The Construction of Triple-Deleted Mutant M18ΔUMS as Cell Factory in Order to Improve PCA Production

Abstract

Phenazines are secondary metabolites chiefly known for their broad-spectrum anti-microbial property. Phenazine-1-carboxylic acid (PCA) produced by Pseudomonas aeruginosa, a gram-negative bacterium, is an effective biocontrol agent against a number of plant-related pathogens. This experiment aimed to increase the production of PCA by deleting genes UTR, phzM and phzS in Pseudomonas aeruginosa M18, thus rendering the new strain more suitable for commercial use. The triple-deleted mutant M18ΔUMS shows significantly increased performance in PCA production compared to wild-type M18 strain. The new strain should be more applicable for commercial usage. However, M18ΔUMS exhibits a decreased growth rate compared to M18, indicating a inhibitory effect caused by the excess PCA.

Full text

*Corresponding author: Yangmike24@gmail.com
yawenhe@sjtu.edu.cn
The Construction of Triple-Deleted Mutant M18∆UMS as Cell
Factory in Order to Improve PCA Production
Da Yang*, Yawen He
School of Life Sciences and Biotechnology, Shanhghai Jiao Tong University, Shanghai 200240, China
Abstract: Phenazines are secondary metabolites chiefly known for their broad-spectrum anti-microbial
property. Phenazine-1-carboxylic acid (PCA) produced by Pseudomonas aeruginosa, a gram-negative
bacterium, is an effective biocontrol agent against a number of plant-related pathogens. This experiment
aimed to increase the production of PCA by deleting genes UTR, phzM and phzS in Pseudomonas
aeruginosa M18, thus rendering the new strain more suitable for commercial use. The triple-deleted mutant
M18∆UMS shows significantly increased performance in PCA production compared to wild-type M18
strain. The new strain should be more applicable for commercial usage. However, M18∆UMS exhibits a
decreased growth rate compared to M18, indicating a inhibitory effect caused by the excess PCA.
1 INTRODUCTION
1.1 Pseudomonas Aeruginosa
Psedomonas aeruginosa is a gram-negative, rod-shaped
and mono-flagellated bacterium. It can colonize a wild
range of environment including soil, water, plants,
animals and human. Its ubiquitous presence is partially
due to its ability to catabolize a broad-spectrum of
organic molecules. P. aeruginosa is also an opportunistic
human pathogen that frequently infects individuals with
compromised immune system. Infection commonly
occurs among patients with cystic fibrosis, cancer, or
AIDS.
1.2 Phenazines
Phenazines designates to a group of nitrogen-containing,
secondary metabolites. They are chiefly known for their
broad-spectrum capacity of suppressing various plant-
related pathogens. Up to 2017, it has been identified that
there are around 100 different phenazines derivatives and
an addition of 6000 compounds that contain phenazines
(Guttenberger, 2017). Biologically, they function as both
antibiotics and signaling molecules within bacterials. In
addition to their agricultural application as antibiotics for
plants, phenazines also attract the attention of researchers
from areas including microbial fuel cell production,
environmental sensing and antitumoractivity (Du, 2013).
In general, phenazines are secreted by a number of
bacterial genera including, notably, Psedomonas strains
(Mavrodi, 2010). While it is feasible to produce
phenazines through chemical synthesis, the method is
complicated by its low yield and toxic byproducts such
as aniline, azobenzoate, lead oxide or o-
phenylenediamine (Cheluvappa, 2014). Thus,
biosynthesis is employed in many occasion instead. By
uilitizing recombinant DNA technologies, several strains
of psedomonas can be engineered into cell factories that
yield phenazines in large quantity (Bilal, 2017). Among
all phenazine-producing pseudomonads, phenazine
biosynthesis is chiefly controlled by the gene cluster
phzABCDEFG (Blankenfeldt, 2013). Further cross-
species examinations uncover that phzB, phzD, phzE,
phzF, and phzG are conserved in all phenazine-producing
pseudomonads (Mavrodi, 2010). Accessory genes,
including phzO, phzH, phzM and phzS, that flank the
core biosynthetic genes are found within most phenazine-
producing bacteria and these genes are responsible for
many phenazine derivatives (Chin-A-Woeng, 2001).
1.2.1.As antibiotics
Phenazines are known for their anti-microbial property.
They have excellent antifungal capacity. For instance,
phenazine antibiotics produced by Pseudomonas are
proven to be an effective biocontrol against Fusarium
wilt, a lethal fungal infection of planets, on a wild range
of crops (Anjaiah, 1998). Other than fungus, phenazines
are known to control a variety of plant pathogens
including Streptomyces scabies, a bacterial that causes
corky lesions (Arnau, 2015), Pythium spp., which causes
infection among germinating tomatoes (Gurusiddaiah,
1986), Phytophthora infestans, an oomycete that causes
serious potato loss worldwide (Morrison, 2016) as well
as many other pathogens.
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(https://creativecommons.org/licenses/by/4.0/).
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1.2.2.As signaling molecules
Two categories of Psedomonas can be established
according to the copy number of the core phz gene
clusters. Strains such as P. fluorescence 2–79, P.
aureofaciens 30–84 and P. chlororaphis PCL1391 belong
to Psedomonas that possess only one set of core phz gene
cluster. These strains prove to be excellent Plant growth-
promoting rhizobacteria. Another category of
Psedomonas, on the other hand, possess two sets of
nearly identical core phz gene clusters: phzA1-G1 and
phzA2-G2. P. aeruginosa PAO1 and P. aeruginosa M18,
belong to this group. It is discovered that among
Pseudomonas sp. M18, phzA2-G2 only produces fairly
small amount of PCA while the majority of PCA is
produced from phzA1-G1. Moreover, the transcription of
phzA1-G1 is in fact induced by the small amount of PCA
produced from phzA2-G2. In this case, phenazines act as
both the product and the signaling molecule (Li, 2011).
1.2.3.Phenazine Derivates
Phenazine-1-carboxylic acid (PCA) designates to an
aromatic carboxylic acid. It substitutes a carboxy group
at C1 position of phenazine molecule. PCA is an
effective antibiotic agent and it has been used as the main
ingredient in an environment friendly fungicide named
Shenqinmycin, which is registered by the ministry of
Agriculture of China in 2011 (Du, 2015). The 1%
Shenqinmycin solution has been proven efficacious
against Rhizoctonia solani and Fusarium oxysporum
among a variety of rice, wheat and vegetable (Du, 2015).
Pyocyanin (PYO) is a metabolite produced from
Pseudomonas. It is involved with a variety of important
cellular activities. In P. aeruginosa, it is a virulence factor
ad well as a signaling molecule for quorum sensing. It is
also responsible for the blue-green colouration in P.
aeruginosa. Moreover, it sometimes functions as a
antimicrobial agent.
Phenazine-1-carboxamide (PCN) is an aromatic
amide. It substitutes a carbamoyl group at C1 position of
phenazine molecule. Like PCA, it is a strong antibiotic
against fungal pathogens like Fusarium oxysporum
(Chin-A-Woeng, 1998). It is also know to lessen the
effect of root disease caused by Gaeumannomyces
graminis var. tritici (Daval, 2011).
1.3 Phenazine Biosynthetic Pathway in M18 Phz
Cluster
A comprehensive graph of Phenazine biosynthetic
pathway from “Engineering Pseudomonas for phenazine
biosynthesis, regulation, and biotechnological
applications: a review” is presented below (Bilal, 2017).
Figure 1: The proposed PCA biosynthetic pathway (Bilal, 2017)
As illustrated in Figure. 1, Phenazines biosynthesis
starts with Shikimate pathway. At first,
phosphoenolpyruvate (PEP) and erythrose 4-phosphate
(E4P) is condensed in order to form 3-deoxy-d-ara-bino-
heptulosonate-7-phosphate (DAHP). A three-step
enzymatically catalyzed reactions took place and
transform DAHP into chorismate, which can be
transform into a wild variety of three-ringed aromatic
core phenazine structures. Through a series of phz genes
(A1-G1, or A2-G2), chorismate is eventually transformed
into PCA. Then a number of accessory genes that flank
the phz genes transform PCA into other phenazine
derivates in accordance to a number of factors. These
accessory genes includes phzO, phzH, phzM and phzS.
PhzM can transform PCA into 5MPCA, which in turn
transformed into PYO by phzS. PhzS alone can change
PCA into 1-OH-PHZ while phzH can produce PCN from
PCA.
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1.4 Aim of the Study
The aim of the study is to increase the PCA production of
Pseudomonas aeruginosa M18. Firstly, the object is to
knock out UTR gene, which help regulating the
production of PCA. PCA production is expected to
increase without functioning regulatory system. Then, the
study aim to delete phzS and phzM. These accessory
genes are responsible for further conversion of PCA into
PYO and other phenazine derivates. Their deletion can
result in PCA accumulation. Thus, the goal is to
construct the triple-deleted mutant M18∆UMS that yield
greater amount of PCA than the wild type. As mentioned
before, the antibiotic quality against plant pathogens
make PCA an ideal biocontrol agent for commercial use
in agriculture. This study aims to further improve the
efficiency of PCA production and explore more cost-
effective, safer methods of biosynthesis.
2 MATERIALS & METHODS
2.1 Strains and Plasmids used in this Study
Pseudomonas aeruginosa M18 (M18) is a strain isolated
from the rhizosphere soil of sweet melon in Songjiang,
Shanghai, China.
1-aminocyclopropane-1-carboxylate (ACC) is used as
a sole nitrogen source.
E.coli DH5a is a strain of E.coli commonly used for
cloning and related application. It is quite versatile and
have a wild range of application.
E. coli S17-1lpir contain the pir gene. It is commonly
used as the host strain that houses the transposon vector
DNA through biparental mating.
pk18 mobsacB Plasmid pk18 mobsacB is a cloning
vector that can mobilize into a variety of gram negative
and gram positive bacteria. It can integrate into host
chromosome via homologous recombination. Medium
containing 10% sucrose can facilitate selection against
bacterial containing this plasmid.
2.2 Bacterial Growth
M18 is incubated on LB nutrient medium with
Spectinomycin antibiotic at 28ºC. LB nutrient medium is
created by a mixture of 5g yeast extract, 10g tryptone, 5g
NaCI and a 1 liter of distilled water.
2.3 DNA Manipulation
M18 is incubated on LB nutrient medium with
Spectinomycin antibiotic at 28ºC. LB nutrient medium is
created by a mixture of 5g yeast extract, 10g tryptone, 5g
NaCI and a 1 liter of distilled water.
2.3.1.The Construction of M18∆UTR
A. Design Primers
Firstly, two pairs of primers, UTR F1/R1, UTR
F2/R2 are designed to knock the UTR sequence. Then,
genome is extract from the strain Pseudomonas
aeruginosa M18.
B. PCR amplification
UTR-1 and UTR-2 are amplified through polymerase
chain reaction (PCR) according to the following table
and procedure. DNA is purified afterward.
PCR Sequence: 95˚C 5min, (95˚C 30s, 55˚C 40s,
72˚C 40s)*25, 72˚C 5min, 12˚C infinity
Table.1: Materials for PCR amplification
Materials
ul
10x Taq buffer with Mg2+
2.5*8=20
Taq
0.15*8=1.2
dNTP (2.5mM)
5*8=40
UTR F1/F2
1
UTR R1/R2
1
M18 delta u geomic DNA
0.5*8=4
ddH2O
16*8=128
Figure 2: Agarose gel analysis to show the PCR products
As shown in Figure. 2, 4 liens above are UTR1 and 4
liens below are UTR2.
C. Fragment Alignment
UTR-1 and UTR-2 are aligned by performing another
PCR. DNA is purified afterward.
PCR Sequence: 95˚C 5min, (95˚C 30s, 55˚C 40s,
72˚C 1min30s)*25, 72˚C 5min, 12˚C infinity
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Table. 2 Materials for fragment alignment
Materials
ul
10x KOD buffer
5*8=40
KOD
0.25*8=2
MgSO4
4*8=32
dNTP (2.5mM)
5*8=40
UTR F1
2*8=16
UTR R2
2*8=16
UTR1 product
0.25*8=2
UTR2 product
0.25*8=2
ddH2O
33*8=264
Figure 3: Agarose gel analysis to show the fusion PCR
products
As shown in Figure.3, all 16 lines are UTR-12.
D. Enzyme digestion
The aligned UTR fragment are treated with enzyme
EcoRI and XbaI, then placed in 37˚C water bath for 4
hours, followed by 65˚C water bath for 10 min.
E. Plasmid digestion
UTR fragment is digested with plasmid Pk18
mobsacB (Ecor-, Xba) and placed in 4˚C refrigerator
over night.
F. Plasmid transformation and bacterial colony
PCR
E.coli DH5a and the 5 ul product are mixed waited
upon for 30 minutes while keeping E.coli DH5a on ice
for all time. Then the mixture is placed in 42˚C water
bath for 90s and return it to ice for 2 min. On Clean
Bench, 800 ul liquid LB nutrient medium is added to the
mixture. Then the mixture is put in 37˚C, 2000 rpm
shaker for 1h. 100 ul liquid is extracted from the mixture
and plastered on petri dish with LK nutrient medium.
Then the petri dish is left in 37˚C shaker to incubate
overnight. Bacterial colony PCR is performed according
to the following table and procedure in order to confirm
the the success of plasmid conjugation.
PCR Sequence: 95˚C 5min, (95˚C 30s, 55˚C 15s,
72˚C 1min30s)*25, 72˚C 5min, 12˚C infinity
Table. 3 Materials for bacterial colony PCR
Materials
ul
10x Taq buffer with Mg2+
2.5*32=80
Taq
0.2*32=6.4
dNTP (2.5mM)
2*32=64
M13F
0.5*32=16
M13R
0.5*32=16
ddH2O
19.3*32=617.6
Figure 4: Agarose gel analysis to show the plasmid conjugation
As shown in Figure.4, bright lines in the middle
represent successful plasmid conjugation.
G. Gene sequencing
Then the samples are sent for gene sequencing.
Plasmid is extracted and transformed into E. coli S17-
1lpir.
H. Plasmid transformation into E. coli S17-1lpir
E. coli S17-1lpir and the 1 ul product are mixed and
waited upon for 30 minutes while keeping E. coli S17-
1lpir on ice for all time. Then the mixture is placed in
42˚C water bath for 90s and on ice for 2 min. On Clean
Bench, 800 ul liquid LB nutrient medium is added to the
mixture. Then put the mixture in 37˚C, 2000 rpm shaker
for 1h. 100 ul bacterial liquid is extracted from the
mixture and plastered on petri dish with LK nutrient
medium. Then left the petri dish to incubate at 37˚C
overnight. Next, 10 ml LB and 5 ul Kanamycin are added
to conical flask, then one colony is selected on the petri
dish and dipped in the flask using inoculating stick. Then
the flask is left in 37˚C shaker to incubate overnight. 8
lines are draw with the bacterial fluid in conical flask on
a new petri dish with LS nutrient medium. The petri dish
is incubated at 37˚C overnight. 10 ml LB and 5 ul
Kanamycin are added to the conical flask, then one line
is scratched on the petri dish and dipped in the flask
using inoculating stick. Then the flask is left in 37˚C
shaker to incubate overnight.
I. Preparing M18
Firstly, Pseudomonas aeruginosa M18 is retrieved
from refrigerator, then the bacterial fluid is plastered on
petri dish with LS nutrient medium. It is incubated at
28˚C overnight. 8 bacterial colonies are selected on the
incubated petri dish. 8 lines are draw on a new petri dish
with LS nutrient medium, the dish is incubated at 28˚C
overnight. 10 ml LB and 5 ul Kanamycin are added to the
conical flask, then one line is scratched on the petri dish
and dipped in the flask using inoculating stick. Then the
flask is left in 28˚C shaker to incubate overnight.
J. Mating
1 ml of Pseudomonas aeruginosa M18 and 1 ml of E.
coli S17-1lpir are extracted and placed in separate 1.5 ml
plastic tubes. The tubes is centrifuged at 5000 rotation,
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25˚C for 2 minutes. Then 1 mL LB is added and mixed
after removing the liquid. The process is repeated once.
600 ul LB is added to E. coli S17-1lpir and 300 ul LB to
Pseudomonas aeruginosa M18, they are mixed in one
tube and the bacterial liquid is dipped on a petri dish with
LK nutrient medium. The petri dish is dried and
incubated at 28 ˚C for 6-12 hours.
K. 1st round of selection
1 ml of PBS buffer is mixed with 2 loops of colonies.
Then two dilution (3* and 10*) are made. 100 ul liquid is
took from each dilution and plastered on petri dishes with
LSK nutrient medium. The dishes are incubated at 28˚C
for 36 hours. 8 colonies are selected from the petri dish
3* dilution and 8 lines are draw on a new petri dish.
Incubate the new petri dish at 28˚C for 1 day.
L. 2nd round of selection and bacterial colony
PCR
2 loops are scooped from each lines and mixed with 1
ml PBS buffer. Then 2 dilutions (10^-2, 10^-3) are made.
Each dilution is plastered on a petri dish with LBS
nutrient medium. They are incubated at 28 ˚C for 36
hours. Two petri dishes are prepared with LK and LS
nutrient medium and the following markings. Colonies
are selected from the petri dish with 10^-3 dilution and
draw a short line beneath each marks. Both dishes are
incubated at 28˚C for 1 day. The picture below shows the
result.
Figure 5: Colonies in the selection plates
As shown in Figure.5, number 2,5,6,7,9,10,13,15
show positive results. Colonies 1-15 on LS plate are
chosen for bacterial colony PCR. Steps are performed
according to the following table and procedure in order
to confirm the the existence of M18∆UTR.
PCR Sequence: 95˚C 5min, (95˚C 30s, 55˚C 40s,
72˚C 40s)*25, 72˚C 5min, 12˚C infinity
Table. 4 Materials for bacterial colony PCR
Materials
ul
10x Taq buffer with Mg2+
2.5*16=40
Taq
0.2*16=3.2
dNTP (2.5mM)
2*16=32
UTR-F1
0.5*16=8
UTR-F2
0.5*16=8
ddH2O
19.3*16=308.8
Figure 6: PCR analysis to identify the deletion mutants
As shown in Figure.6, lines at the lower positions are
positive. Number 5,6,7,9,10,13,15 show positive results
with the line at 2 being too faint.
The colonies with positive results (which are
5,6,7,9,10,13,15) are selected. 10 ml LB and 5 ul
Kanamycin are added to the conical flask, then the
selected colonies are scratched and dipped in the flask
using inoculating stick. Then left the flask in 28˚C shaker
to incubate overnight.
The constructed M18∆UTR are preserved by adding
500 ul bacterial fluid and 500 ul glycerinum in a 1.5 ml
test tube, briefly showered by liquid nitrogen. The frozen
test tube is place within refrigerator.
2.3.2.phzM knockout
The general procedure is the same as UTR knockout.
Two pairs of primers, phzM F1/R1, phzM F2/R2 are
designed to knock the phzM sequence. Then, genome is
extract from the strain M18∆U. In the following steps,
taq is used in PCR amplification, KOD is used for
fragment alignment, and enzymes EcoRI and BamHI are
used in enzyme digestion. Pk18 mobsacB (Ecor-, BamH-
) is constructed for plasmid digestion. Lastly, phzM-F1
and phzM-F2 are used in bacterial colony PCR. The
constructed M18∆UM is preserved in a 1.5 ml test tube
showered by liquid nitrogen. The frozen test tube is place
within refrigerator.
2.3.3.phzS knockout
The general procedure is the same as UTR knockout.
Two pairs of primers, phzS F1/R1, phzS F2/R2 are
designed to knock the phzS sequence. Then, genome is
extract from the strain M18∆UM. In the following steps,
taq is used in PCR amplification, KOD is used for
fragment alignment, and enzymes EcoRI and BamHI are
used in enzyme digestion. Pk18 mobsacB (Ecor-, BamH-
) is constructed for plasmid digestion. Lastly, phzS-F1
and phzS-F2 are used in bacterial colony PCR. The
constructed M18∆UMS is preserved in a 1.5 ml test tube
showered by liquid nitrogen. The frozen test tube is place
within refrigerator.
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3 RESULTS
3.1 M18 Colony phenotypes/colors
M18 appear to be yellow. M18∆U was deep blue/green,
PYO is responsible for this coloration. It can be inferred
that a great amount of PYO is produced once UTR is
deleted. M18∆UM was also blue/green. M18∆UMS
appear to be gold, indicting PYO is no longer present
once genes (phzM, phzS) are both knocked out.
3.1.1.Rates of Growth according to OD
In order to measure rate of growth for strain each strain,
first 500 ul bacterial liquid is added to two 1.5 ml test
tubes respectively. Then they are centrifuged at 12000
rotation for 5 minutes, the liquid is discarded and 500 ul
water is added. Solution is mixed well. 100 ul solution is
moved to a new 2 ml tube, then 900 ul water is added
and the OD value is measured. This process is repeated
for M18, M18∆U, M18∆UM, M18∆UMS.
Table. 5 Initial OD values of M18 strains
M18
M18∆U
M18∆UM
M18∆UMS
0.571A
0.592A
0.567A
0.491A
0.562A
0.610A
0.564A
0.491A
Formula A=OD*(x+2) is used to calculate x, which is
the volume of ppm. “OD” beng the average for each
strain and times it by 10. “A” being the volume of
bacterial liquid added, in this case, is 2 ml.
The calculated ppm value being:
Table. 6 ppm of M18 strains
M18
M18∆U
M18∆UM
M18∆UMS
9.33 ml
10.02 ml
9.31 ml
7.82 ml
Then 50 ul Spectinomycin and 2 ml bacterial liquid
are added, calculated volume of ppm is added into a 250
ml conical flask for each strain. The flasks are incubated
in 28˚C shaker. The OD values of each strain are
measured at 12 hour, 24 hour and 36 hour. The final
average measurement is listed in the following table and
a graph is plotted accordingly.
Table. 7 OD values of M18 strains at 12, 24, and 36h
0h
12h
24h
36h
M18
0.02
0.2385
0.409
0.585
M18∆U
0.02
0.2
0.398
0.542
M18∆UM
0.02
0.207
0.3825
0.5255
M18∆UMS
0.02
0.1745
0.373
0.5285
Figure 7: Growth Curves of M18 Strains
As shown on the graph, for all four strains, the
growth rate remain approximately positive and steady
throughout 36 hours of growth. This means all four
strains are growing at a constant rate. The slope of M18
appears to be steepest among the four strains while the
slope of M18∆UMS seems to be the flattest. This
indicates that M18 is growing faster than all other strains,
and M18∆UMS is the slowest.
3.1.2.PCA Yield
3 test tubes are prepared for each strain. Each tube is
filled with 180 ul bacterial liquid and 20 ul hydrochloric
acid, they are shaken vigorously for 10 seconds. 540 ul
chloroform is added and shaken for another 2 minutes.
The tubes are centrifuged at 12,000 rotation for 10
minutes. The lower clear fluid id moved into new tubes.
This procedure is performed at 24 hour and 36 hour.
Next, the tubes are placed in rotating spiral heater at
30˚C for 30 minutes. 100 ul of methyl alcohol is added
and mixed. Then 70 ul solution is moved into designated
glass tubes. HPLC is performed. The measured PCA
production is listed below and a graph is plotted
accordingly.
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Table. 8 PCA yield at 24h and 36h
M18
M18∆U
M18∆UM
M18∆UMS
24h
24.2580568
117.114612
176.8403066
242.098137
36h
35.77176314
205.0600281
262.2362896
294.745135
Figure 8: PCA Production of M18 Strains
From this graph, we can see that at both 24h and 36h,
the production of PCA increases as more targeted genes
are knocked out. M18 produce the least amount of PCA
while M18∆UMS produce the most amount of PCA.
4 DISCUSSIONS
4.1 Increased PCA production of M18∆UMS
As expected, the triple-deleted mutant produce the
highest amount of PCA, while the wild type produce the
lowest amount. The construction of triple-deleted mutant
successfully improve the production of PCA.
4.2 Toxic Inhibition Effect of PCA
It can be noticed that M18∆UM produce the highest
amount of PCA and the least amount of growth.
Conversely, the wild type M18 produce the least amount
of PCA and the highest amount of growth. It can be
inferred that the production of PCA might be negatively
correlated with the growth rate of M18 strain. Another
study also discovers that the when phz genes become
inactivated, mutant stains of M18 produce less PCA and
show a greater amount of growth compared to the wild
type (Li, 2011). The researchers suggest that a potential
toxic inhibition effect on bacterial might emerge under
certain PCA concentration (Li, 2011). Thus it is
reasonable to infer that the excessive concentration of
PCA caused by deletions might have negatively impact
the growth of bacterials in M18∆UMS, M18∆UM, and
M18∆U. Although it is possible that the deletion of genes
might have affect other mechanisms. PYO has a range of
biological functions including being electron shuttle for
cell respiration.
4.3 The Regulatory Function of UTR Genes
The PCA production increases sharply after the deletion
of UTR genes. This elucidates that UTR genes play a
negative regulatory role in the production of PCA. The
regulation of phenazine production is maintained by a
complex network of QS system, GacS/GacA two-
component signal transduction, small non-coding RNAs
including RsmX, RsmY, and RsmZ as well as some other
regulators in response to the immediate environmental
condition (Morrison, 2016). In P. aeruginosa,
GacS/GacA signal transduction system performs its
function exclusively by controlling the transcription of
the these small RNAs (Brencic, 2009). These RNAs bind
to the mRNA-repressor proteins of the RsmA/CsrA
family, which themselves bind to UTR regions (Karine,
2008). The precise mechanism of entire regulatory
system and its connection to UTR are still wanting, but it
is definite that UTR inhabit the production of PCA.
4.4 Expected PYO Production
Both M18∆U and M18∆UM show deep blue/green
coloration, indicating the presence of PYO. The deep
blue/green coloration of M18∆U suggests an
overabundance of PCA will lead to an overproduction of
PYO as well. M18∆UM’s color indicates that there are
two pathways that can convert PCA into PYO. As
mentioned before, phzM can transform PCA into
5MPCA, which I can be converted into PYO by phzS.
Another pathway seems to be functioning when phzM is
knocked out. It converts PCA into 2-OH-PHZ through
phzO, then 2-OH-PHZ changes into 5MPCA, which can
be covered into PYO in the presence of phzS. Thus, the
deletion of phzM and phzS should eliminate the
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production of PYO altogether. Since these genes are
primely involved in the conversion from PCA to PYO.
PYO is a major virulence factor, thus the reduction of
PYO should decrease the pathogenicity against human
and animals (Du, 2013). The wild type M18 already
produces less PYO than other P. aeruginosa strains
(Huang, 2009). However, mutant M18∆UMS should not
produce any PYO at all. This would render M18∆UMS a
safer agent than M18 and more suitable for commercial
use.
4.5 Expected PCN Production
PhzS also promote the conversion from PCA to 1-OH-
PHZ. So, ideally a decrease or elimination of 1-OH-PHZ
is expected after the deletion of phzM. The conversion
from PCA to PCN is mostly depended on phzH, which
remain intact. So, the production of PCN is expected to
stay unchanged. Although the deletion of phzS and phzM
will result in the accumulation of extra PCA, it is
unknown whether the excess will result in increased PCN
production.
5 CONCLUSION
To conclude, the construction of triple-deleted mutant
M18∆UMS is a success. The mutant strain produces
more PCA than the wild type, presumably cost-effective.
Thus, it can be a potential candidate for commercial use.
However, more improvements might be made if a better
understanding of the complex interaction between
various regulatory systems is gained. So further studies
should focus on the regulatory mechanisms go phenazine
production.
ACKNOWLEDGMENTS
This project was supported by Shanghai Jiao Tong
University. We are grateful to Dr. Ya-Wen He and Dr.
Run-Xian Yao for their support and assistance.
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