An official website of the United States government.
Objective 1. To define the effect of the pectynolityc activities of P. carotovorum on Salmonella fitness and characterize its physiological response to pectinolysis. Because Salmonella is not able to degrade plant pectins, we hypothesize that it benefits directly or indirectly from the pectinolytic activities of phytopathogens. Specifically, we will test the hypothesis that Salmonella directly benefits by scavenging uronic acid mono- or oligomers that result from pectin hydrolysis. Alternatively, we will test the hypothesis that the pectinolytic activities of P. carotovorum indirectly facilitate proliferation of Salmonella by liberating starches, which are not utilized by Pectobacterium. To test the first hypothesis, P. carotovorum export of degradative exoenzymes will be interrupted by disrupting its Out type-II secretion system and Salmonella uptake and utilization of these mono- and oligo-mers will be disrupted using a KdgR regulator mutant (45). The competitive fitness of the Salmonella mutant will be compared to that of its wild-type during colonization of tomato soft rots caused by the virulent tomato strain P. carotovorum strain SR38 or its isogenic export mutant. As a control, Salmonella infections will be supplemented with applications of recombinant hexahistidine-tagged pectinases. To test whether Salmonella preferentially utilizes starches liberated by pectinolytic activities of P. carotovorum, the competitive fitness of ?-amylase deficient S. enterica mutants will be assessed in tomatoes and within soft rots. <P>Objective 2. To determine if Salmonella utilizes its ability to synthesize the auxin indole-3-acetic acid (IAA) to respond to and alter its environment during colonization of tomatoes. We will test the hypothesis that the IAA produced by Salmonella serves as a signal molecule that increases Salmonella's fitness during tomato infection. To this end, an isogenic mutant of STM2405 (which encodes a predicted IAA synthase IdpC) will be made. The mutant will be characterized in vitro and its fitness in tomato (in the presence or absence of P. carotovorum) will be tested. Recombination-based In Vivo Expression Technology (RIVET) mutants or qRT-PCR will be used to define the regulation of ipdC under different environmental conditions. To determine if Salmonella responds to the products of ipdC, Salmonella's IAA regulon will be determined using mRNA-seq. Because Salmonella is hypothesized to benefit from the plant tissue degradative abilities of phytobacteria, the influence of Salmonella-synthesized IAA on P. carotovorum will be determined by examining the timing and level of P. carotovorum production of pectinolytic enzymes and the expression of the corresponding genes.
Non-Technical Summary:<br/>
The contamination of fresh produce with harmful bacteria such as Salmonella is an increasing public health problem. To infect crops, Salmonella must interact with the naturally occurring bacteria already on the plant. One of these bacteria, Erwinia carotovora, is responsible for causing soft rot or sunken black spots of degraded tissue in a variety of produce. A clear link has been established between the presence of soft rot and Salmonella contamination of produce. However, the mechanisms involved in these interactions are currently unknown. The purpose of this project is to understand how Salmonella exploits or manipulates soft rot to reach large population sizes that may put consumers at risk of enteric disease. Soft rot is caused by the bacterial enzymes which degrade un-digestible plant polymers into usable sugars or acids. This project will determine if Salmonella benefits by competing for these simple molecules or scavenging for the starches which Erwinia can't digest. Erwinia regulates enzyme production via the plant hormone indole-3-acetic acid (IAA). Salmonella is also capable of producing an indole derivative, which is unusual for a human enteric pathogen, although the role of this signal in Salmonella is unknown. This study will also examine the genetic regulatory changes induced by IAA in both Salmonella and Erwinia. Understanding the metabolic and signaling interactions between the relevant pathogens will allow the selection of intervention strategies to improve the management of produce safety.
<P>
Approach:<br/>
Objective 1. To determine if Salmonella directly benefits from pectinolysis, the export of P. carotovorum degradative exoenzymes will be interrupted by disrupting the Out type-II secretion system. Pectate lyase concentrations will be measured in vitro prior to tomato experiments via the spectrophotomeric methods. To determine if Salmonella interacts with P. carotovorum as a competitor or niche specific specialist, competitive co-infections with 14028 and its isogenic carbon utilization mutants will be established by co-inoculation with SR38 or its outS mutant in market ready green tomatoes. Whole tomatoes will be harvested by stomaching in PBS and the homogenate serially plated on XLD to determine Salmonella densities. 50 recovered colonies will be patched onto LB agar with appropriate antibiotics to quantify the ratio of mutant to wild-type Salmonella and calculate a competitive index. Significance will be determined by t-test comparison to unmarked-marked Salmonella wild type co-infections. Mutants of interest will be tested for complementation and with antibiotic swaps to control for confounding factors. To remove potential confounding interactions of other pathways, hexahistidine-tagged pectate lyases produced by P. carotovorum will be used as a control to cause soft rot.
<P>
Objective 2. Salmonella's IAA regulon will be determined by comparing the transcriptome of wild-type 14028 and its ipdC mutant in vitro using mRNA-seq. Optimized IAA production conditions will be determined using an ipdC RIVET reporter. RIVET uses a heritable marker of gene activation and "records" activation events which occur at anytime during the assay. Production of IAA under favorable conditions will be confirmed via TLC, quantified via HPLC and transcript levels confirmed via qRT-PCR prior to mRNAseq. To help determine the relevance of IAA signaling in planta, the transcriptomes of both S. enterica 14028 and P. carotovorum SR38 will be also determined from tomato soft rot samples. To determine if Salmonella influences P. carotovorum pectinase activity via IAA production the expression of Pectobacterium Pel genes and concentrations of pectate lyases will be determined in the presence of pure IAA, wild-type Salmonella or its ipdC mutant both in vitro and in soft rot lesions. Competitive co-infection of green market tomatoes using defined ipdC mutants of both S. enterica 14028 and P. carotovorum SR38 will be used to determine in vivo relevance. To determine if Salmonella directly influences tomato auxin signaling via IAA production, transcript levels of tomato genes involved in auxin production of signaling will be determined via qRT-PCR in response to wild type Salmonella, its isogenic ipdC mutant and heat killed 14028. All mutants will be constructed via lambda-red mutagenesis and will be based on Salmonella strain 14028 or P. carotovorum SR 38, a virulent strain used extensively in our lab for tomato co-infections. Competitive co-infections will be monitored for 3 days, under normal conditions soft rotted lesions appear after 24 hours and reach a dime size in 2 days.