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Introduction

A recent survey (footnote: Del Rio) found that two bacterial strains LD1 and LD2 (later identified as Pseudomonas fluorescens and putida) were capable of almost completely degrading a mixture of naphthenic acids when co-cultured together.

Figure 1. Source: (Del Rio)

Figure 1 shows the percentage of remaining naphthenic acids in a solution after four weeks of bacteria culturing. Both LD1 and LD2 may be capable of some degradation on their own, but the effect of co-culturing them together is to achieve a near total degradation of all samples in the mixture.

Figure 2.

Figure 2 shows how the degradation affected the concentrations of specific naphthenic acids of varying carbon number and saturation, as measured by GC-MS. The chart on the left of figure 2 shows the initial concentrations of each of the naphthenic acids, and the chart on the right shows the concentration of the naphthenic acids after four weeks of a LD1-LD2 co-culture. Based on the decreased height and scale, the figure demonstrates that naphthenic acids were more or less indiscriminately degraded by the co-culture.

When both figure 1 and 2 are taken together, the most likely conclusion is that the genes responsible for the degradation are spread across the two species. Furthermore, there exists a collection of essential genes in this degradation pathway which are unique to one species and not present in the other. The degradation pathway has not been experimentally confirmed, but it is believed to be some sort of metabolic pathway which involves beta-oxidation (footnote: Del Rio). In any case, the promoters upstream of these essential genes would be a likely place to find a naphthenic acid promoter - and indeed, our later experiments found that they were. A weakness of the bioinformatics search was that LD1 and LD2 have not yet been sequenced, and therefore we assume similarity between them and corresponding strains that have been sequenced; this weakness complicated primer design but was not prohibitive to the project.

Abstract

Three online algorithms (DarkHorse, ProteinWorld DB, and Pseudomonas.com) were used to find target gene candidates in silico, and a fourth (MUMmer ) was used to confirm experimental hypotheses. Using the data from these sources, we were able to construct a short list of gene candidates which were then processed through a quantative Real-Time Polymerase Chain Reaction (qRT-PCR) to verify gene expression as a response to naphthenic acids. The qRT-PCR found that Enoyl CoA Hydratase, from Pseudomonas putida, was expressed as a response to naphthenic acids. The characterization of its promoter can be found below.

DarkHorse Results

DarkHorse is an online database for finding phylogenetically atypical proteins in various microbial species. Generally, phylogenetic atypicality can be useful for predicting the horizontal transfer of genes; in this bioinformatics survey, this also functions to provide a list of relatively unique genes in each of the strains in Pseudomonas putida and fluorescens.

insert the bit here about the bioninformatics search dark horse and all come one pplllllllll!!!!!!!!!!!!!!1

Using RT-qPCR to Detect Upregulation of Genes in Response to NAs

The pseudomonas strains (P. putida and P. fluorescens) were exposed to NAs to see if the genes selected through the bioinformatics search were in any way upregulated when compared with ordinary levels of expression. If they were upregulated it would indicate whether or not they contained NA responsive regions and hence a possible NA responsive promoter.

After treating the cells they were then preserved by exposure to an RNA stabilizing chemical, lysed and the RNA extracted. Next the RNA was converted to cDNA using random short primers and reverse transcriptase. The cDNA was then subjected to RT-PCR with primers that would amplify select genes of interest as well as control primers that amplify 16S-RNA sequences whose levels were expected to be stable.

To date no data has been obtained from the PCRs because the primers have not been amplifying the genes. Trouble shooting and optimization are ongoing at this point to determine the best parameters for the primers to work. MAKE CHANGES HERE NEW DATA HAS BEEN OBTAINED !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Promoter Characterization

THIS SECTION SHOULD CONTAIN CHARACTERIZATION FOR THE PROMOTER, IF IT EXISTS.