Laboratory assessment of E.Coli

The discovery of E. Coli by Theodor Escherichia in 1885 led to a breakthrough in the field of science. Being a facultative anaerobe, certain serotypes of E.coli are harmless. Its virulence factors include production of Shiga toxins, intestinal colonization and possessing putative virulence plasmid referred to as pO157. E.coliO157:H7 causes food borne illness, having severe symptoms on humans, (McGann, 2016 pp 4420). Prevalence of food borne illness are estimated to have a mortality rate of 60 deaths per year, 2300 hospitalization rate and 73,000 incidence rate as per the year in USA, (Hasman etal, 2016).

1. coli can be transmitted though contamination of water and soils. Further its transmission can take place through plants and animals through the usage of flagella into the cell walls of plants, (Sharapov et al, 2016 pp 2025).
2. coli presences have been found in plant surface and contaminate fresh vegetables such as spinach, lettuce and sprout seed. The resistance has been noticed even after washing the vegetable making it hazardous. Further bit can penetrate through plant cells through the root system and contaminate food plants thus causing diseases. In plants such as the lettuce, there surfaces can harbor E. coli which is resisting on the surface of the surface even after washing, it is spread to the stomata and on the leaves. Further they can penetrate through their root systems which eventually get onto the sizable parts. Further soils, water and animals can contribute to or lead to contamination in the farm.. Thus the strain can be found on the fruits parts of the plants.  Further the strain has been observed t enter through leaf structure of plants, causing disinfection of plants, (Schmidt et al, 2016).

Lab assessment of E coli has been ongoing in the Holden lab of James Hutton University taking the lead. The culture of E coli Sakai was made in minimal media refer to as LB broth and shown splitting process into various minimal media culture which has a leaf lysate. After full growing of the E coli, in the spinach leafy state with the minimal media, RNA sequence was performed in 3 parts and the readings taken to understand the various interactions which have been initiated on the interaction between bacteria and plant host in order to minimize the risks of acquiring food borne illness.

In assessing this experiment, principal component analysis was performed, where maximum variables of samples were done. This ensured visualization of the samples relationships. Principle component analysis allows for transformations of PC 1 in horizontal axis while the PC 2 on the vertical axis. This data indicated PC1 variation o the right side with 86.03% while PC2 had 6.81%.

On performances of cluster analysis, the two culture media were separated into leafy lysate and minimal media. This illustration indicated more closely related values. This signifies that the individual; replicates exhibit almost no formal structure. The graphs of the leaf lysate and minimal media are closely separated. This indicates that the replication is almost similar. The p-values indicating of approximate unbiased value, which is reflective of bootstrap value which has overall measurement of the branching pattern.

Scatter plot analysis using the FDA seq package using principle component analysis and clustering was done. Using Edge R, the estimate of the variation was assessed, with high biological value affecting the ability to identify the expressed genes. Further the use of edger was used in identification of differential expression of genes. Top genes were significantly expressed. Edge R is effective on identification of differential expression. Each of the 10 genes has been expressed as shown by the P-values. Each has a special description of the specific gene. Evaluation of large scale differential expression having three biological values provides clerical fold change. For many cases, log count per million signifies fold change which is appropriate and reliable.  These results illustrate the fold change being 2, which has high relativity value. Fold change is often high or low. As log changes, and then there is biological significant difference which exist. Thus as the read count is high so do the log fold change, which confirms the significance of the genes, (Wallden et al, 2016). Dispersion reflects the dispersion of data over time; the genes are trended having similar expression as those of dispersions in the gene direction. Display tag wise dispersion often indicates the common value. This justifies the use of log changes which predicts significant difference in the gene expression.

Volcano plot an functional annotation analysis

When the two media are assessed on a volcano, there exhibit high points of positive and negative values, while those at the lower bottom have nice folder change however indicating false discovery rate, thus indicating low significance. Expressions of the significant genes on mapping showed 41% of the presence. Green gene halos showed down regulation while the red halo genes were up regulated while genes with halo yhcN and bright green gen expressed could have significance based on its high level expression, (Liu et al, 2016). Thus the reliance of fold change can be high or low, thus justifying the need for log field changes which have high representation through biological significance in terms of expressions. Hence the investigation of biological differences as indicated in the read count can be high while the log change further signifies high values, thus confirming high significant biological genes.

Expression on KEGG pathway revealed important functions on the metabolic pathway. Presence of ion sensor and nutrient transporters found on the leafy state, further sulfur and carbon metabolism enzymes were over expressed.

The pathway of copper on the KEGG pathway shows that there is a display on ABC transporters. Copper is under expressed in the pathway, while there is an up regulation of the ion sensors on the nutrient transporters in the leafy state. The association of the up regulation ion sensors and nutrient linkages in the leaf lysate have shown to be illustrative of gene expression. On the other end sulfur and carbon metabolisms enzymes shows over expression. Thus sulfur and carbon have been shown to have enzymes which are metabolically over expressed.

E coli strains have been shown to utilize plants being host and becoming internalized causing interaction with the plant o a molecular level.  Based on this investigation on the responses of E coli O157:H7 Sakai growth in spinach leaf lysate solution was compared with the minimal media, (Rasmussen & Bell, 2017). Its performance was made using RNA seq with different expression analysis for determination of genes which are essential for the bacterium to form symbiotic relationship with the plant. Performance of differential expression exhibited low performance with low replicability of less than 12.

The findings show that the gene had more up regulation in spinach leaf lysate solution; having yhcN .This gene is preserved in between the E. coli Sakai and the K12. I t is not much known on its functionality of Sakai gene. Few studies have investigated on the functionality of Sakai gene, yhcN has been shown to be involved in stress response attributed to Cadmium and hydrogen peroxide.  Further it is essential in bio film formation in E.coli BW25113, derived from K-12, (Arthur et al, 2017 pp 18-21). This is explained on the basis that plants which have been wounded often exhibit production of oxidative bursts such as hydrogen peroxide and superoxide. The formed indication leads to expression of yhcN in the E.coli which serves as a protective measure for organism against the harmful effects of ROS which is produced from the wounded plant.  Further the yhcnN expression has also been linked to induced response in plant phenolics like ferulic acid and furfural plants which are both present in spinach. This display is illustrated y the action of the plants to be able to produce oxidative reactive oxygen having combination of hydrogen peroxide and superoxide. The induction process exhibited by the yhcnN in E coli has the ability to form protection from organism against the lethal effects of reactive oxygen substrate, ,(Sharpove et al, pp. 2026).

KEGG pathways visualization

Most down regulated gene that was observed in spinach leaf lysate was cusF. Lesser down regulation was observed on cusC and cusB. These genes are important in maintained of homeostasis in the cells. The homeostasis generated are linked to copper production which is essential enzymatic function. CusF which binds copper is found in cytoplasm of the E coli as encoded in cusCFB operon. Further it plays crucial function on enzymes activity, but be harmful in the cytoplasm of E. coli. Cus B is a fusion protein while Cus C forms the outer membrane protein, (Saldana-Meyer et al, 2014). The two proteins are essential in formation of efflux pump system which offers transport to the copper and silver in cytoplasm membrane to the extra cellular membranes. CusSR controls the two complete systems though peri plasmid sensors and response factor, (Uhlen et al, 2015).

The expression of cusCFB in the minimal media is key and must have copper. An example of this is the MOPS media, which has copper IV sulfate. Further sucrose which is also found on the spinach leaf lysate is not present in the minimal media.  Also the sucrose can have direct impacts through reduction of copper toxicity which improves the tolerance of E.coli in presence of copper. The activation of Cus R drives the transcription of cusCFB, thus being dependent on the extracellular copper concentrations. The minimal media expression illustrated in cusCFB shows that the medium have copper. Further presence of sucrose in the case of leaf lystat solution is directly involved in reduction of copper toxicity which increases E. coli tolerance. This is linked to down regulation display of cus CFB operon in the spinach leafy lysate, (Cho et al., 2014 pp. 4).  When there is present in the spinach leafy state , it is not detrimental in any way on the cells culture in the minimal media, thus cells won’t be able to pump and thus would need cusCFB to ne normally regulated and expressed in the media.

The enrichment of KEGG pathways has been shown to have ABC transporter which is significantly expressed. The binding of ATP on the ABC forms large super family having integral membranes which uses hydrolyses to enable catalysis of transport substrates across the lipid membranes and against concentration gradient, (Gu, Qiano & Wu, 2017). These Transported compound include substrates such as anti biotic and oligo peptides. The down regulation of Sakai shows that it is differentially expressed in genes which belong to the ABC family, which respond to leafy growth in lysate solution. These expressions occurred in presence of sugar, peptide, lipid and ion transport which is decreased in these states, (Scholaz et al, 2015 pp. 1930-1935). The ultimate function of E coli in such systems is to promote the molecules from the extracellular environment to the cells. Sakai exhibits down regulation of the majority of genes which belong to ABC response to this growth demand in the lystae solutions. Gene expressions were dramatically reduced by the expression of sugar, organic transport, sugar and peptide compounds.

The outer membrane of the bacteria has porins which are essential for allowing substrates such as amino acids and sugars to be permeable through the concentration gradient. The dependency of influx substrates relies on the intra and extra cellular concentrations, which allows the bacteria to get essential compounds such as sugars, amino acids and minerals. During low concentration, active transport is initiated for the nutrients, (Houser et al, 2015). This offers an explanation of the results obtained which indicate poor nutrients sin the minimal media, which requires ABC transporters to facilitate the nutrients though hydrolyses. In the leafy growth process, the solution is rich in nutrients, thus the bacteria organism is expected to get nutrients through passive means and down the concentration gradient which offers transport thus an expression of transporter genes may not be necessary in the process,, (Locher, 2016 pp. 487).

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