Lab Assessment of E. Coli Sakai Growth in Spinach Leaf Lysate Solution

This article discusses the lab assessment of E. Coli Sakai growth in spinach leaf lysate solution and its performance using RNA seq with different expression analysis for determination of genes which are essential for the bacterium to form symbiotic relationship with the plant. It also covers the results of the experiment and their analysis, including principal component analysis, cluster analysis, scatter plot analysis, volcano plot and functional annotation analysis, and KEGG pathways visualization. The article concludes with a discussion of the findings and their implications.

Introduction
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).
E. 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).
E. 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).
Laboratory assessment of E.Coli
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.
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Lab results and analysis
Data analysis
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%.
Cluster analysis
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
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
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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.
KEGG pathways visualization
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.
Discussion
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
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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).
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
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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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