Use of xylosidase 3C from Segatella baroniae to discriminate xylan non-reducing terminus substitution characteristics.

OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) ß-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of ß-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

Effect of Protein Surface Hydrophobicity and Surface Amines on Soy Adhesive Strength.

Soy is considered one of the most promising natural materials for manufacturing wood adhesives due to its low cost, high protein content, and ready availability. However, more cost-effective ways of improving its wet shear strength are needed to achieve wider market acceptance. Protein adhesive wet strength depends on the use of (typically expensive) crosslinking additives as well as the processing/denaturation of the protein. It has been commonly stated in the literature that protein denaturation leads to higher bond strength by activating the surface and exposing the reactive groups. Therefore, we investigated how differences in surface reactive groups (surface hydrophobicity and reactive amine groups) brought on with different denaturation treatments relate to bonding performance. Fourteen soy protein isolates (SPIs) with different denaturation histories were investigated. Characterization of the SPIs included surface hydrophobicity, surface amine content, extent of protein hydrolysis, and bond strength (wet and dry, with and without polyamidoamine epichlorohydrin (PAE) crosslinking agent) by ASTM D7998. The molecular weight patterns showed that proteins denatured by extensive hydrolysis had very low bond strengths. Adding the crosslinker, PAE, improved all the shear strength values. We found that the number of water-accessible reactive amine groups on protein surfaces had no impact on the adhesive strength, even with the amine-reactive crosslinker, PAE. Conversely, increased surface hydrophobicity was beneficial to adhesive strength in all cases, though this correlation was only statistically significant for wet strength without PAE. While, in general, denatured proteins are typically thought to form better bonds than native state proteins, this work suggests that it matters how proteins are denatured, and what surfaces become exposed. Denaturation by hydrolysis did not improve bond strength, and extensive hydrolysis seemed highly detrimental. Moreover, exposing hydrophobic surface groups was beneficial, but exposing covalent bond-forming reactive amine groups was not.

The first crystal structure of a xylobiose-bound xylobiohydrolase with high functional specificity from the bacterial glycoside hydrolase family 30, subfamily 10.

Xylobiose is a prebiotic sugar that has applications in functional foods. This report describes the first X-ray crystallographic structure models of apo and xylobiose-bound forms of a xylobiohydrolase (XBH) from Acetivibrio clariflavus. This xylan-active enzyme, a member of the recently described glycoside hydrolase family 30 (GH30), subfamily 10, phylogenetic clade has been shown to strictly release xylobiose as its primary hydrolysis product. Inspection of the apo structure reveals a glycone region X2 -binding slot. When X2 binds, the non-reducing xylose in the -2 subsite is highly coordinated with numerous hydrogen bond contacts while contacts in the -1 subsite mostly reflect interactions typical for GH30 and enzymes in clan A of the carbohydrate-active enzymes database (CAZy). This structure provides an explanation for the high functional specificity of this new bacterial GH30 XBH subfamily.

A New Subfamily of Glycoside Hydrolase Family 30 with Strict Xylobiohydrolase Function.

The Acetivibrio clariflavus (basonym: Clostridium clariflavum) glycoside hydrolase family 30 cellulosomal protein encoded by the Clocl_1795 gene was highly represented during growth on cellulosic substrates. In this report, the recombinantly expressed protein has been characterized and shown to be a non-reducing terminal (NRT)-specific xylobiohydrolase (AcXbh30A). Biochemical function, optimal biophysical parameters, and phylogeny were investigated. The findings indicate that AcXbh30A strictly cleaves xylobiose from the NRT up until an α-1,2-linked glucuronic acid (GA)-decorated xylose if the number of xyloses is even or otherwise a single xylose will remain resulting in a penultimate GA-substituted xylose. Unlike recently reported xylobiohydrolases, AcXbh30A has no other detectable hydrolysis products under our optimized reaction conditions. Sequence analysis indicates that AcXbh30A represents a new GH30 subfamily. This new xylobiohydrolase may be useful for commercial production of industrial quantities of xylobiose.

CaXyn30B from the solventogenic bacterium Clostridium acetobutylicum is a glucuronic acid-dependent endoxylanase.

OBJECTIVE: We previously described the structure and activity of a glycoside hydrolase family 30 subfamily 8 (GH30-8) endoxylanase, CaXyn30A, from Clostridium acetobutylicum which exhibited novel glucuronic acid (GA)-independent activity. Immediately downstream from CaXyn30A is encoded another GH30-8 enzyme, CaXyn30B. While CaXyn30A deviated substantially in the highly conserved ß7-α7 and ß8-α8 loop regions of the catalytic cleft which are responsible for GA-dependence, CaXyn30B maintains these conserved subfamily 8 amino acid residues thus predicting canonical GA-dependent activity. In this report, we show that CaXyn30B functions as a canonical GA-dependent GH30-8 endoxylanase in contrast to its GA-independent neighbor, CaXyn30A. RESULTS: A clone expressing the catalytic domain of CaXyn30B (CaXyn30B-CD) exhibited GA-dependent endoxylanase activity. Digestion of glucuronoxylan generated a ladder of aldouronate limit products as anticipated for canonical GA-dependent GH30-8 enzymes. Unlike the previously described CaXyn30A-CD, CaXyn30B-CD showed no activity on arabinoxylan or the generation of appreciable neutral oligosaccharides from glucuronoxylan substrates. These results are consistent with amino acid sequence comparisons of the catalytic cleft and phylogenetic analysis.

Draft Genome Sequence of Burkholderia cepacia ATCC 17759, a Polyhydroxybutyrate-Co-Valerate Copolymer-Producing Bacterium.

Burkholderia cepacia ATCC 17759, isolated from forest soils in Trinidad, accumulates large amounts of polyhydroxyalkanoate copolymers when grown on xylose, mannose, arabinose, other carbohydrates, and organic acid cosubstrates. This 8.72-Mb draft genome sequence of B. cepacia ATCC 17759 will provide better insight into this organism's utility in lignocellulose bioconversion.

A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium.

Glycoside hydrolase family 30 subfamily 8 (GH30-8) ß-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the -3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains.

Genomic and transcriptomic analysis of carbohydrate utilization by Paenibacillus sp. JDR-2: systems for bioprocessing plant polysaccharides.

BACKGROUND: Polysaccharides comprising plant biomass are potential resources for conversion to fuels and chemicals. These polysaccharides include xylans derived from the hemicellulose of hardwoods and grasses, soluble ß-glucans from cereals and starch as the primary form of energy storage in plants. Paenibacillus sp. JDR-2 (Pjdr2) has evolved a system for bioprocessing xylans. The central component of this xylan utilization system is a multimodular glycoside hydrolase family 10 (GH10) endoxylanase with carbohydrate binding modules (CBM) for binding xylans and surface layer homology (SLH) domains for cell surface anchoring. These attributes allow efficient utilization of xylans by generating oligosaccharides proximal to the cell surface for rapid assimilation. Coordinate expression of genes in response to growth on xylans has identified regulons contributing to depolymerization, importation of oligosaccharides and intracellular processing to generate xylose as well as arabinose and methylglucuronate. The genome of Pjdr2 encodes several other putative surface anchored multimodular enzymes including those for utilization of ß-1,3/1,4 mixed linkage soluble glucan and starch. RESULTS: To further define polysaccharide utilization systems in Pjdr2, its transcriptome has been determined by RNA sequencing following growth on barley-derived soluble ß-glucan, starch, cellobiose, maltose, glucose, xylose and arabinose. The putative function of genes encoding transcriptional regulators, ABC transporters, and glycoside hydrolases belonging to the corresponding substrate responsive regulon were deduced by their coordinate expression and locations in the genome. These results are compared to observations from the previously defined xylan utilization systems in Pjdr2. The findings from this study show that Pjdr2 efficiently utilizes these glucans in a manner similar to xylans. From transcriptomic and genomic analyses we infer a common strategy evolved by Pjdr2 for efficient bioprocessing of polysaccharides. CONCLUSIONS: The barley ß-glucan and starch utilization systems in Pjdr2 include extracellular glycoside hydrolases bearing CBM and SLH domains for depolymerization of these polysaccharides. Overlapping regulation observed during growth on these polysaccharides suggests they are preferentially utilized in the order of starch before xylan before barley ß-glucan. These systems defined in Pjdr2 may serve as a paradigm for developing biocatalysts for efficient bioprocessing of plant biomass to targeted biofuels and chemicals.

Transcriptomic analysis of xylan utilization systems in Paenibacillus sp. strain JDR-2.

Xylans, including methylglucuronoxylans (MeGX(n)) and methylglucuronoarabinoxylans (MeGAXn), are the predominant polysaccharidesin hemicellulose fractions of dicots and monocots available for conversion to biofuels and chemicals. Paenibacillus sp. strain JDR-2 (Pjdr2) efficiently depolymerizes MeGX(n) and MeGAX(n) and assimilates the generated oligosaccharides, resulting in efficient saccharification and subsequent metabolism of these polysaccharides. A xylan utilization regulon encoding a cellassociated GH10 (glycoside hydrolase family 10) endoxylanase, transcriptional regulators, ABC (ATP binding cassette) transporters, an intracellular GH67 -glucuronidase, and other glycoside hydrolases contributes to complete metabolism. This GH10/GH67 system has been proposed to account for preferential utilization of xylans compared to free oligo- and monosaccharides. To identify additional genes contributing to MeGX(n) and MeGAXn utilization, the transcriptome of Pjdr2 has been sequenced following growth on each of these substrates as well as xylose and arabinose. Increased expression of genes with different substrates identified pathways common or unique to the utilization of MeGX(n) or MeGAX(n). Coordinate upregulation of genes comprising the GH10/GH67 xylan utilization regulon is accompanied with upregulation of genes encoding a GH11 endoxylanase and a GH115 -glucuronidase, providing evidence for a novel complementary pathway for processing xylans. Elevated expression of genes encoding a GH43 arabinoxylan arabinofuranohydrolase and an arabinose ABC transporter on MeGAX(n) but not on MeGX(n) supports a process in which arabinose may be removed extracellularly followed by its rapid assimilation.Further development of Pjdr2 for direct conversion of xylans to targeted products or introduction of these systems into fermentative strains of related bacteria may lead to biocatalysts for consolidated bioprocessing of hemicelluloses released from lignocellulose.

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark ß8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved ß8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily ß-1,4-xylanase function.

Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava.

BACKGROUND: The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose. FINDINGS: We examined the sensitivity of seven polyhydroxyalkanoate producing bacteria: Azohydromonas lata, Bacillus megaterium, Bacillus cereus, Burkholderia cepacia, Pseudomonas olevorans, Pseudomonas pseudoflava and Ralstonia eutropha, against seven fermentation inhibitors produced by the saccharification of lignocellulose: acetic acid, levulinic acid, coumaric acid, ferulic acid, syringaldehyde, furfural, and hyroxymethyfurfural. There was significant variation in the sensitivity of these microbes to representative phenolics ranging from 0.25-1.5 g/L coumaric and ferulic acid and between 0.5-6.0 g/L syringaldehyde. Inhibition ranged from 0.37-4 g/L and 0.75-6 g/L with acetic acid and levulinic acid, respectively. B. cepacia and P. pseudoflava were selected for further analysis of polyhydroxyalkanoate production. CONCLUSIONS: We find significant differences in sensitivity to the fermentation inhibitors tested and find these variations to be over a relevant concentration range given the concentrations of inhibitors typically found in lignocellulosic hydrolysates. Of the seven bacteria tested, B. cepacia demonstrated the greatest inhibitor tolerance. Similarly, of two organisms examined for polyhydroxybutyrate production, B. cepacia was notably more efficient when fermenting pentose substrates.

Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood.

Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these--two in glycoside hydrolase family 5, one in family 10, and one in family 12--have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

Pseudomonas sax genes overcome aliphatic isothiocyanate-mediated non-host resistance in Arabidopsis.

Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

Abscisic acid has a key role in modulating diverse plant-pathogen interactions.

We isolated an activation-tagged Arabidopsis (Arabidopsis thaliana) line, constitutive disease susceptibility2-1D (cds2-1D), that showed enhanced bacterial growth when challenged with various Pseudomonas syringae strains. Systemic acquired resistance and systemic PATHOGENESIS-RELATED GENE1 induction were also compromised in cds2-1D. The T-DNA insertion adjacent to NINE-CIS-EPOXYCAROTENOID DIOXYGENASE5 (NCED5), one of six genes encoding the abscisic acid (ABA) biosynthetic enzyme NCED, caused a massive increase in transcript level and enhanced ABA levels >2-fold. Overexpression of NCED genes recreated the enhanced disease susceptibility phenotype. NCED2, NCED3, and NCED5 were induced, and ABA accumulated strongly following compatible P. syringae infection. The ABA biosynthetic mutant aba3-1 showed reduced susceptibility to virulent P. syringae, and ABA, whether through exogenous application or endogenous accumulation in response to mild water stress, resulted in increased bacterial growth following challenge with virulent P. syringae, indicating that ABA suppresses resistance to P. syringae. Likewise ABA accumulation also compromised resistance to the biotrophic oomycete Hyaloperonospora arabidopsis, whereas resistance to the fungus Alternaria brassicicola was enhanced in cds2-1D plants and compromised in aba3-1 plants, indicating that ABA promotes resistance to this necrotroph. Comparison of the accumulation of salicylic acid and jasmonic acid in the wild type, cds2-1D, and aba3-1 plants challenged with P. syringae showed that ABA promotes jasmonic acid accumulation and exhibits a complex antagonistic relationship with salicylic acid. Our findings provide genetic evidence that the abiotic stress signal ABA also has profound roles in modulating diverse plant-pathogen interactions mediated at least in part by cross talk with the jasmonic acid and salicylic acid biotic stress signal pathways.

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum.

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5'UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE.

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

The Tat pathway of the plant pathogen Pseudomonas syringae is required for optimal virulence.

Pseudomonas syringae is a gram-negative bacterium that infects a number of agriculturally important plant species. The ability of the organism to deliver virulence factors across the plant cell wall is a key to its pathogenicity. Deletion mutants in the twin arginine translocation (Tat) pathway of two pathovars of P. syringae, pvs. tomato DC3000 and maculicola ES4326, displayed a range of pleiotropic phenotypic changes, such as defects in fluorescent siderophore production, a decrease in sodium dodecyl sulfate and copper resistance, and a significant loss in fitness using Arabidopsis thaliana or tomato as plant hosts. The genome sequence of P. syringae pv. tomato DC3000 encodes a number of potential virulence factors that are predicted to be translocated via the Tat pathway, including several proteins involved in iron scavenging (two siderophore receptors, PSPTO3474 and PSPTO3294, and an aminotransferase, PSPTO2155, involved in siderophore biosynthesis). Further candidates for Tat-dependent pathogenicity determinants include the homologs of a cell wall amidase (PSPTO5528), an enzyme involved in periplasmic glucans biosynthesis (PSPTO5542), and two putative phospholipases (PSPTO3648 and PSPTOB0005). Translocation of the putative amidase, aminotransferase, glucans biosynthetic enzyme, and the two phospholipases, but not the two siderophore receptors, is shown to be dependent on the Tat pathway. Strains deleted for the genes encoding the probable aminotransferase and amidase enzymes are significantly less infectious than the wild type. We conclude that the incremental effects due to the failure to correctly localize at least two, and possibly more, Tat substrates gives rise to the attenuated fitness phenotype of the P. syringae pv. tomato DC3000 tat strain.