), and pyoverdine biosynthesis mutants of this pathogen exhibit reduced virulence (20, 33). For the regulation of iron homeostasis, it was reported that Fur (ferric uptake regulator) is a global regulator that controls the expression of siderophore-mediated iron uptake in P. syringae pv. tabaci 11528 (6).In the present study, two genes predicted to be involved in pyoverdine synthesis, encoding the pyoverdine side chain peptide synthetase III l-Thr-l-Ser component (pvdJ) and the pyoverdine chromophore synthetase (pvdL), and the TonB-dependent ferripyoverdine receptor gene (fpvA) were disrupted in order to elucidate the roles of the pyoverdine-mediated iron acquisition system in the virulence of P. syringae pv. tabaci 6605. By use of these mutants, several important virulence factors, flagellum-dependent motility, the production of tabtoxin and extracellular polysaccharide (EPS), and biofilm formation were investigated. Although the ΔpvdJ and ΔpvdL mutants had reduced ability to produce EPS, the antibiotic tolerance of these mutants was drastically increased. The correlations among the pyoverdine-mediated iron uptake system, quorum-sensing regulation, and multidrug efflux are also discussed.     

Mqo,a Tricarboxylic Acid Cycle Enzyme,Is Required for Virulence of Pseudomonas syringae pv. tomato Strain DC3000 on Arabidopsis thaliana

Plant pathogenic bacteria, such as Pseudomonas syringae pv. tomato strain DC3000, the causative agent of tomato bacterial speck disease, grow to high levels in the apoplastic space between plant cells. Colonization of plant tissue requires expression of virulence factors that modify the apoplast to make it more suitable for pathogen growth or facilitate adaptation of the bacteria to the apoplastic environment. To identify new virulence factors involved in these processes, DC3000 Tn5 transposon insertion mutants with reduced virulence on Arabidopsis thaliana were identified. In one of these mutants, the Tn5 insertion disrupted the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the tricarboxylic acid cycle. mqo mutants do not grow to wild-type levels in plant tissue at early time points during infection. Further, plants infected with mqo mutants develop significantly reduced disease symptoms, even when the growth of the mqo mutant reaches wild-type levels at late stages of infection. Mutants lacking mqo function grow more slowly in culture than wild-type bacteria when dicarboxylates are the only available carbon source. To explore whether dicarboxylates are important for growth of DC3000 in the apoplast, we disrupted the dctA1 dicarboxylate transporter gene. DC3000 mutants lacking dctA1 do not grow to wild-type levels in planta, indicating that transport and utilization of dicarboxylates are important for virulence of DC3000. Thus, mqo may be required by DC3000 to meet nutritional requirements in the apoplast and may provide insight into the mechanisms underlying the important, but poorly understood process of adaptation to the host environment.One important aspect of interactions between plant pathogens and their hosts is the ability of the pathogen to obtain nutrients within the plant tissue. Nutrient acquisition is essential for growth within the host, since both cell division and DNA replication can be influenced by nutrient availability. Bacterial plant pathogens differ in the strategies they use to get necessary nutrients during infection. Some pathogens, such as Agrobacterium tumefaciens, elicit production of specific carbon and nitrogen sources by the plant (1). Other pathogens may rely on metabolites that are readily available in the plant apoplast or may stimulate the release of water or nutrients from surrounding plant cells (27).Little is known about how pathogenic Pseudomonas syringae strains acquire nutrients when growing in their hosts. P. syringae strains are gram-negative gammaproteobacteria, which as a group cause disease on many agriculturally important plants. For example, P. syringae pv. tomato strain DC3000 causes disease on tomato, A. thaliana, and several agriculturally important Brassicas, such as turnip, mustard, collard, and cauliflower (8, 51). Initially, DC3000 colonizes plant surfaces and then enters the plant tissue through natural openings (such as stomata) or wounds (34, 38). DC3000 then establishes itself in the plant apoplast, the intercellular space between plant cells (38). Once in the apoplast of susceptible hosts, DC3000 multiplies to high levels, and the infected plants develop disease symptoms, including chlorosis (yellowing) of the leaf tissue and necrotic spots or patches called lesions (38, 49). Pseudomonads, such as P. aeruginosa and P. fluorescens, preferentially utilize tricarboxylic acid (TCA) cycle intermediates (20, 29, 33, 44), and DC3000 utilizes these carbon sources in culture (19). Some studies to investigate nutrient acquisition of DC3000 have been carried out (3, 7, 40); however, it is not clear what carbon sources DC3000 utilizes when growing in plant tissue.Several virulence factors are necessary for DC3000 to enter, grow inside the plant, and cause disease. Like many other bacterial pathogens, DC3000 uses a type III secretion system (TTSS) (15), which is encoded by the hrp/hrc genes, to inject effector proteins into plant cells (16, 27). Many of these effectors suppress host defenses, and it is likely that some may be involved in modulating the apoplastic environment or nutrient acquisition (16). DC3000 also produces the phytotoxin coronatine, which promotes entry of the bacteria into the plant apoplast by stimulating the opening of stomata (34) and is required for bacterial growth in the apoplast by suppressing salicylic acid (SA)-dependent host defenses (4, 45). Coronatine also promotes disease symptom development via an SA-independent mechanism (4). While much emphasis has been placed on exploring how type III-secreted effectors and coronatine promote DC3000 virulence, other factors are also likely to be important during pathogenesis.To identify additional factors involved in pathogenesis, we undertook a genetic screen to identify novel virulence factors (5, 24). DC3000 mutants with reduced virulence were identified by assaying for their ability to elicit disease symptoms on A. thaliana and tomato plants (24, 37). One of these mutants, AK4C9, had reduced virulence on both hosts. The gene disrupted in this mutant is the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the TCA cycle. mqo mutants grow more slowly than wild-type DC3000 in planta and in culture when dicarboxylates are the only carbon source, suggesting that dicarboxylates are important for the growth of DC3000 in the apoplast. In the present study, we explore the role of Mqo and a dicarboxylate transporter, DctA1, in DC3000 pathogenesis.     

Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.Many proteobacterial pathogens use a type III secretion system (T3SS) as their primary mechanism to overcome and infect eukaryotic hosts. T3SSs are complex macromolecular machines that span both the bacterial cell envelope and host cell barriers to deliver proteins, commonly termed effectors, from the bacterial cytoplasm into the host cytoplasm (13, 19). After delivery into the host, effector proteins manipulate host cell function and suppress host defenses, allowing bacterial proliferation and disease development (6, 20). Bacteria that rely on T3SS to cause disease include plant pathogens such as Pseudomonas syringae, Ralstonia solanacearum, Erwinia and Xanthomonas species and animal pathogens in the genera Yersinia, Salmonella, Shigella, Escherichia, and Pseudomonas. While the repertoire of effectors delivered by a given T3SS is unique, the T3SS machinery is more universal (13). T3SS includes a core set of eight conserved proteins. These proteins, which are also conserved in bacterial flagellar biogenesis machines, make up the multiringed base structure, or basal body, that spans the bacterial membranes and cell wall. T3SS machines are also comprised of less-conserved and unique proteins that vary between systems. These include regulatory proteins that orchestrate construction of the machine and the extracellular components that function to translocate effectors across host barriers.The extracellular portion of the T3SS is comprised of the pilus or needle appendage (in plant or animal pathogens, respectively), which acts as a conduit for effector delivery, and the translocon complex, which creates the pore in the host cell membrane. These substructures vary between different T3SSs; presumably these external structures have adapted to allow different bacteria to infect different types of host cells. For Yersinia enterocolitica to infect macrophage cells, the T3SS needle must be a particular length (∼58 nm) to bridge the lipopolysaccharides extending from the bacterial outer membrane and reach the host cell membrane (35). Several other animal pathogens have T3SS needles of a defined length (48). Enteropathogenic Escherichia coli also has an additional extension beyond the needle called the EspA filament that functions to span the mucous layer found outside enterocyte cells (13). In plant pathogens, however, the extracellular gap between a bacterium and a plant cell includes a thick plant cell wall that is variable in width between plant species. Consequently, plant pathogenic Pseudomonas syringae has a pilus that can measure over 1 μm in vitro (25).Another major difference between the T3SS machineries of animal and plant pathogens is their translocon complexes. In animal pathogens, these are typically comprised of three essential proteins, but there is growing evidence that plant pathogen translocons employ diverse, functionally redundant components (28). There is growing interest in understanding the regulatory players that orchestrate the construction of diverse machinery. It is hypothesized that the assembly of the T3SS must involve several tightly regulated steps that allow secretion of the required components, followed by that of effectors upon completion. Of particular interest here is the control of pilus/needle subunit secretion, which is necessary when the pilus/needle is being constructed but would presumably compete with translocon and effector secretion after the T3SS is complete.We study the model plant pathogen P. syringae pv. tomato (Pto) DC3000, the causal agent of bacterial speck of tomato and Arabidopsis thaliana (8). DC3000 has a T3SS that delivers ca. 28 effectors and is essential for pathogenesis (11, 12, 30, 43). The P. syringae T3SS is encoded by hrp and hrc genes (hypersensitive response and pathogenicity/conserved), which are located in a pathogenicity island on the chromosome (4). hrc genes encode the conserved core components present in every T3SS. hrp genes encode T3SS components that are divergent or unique to P. syringae and enterobacterial plant pathogens, which also possess Hrp1 class T3SS (13). In contrast, plant pathogenic Ralstonia and Xanthomonas spp. have Hrp2 class T3SS, as indicated by several different Hrp proteins and distinct regulatory systems.To better understand the T3SS machinery, we previously conducted a survey of the hrp genes of P. syringae pv. syringae (Psy) 61 to complete the inventory of all those encoding proteins capable of traveling the T3SS into plant cells when expressed from a constitutive promoter (39). We hypothesized that these proteins might aid in pilus or translocon construction or regulate the construction process. HrpP was one protein found to be a T3SS substrate and important for secretion and translocation of the model effector AvrPto. Importantly, HrpP is related to a well-studied protein from Yersinia enterocolitica, YscP, which is a T3SS-secreted protein and a regulator responsible for switching the T3SS from secreting needle subunits to secreting effector proteins (15, 38, 47). It has also been shown that secretion of YscP into the culture medium is not essential for the switch function and that there may be two type III secretion signals embedded in YscP (2).The phenotype of a yscP mutant is unregulated secretion of the needle subunit, no secretion of effectors, and production of needles of indeterminate length. The switching phenotype requires a domain at the C terminus of YscP called the type III secretion substrate specificity switch (T3S4) domain, which is a conserved feature unifying its homologs (1). YscP has been proposed to act as a molecular ruler because the length of the YscP protein is directly correlated with the length of the Ysc needle (26). According to this model, when the needle has reached its proper length, YscP signals to the T3SS machinery to stop secreting needle subunits and begin secreting effector proteins. However, other functional models have been hypothesized for homologs of YscP. A recent study of the Salmonella enterica serovar Typhimurium YscP homolog InvJ showed that an invJ mutant lacked an inner rod. When the inner rod protein PrgJ was overexpressed, the length of the needle decreased relative to that of the wild type, leading the researchers to conclude that InvJ controls the inner rod, which in turn controls needle length (33). Recent evidence in Yersinia has lent more support to this model. YscP was found to negatively control secretion of YscI, the inner rod protein (51). Also, certain YscI mutations affected needle assembly but not effector secretion, implying that YscI may be a key player in substrate switching. Little is known about HrpB, the inner rod homolog in P. syringae (22), other than that the protein can be translocated into plant cells and is essential for T3SS function (39).Other models for length control/substrate switching have been proposed, such as the “C-ring cup model” in flagella, which was based on the observation that certain mutations in proteins that make up the inner membrane C ring of the basal body lead to shorter hooks (the flagellar equivalent of the needle), thus suggesting that C-ring capacity controls hook length (32). A more recent, flagellar “molecular-clock” model suggests that because overexpression of hook subunits leads to longer hooks and hook polymerization-defective mutants make shorter hooks, hook polymerization initiates a countdown, and the timing, in cooperation with the YscP homolog FliK, determines final hook length (34).HrpP is considered a member of the YscP/FliK family due mostly to the presence of a T3S4 domain at its C terminus. HrpP is also proline rich (10.6%), which is considered a characteristic of the family. The most striking feature of HrpP is its small size; the protein is 189 amino acids, compared with YscP from Y. enterocolitica, which is 453 amino acids and 8.4% proline. We were intrigued by how HrpP functions in P. syringae to regulate a pilus that can measure several hundred nanometers in length. Also, unlike animal pathogen needles and flagellar hooks, the pilus of P. syringae is predicted to be indeterminate in length, based on the fact that plant cell walls vary in width between species (40).We hypothesized that HrpP would be a main player in regulating pilus construction in P. syringae by allowing the system to make the transition between secretion of pilus subunits and secretion of translocon or effector proteins, though perhaps by a novel mechanism. In this study, we more precisely define the role of HrpP in the P. syringae T3SS. We show that HrpP is a T3SS substrate in DC3000, is translocated into plant cells at levels equivalent to those of effectors, and is essential for the function of the T3SS. Though it is highly translocated and variable, we found that HrpP from different P. syringae pathovars could complement the DC3000 hrpP mutant. Analysis of truncations of HrpP and an impassible HrpP-green fluorescent protein (GFP) fusion suggests that it has structural similarities to YscP, but surprisingly, HrpP was found to be required for full secretion of the pilus subunit HrpA as well as for translocation of HrpB.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

2-Amino-3-(Oxirane-2,3-Dicarboxamido)-Propanoyl-Valine,an Effective Peptide Antibiotic from the Epiphyte Pantoea agglomerans 48b/90

The epiphyte Pantoea agglomerans 48b/90, which has been isolated from soybean leaves, belongs to the Enterobacteriaceae, as does the plant pathogen Erwinia amylovora, which causes fire blight on rosaceous plants such as apples and leads to severe economic losses. Since P. agglomerans efficiently antagonizes phytopathogenic bacteria, the P. agglomerans strain C9-1 is used as a biocontrol agent (BlightBan C9-1). Here we describe the bioassay-guided isolation of a peptide antibiotic that is highly active against the plant pathogen E. amylovora and pathovars of Pseudomonas syringae, and we elucidate its structure. Bioassay-guided fractionation using anion-exchange chromatography followed by hydrophobic interaction liquid chromatography yielded the bioactive, highly polar antibiotic. The compound was identified as 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine by using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance techniques. This peptide was found to be produced by three of the nine P. agglomerans strains analyzed. Notably, the biocontrol strain P. agglomerans C9-1 also produces 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine. Previously, 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been characterized only from Serratia plymuthica. 2-Amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been shown to inhibit the growth of the human pathogen Candida albicans efficiently, but its involvement in the defense of epiphytes against phytopathogenic bacteria has not been investigated so far.Microbial pathogens pose a major threat to many plants and can cause enormous losses in agriculture. Microorganisms that antagonize pathogens can offer a way to fight plant diseases that is more environmentally friendly than chemical treatment. Such diseases include fire blight, which is caused by Erwinia amylovora and affects many rosaceous plants, e.g., apple and pear (18, 25, 29, 38).Suitable strains for biocontrol agents are often plant-associated microorganisms that are forced to defend their ecological niches under natural conditions and are thus adapted to competition with plant pathogens (2, 3). The species Pantoea agglomerans (formerly Erwinia herbicola) comprises many strains that are promising sources for biocontrol agents (8, 15, 30, 32, 43). P. agglomerans strains are ubiquitous in nature, inhabiting plant surfaces, water, soil, animals, and humans (9, 11). Several Pantoea isolates are known to inhibit E. amylovora efficiently in planta (39, 42). In vitro experiments have revealed some antibiotics from P. agglomerans and uncovered how they act against E. amylovora (22, 43). The known antibiotics produced by P. agglomerans strains, which belong to diverse chemical classes and affect different molecular targets, exhibit both narrow- and broad-spectrum activities (21).For example, P. agglomerans Eh318, isolated from apple leaves, produces two peptide antibiotics, pantocin A and pantocin B; both interfere with amino acid biosynthesis. Pantocin A blocks l-histidinol phosphate aminotransferase (20), and pantocin B acts as an N-acetylornithine transaminase inhibitor (5). Consequently, their inhibitory effects can be compensated for by supplementation with l-histidine and l-arginine, respectively (43). Giddens et al. (2002) described a phenazine antibiotic and its precursors, which were produced by P. agglomerans Eh1087 (10). Andrimid, a hybrid nonribosomal peptide polyketide antibiotic from P. agglomerans Eh335, selectively blocks the carboxyl transfer reaction of prokaryotic acetyl coenzyme A carboxylase; this reaction catalyzes the first committed step of fatty acid biosynthesis (19, 26). P. agglomerans E325 sold as Bloomtime Biological (Northwest Agricultural Products, Pasco, WA) acidifies flower stigmata, thus reducing the growth of E. amylovora. Simultaneously, it produces an antibiotic that has high specificity against E. amylovora and is effective under low-phosphate and low-pH conditions (34).P. agglomerans C9-1, which is registered as the biocontrol agent BlightBan C9-1 (Nufarm Agricultural Inc.), produces two antibiotics, herbicolin O and herbicolin I (16). Like pantocin A, herbicolin O loses its activity in the presence of histidine. However, herbicolin I does not become ineffective in the presence of amino acids (17). Although C9-1 is registered as a biocontrol agent, the chemical nature of herbicolins has remained largely unknown (13, 14).P. agglomerans 48b/90 (Pa48b), an epiphyte from soybean leaves (40), attracted our attention because it strongly inhibits the growth of E. amylovora and Pseudomonas syringae pv. glycinea (27), the pathogen that causes the bacterial blight of soybean. Since the mode of action of Pa48b against plant pathogens, in particular E. amylovora, is elusive, we looked for the molecular basis for the biocontrol potential of Pa48b. Here we describe the isolation, structure elucidation, and bioactivity of a potent antibiotic against plant pathogens that is produced by several P. agglomerans strains. The properties of this antibiotic perfectly match those of the chemically unidentified herbicolin I from P. agglomerans C9-1 (BlightBan C9-1).     

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k_cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K_m) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k_cat = 1.9 × 10² s⁻¹ on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.Aldohexuronate catabolism in bacteria is reported to involve two different pathways, one initiating with an isomerization step and the other with an oxidation step. In the isomerization pathway, aldohexuronate (glucuronate and galacturonate) is isomerized to ketohexuronate by uronate isomerase and ultimately degraded to pyruvate and 3-phosphoglyceraldehyde. The isomerization pathway has been previously reported to occur in bacteria, including Escherichia coli (7), Erwinia carotovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae (9, 23), and Serratia marcescens (28). In the oxidation pathway, aldohexuronate is oxidized to aldohexarate by uronate dehydrogenase (Udh) and further catabolized to pyruvate (2, 5, 7, 9, 18, 19, 24). Uronate dehydrogenase, the key enzyme of this pathway, has been investigated in two plant pathogen bacteria, Pseudomonas syringae and Agrobacterium tumefaciens. To date, only limited studies pertaining to the properties of Udh have been reported in the literature (3, 6, 38, 43), and no sequence has yet been identified. Udh is classified as an NAD-linked oxidoreductase (EC 1.1.1.203), with a total molecular weight of about 60,000. It is a homodimer composed of two subunits with molecular weights of about 30,000 each (38). Udh is a thermally unstable, reversible enzyme, with an optimum pH of about 8.0 (3, 6, 38).In E. coli MG1655 that has the isomerization pathway for aldohexuronate catabolism, glucuronate is transported by an aldohexuronate transporter encoded by exuT and converted to fructuronate by uronate isomerase, encoded by uxaC (22, 30) (Fig. ​(Fig.1).1). Fructuronate is transferred to the Entner-Doudoroff pathway to be utilized as an energy source via 2-keto-3-deoxy-6-phospho-gluconate (7, 27, 31, 32). Therefore, E. coli MG1655 with a uxaC deletion cannot use glucuronate as a carbon source. In this strain, glucarate is converted to 5-keto-4-deoxy-d-glucarate by d-glucarate dehydratase, encoded by gudD, and then transferred to glycolysis via pyruvate or 2-phosphoglycerate (27, 33). Recently, a number of bacterial genome sequences have been published, including those of the Udh-containing P. syringae pv. tomato strain DC3000 and A. tumefaciens strain C58 (4, 10). A shotgun library of P. syringae was constructed to identify the gene encoding Udh. Screening for Udh was conducted in E. coli MG1655 ΔuxaC. Since uronate dehydrogenase converts glucuronate to glucarate, uxaC deletion strains of E. coli harboring the shotgun library of P. syringae that can grow in a minimal medium containing glucuronate as a sole carbon source may carry the gene encoding Udh (Fig. ​(Fig.1).1). Once an initial Udh is identified from P. syringae, a BLAST homology search may lead to the identification of Udhs from other bacteria.Open in a separate windowFIG. 1.Catabolism of glucuronate and glucarate in bacteria. Glucuronate consumption is prevented by knockout of the uxaC gene. The presence of uronate dehydrogenase in a uxaC knockout enables growth of E. coli on glucuronate.     

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.Molecular hydrogen (H₂) is important in intermediary ecosystem metabolism (i.e., processes that link input to output) in wetlands (7, 11, 12, 33) and other anoxic habitats like sewage sludges (34) and the intestinal tracts of animals (9, 37). H₂-producing fermenters have been postulated to form trophic links to H₂-consuming methanogens, acetogens (i.e., organisms capable of using the acetyl-coenzyme A [CoA] pathway for acetate synthesis) (7), Fe(III) reducers (17), and sulfate reducers in a well-studied moderately acidic fen in Germany (11, 12, 16, 18, 22, 33). 16S rRNA gene analysis revealed the presence of Clostridium spp. and Syntrophobacter spp., which represent possible primary and secondary fermenters, as well as H₂ producers in this fen (11, 18, 33). However, H₂-producing bacteria are polyphyletic (30, 31, 29). Thus, a structural marker gene is required to target this functional group by molecular methods. [Fe-Fe]-hydrogenases catalyze H₂ production in fermenters (19, 25, 29, 30, 31), and genes encoding [Fe-Fe]-hydrogenases represent such a marker gene. The objectives of this study were to (i) develop primers specific for highly diverse [Fe-Fe]-hydrogenase genes, (ii) analyze [Fe-Fe]-hydrogenase genes in pure cultures of fermenters, acetogens, and a sulfate reducer, (iii) assess [Fe-Fe]-hydrogenase gene diversity in H₂-producing fen soil enrichments, and (iv) evaluate the limitations of the amplified [Fe-Fe]-hydrogenase fragment as a phylogenetic marker.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.