Foo_Final_f14

Final Report: Characterization of Unknown Pseudomonas aeruginosa
Strains
Name: Marcus Foo
Table of Contents:
Abstract ................................................................................................................... 2
Introduction ............................................................................................................. 3
Methods/Approach................................................................................................... 3
Results ..................................................................................................................... 4
Discussion ............................................................................................................... 17
Bibliography............................................................................................................ 18
Appendix ................................................................................................................. 20

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Using multi-locus sequence analysis, the relatedness of 16 unknown Pseudomonas
aeruginosa strains obtained from cystic fibriosis patients and 5 various known strains of bacteria
were determined. Four of the 16 unknown P. aeruginosa strains (AU1314, AU11103, AU14022, and
AU17465) were further analyzed using blastn test to determine the absence or presence of certain
genes and essentially, the absence and presence of certain characteristics. These properties include
lactose utilization, virulence factors, alginate production, biofilm formation, quorum sensing and
flagellin biosynthesis. P. aeruginosa strain AU1314 was found to be the most virulent of the four due
to the presence of type IV pili; P. aeruginosa strain AU11103 was the only one capable of stable
flagellin synthesis. All strains were predicted to be able to produce alginate, form biofilms, and
undergo quorum sensing but are unable to metabolize lactose.

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Introduction
Identification and classification of an unknown strain of bacteria can sometimes be difficult;
if said bacteria cannot be grown in a laboratory setting, it is difficult to assess biochemical properties
of the bacteria such as its ability to metabolize certain molecules. In other cases, it might be useful to
predict a bacteria’s properties without having to waste time and resources conducting experiments;
for example, it would be difficult and dangerous analyzing an unknown bacteria’s pathogenicity in
humans by ‘innoculating’ a human host with it. One of the ways to circumvent these problems is by
analyzing the unknown bacteria’s genome and comparing the obtained sequences with genes of
known functions. This method of analyzing and comparing genetic sequences serves as the basis of
this report.
Using genetic sequences obtained from unknown strains of Pseudomonas aeruginosa
sampled from cystic fibriosis patients, the aim of this report is to predict any virulence factors,
biochemical properties or any other characteristics of these unknown strains. Some of these
properties include the ability to produce alginate, form biofilms, synthesize flagella and metabolize
lactose as an energy source. Genes related to these processed were first identified in a control
bacterial species and were used as the query when searching for similar genes in the unknown
strains. Their genetic relatedness among one another and compared to known bacterial strains can
also be analyzed using their genome; the technique used here is called multi-locus sequence analysis.
Relatively conserved genetic sequences that are truly essential for the growth and survival, or
housekeeping genes, are used for this technique.
Methods/ Approach
Construction of Phylogenetic-like Trees Using Multi-Locus Sequence Analysis
For the 16 unknowns (including the four specimens that will be further analyzed later) and 5
knowns (P. aeruginosa PAO1, PA14, PA7, Streptococcus pneumonia TCH8431/19A and Klebisella
pneumonia MGH78578), the DNA sequences of the following housekeeping genes were
concatenated in this order: DnaE (DNA Polymerase III alpha subunit, PpsA (Phosphoenolpyruvate
synthase), RecA (Recombinase A), RpoB (RNA Polymerase beta subunit) and GuaA (GMP
synthase). Using the website http://mafft.cbrc.jp/alignment/server, a multi-locus sequence analysis
was conducted using said sequences and a phylogenetic-like tree was obtained, showing the
relatedness between the known and unknown bacterial species. Gene sequences were obtained using
NCBI for known specimens while local blastn was used for unknown specimens, using P.
aeruginosa PAO1 as the query.
Comparison of protein sequences (genes)
Using local blast, the protein sequences that corresponded to the virulent factor genes were
compared to that of the control strain, P. aeruginosa PAO1, which had nearly all of the chosen
genes. In cases where P. aeruginosa PAO1 could not be used as a control, the species used as the
query sequence will be shown. Relevant information from the blastp results that were included are
scores, query values, e-values and identities. These values were used to determine the potential
virulence of each strain.
Determining absence or presence of genes

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For a gene to be considered present in a particular strain, the score value, query value and
identity value must be as high as possible while the e value should be as long as possible. Should any
one of these values not be within a reasonably high or low range (depending on parameter), then we
considered the gene to be absent instead.
Results
MLSA
Diagram 1: Results from the MLSA using all 21 bacterial specimens.
Diagram 2: Closer examination of the unresolved section of the overall tree.
Based on diagram 1, S. pneumoniae TCH8431/19A was the least related to the other bacterial
specimens, followed by Klebisella pneumoniae MGH78578. While all three known P. aeruginosa
strains were relatively unrelated to the unknown P. aeruginosa strains, this could be expected as all
the unknowns came from a similar microenvironment. As for the unknown strains, different strains

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could be grouped together by slight differences but overall, based on the entire tree obtained, most of
the unknowns were closely related with the exception of two: AU11447 and AU 12215.
Lactose Utilization and Glycolysis
In order for a bacterial cell to utilize lactose for energy metabolism, it must first be able to
uptake lactose; as lactose is a relatively large molecule, the cell membrane must have permeases to
allow movement of lactose into the cell. Two genes that encode for lactose permeases are lacS and
lacY; lacS is also able to function as a galactose permease. Before lactose is able to move on to
glycolysis, it must be broken down into glucose and galactose; this can be achieved through the
enzyme β-galactosidase that is encoded by the gene lacZ. Diagram 3 shows the entry of lactose into
the cell and the conversion of lactose into glucose and galactose. A summary of the presence/absence
of the lactose utilization genes mentioned is listed at the end of this section (Table 1).
In the glycolysis pathway, the enzymes (for which the genes that encode them should be the
same for all species) analyzed are as follows: glucose-6-phosphate isomerase, phosphofructokinase,
fructosebisphosphate aldolase, pyruvate kinase and L-lactate dehydrogenase. Glucose-6-phosphate
isomerase convers glucose-6-phosphate into fructose-6-phosphate while phosphofructokinase
converts the latter into fructose-1,6-diphosphate. Fructosebisphosphate aldolase converts fructose-
1,6-diphosphate into dihydroxyacetone phosphate and glyceraldehydes 3-phosphate. Pyruvate kinase
converts phosphoenolpyruvate into pyruvate and finally, L-lactate dehydrogenase converts private
into lactate. Diagram 3 shows the function of fructose-bisphosphate aldolase, pyruvate kinase and L-
lactate dehydrogenase; diagram 11 in the appendix shows the remaining enzymes in a complete
glycolysis pathway. A summary of the presence/absence of the glycolysis genes mentioned is listed
at the end of this section (Table 1).

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Diagram 3: Lactose utilization pathway. (Note the enzymes lacS, lacY and lacZ; the first two encode
for lactose permeases while the latter encodes β-galactosidase)

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Pathway/System Gene/Name
Presence/Absence in Unknown Specimens
AU1314 AU11103 AU14022 AU17465
Lactose Utilization
LacS - - - -
LacY - - - -
LacZ - - - -
Glycolysis
PFK-1 + + + +
Fructose-
bisphosphate
aldolase
+ + + +
Glucose-6-
phosphate
isomerase
+ + + +
Pyruvate
Kinase
+*
+ + +
L-Lactate
Dehydrogenase
+ + + +
Table 1: Summary of presence/absence of genes/protein products related to lactose utilization and
glycolysis in the unknown P. aeruginosa specimens (+ indicates presence, +*
indicates weak
presence and - indicates absence; actual data listed in appendix in tables 8-11)
Virulence-Related Genes
Type III Secretion System
The type III secretion system (T3SS) forms complex, needle like machine on the surface of
bacteria which function in a highly regulated manner to transport effectors protein, such as toxins
exoU, exoS and exoT into the host cell (1). These machines can be separated into 5 parts: needle
complex, translocation apparatus, regulatory proteins, chaperone proteins and effector proteins.
Diagram 4 depicts the type III secretion system and the genes that encode each component; the
component encoded by yscS is shown in diagram 12 in the appendix. A summary of the
presence/absence of the set of T3SS genes mentioned below is listed at the end of this section (Table
2).
The pscC and pscJ genes encode for protein structures that make up the needle complex (1);
the yscS gene also encodes for a structural component of the needle complex (2). SpcS and SpcU
genes encode for chaperone proteins, which facilitates storage of their protein partners (exoS/exoT
and exoU, respectively) and aid in delivery to the secretion apparatus (1). ExsA encodes for a
transcriptional activator that binds to the promoters of the T3SS genes; the complete mechanism of
regulation includes additional proteins, exsC, exsD and exsE (not analyzed in this report) (1). TypA,
a type of ribosome-binding GTPase, has a regulatory function with respect to T3SS and other
virulence factors such as antibiotic resistance; it was shown that genes coding for T3SS were
significantly down-regulated in a typA mutant P. aeruginosa PA14 (3).
As for the toxins, the exoU gene encodes for a phospholipase, capable of causing rapid loss
of membrane integrity (1). The exoS protein is bifunctional toxin that has GTPase-activating protein
(GAP) activity and ADP ribosyl transferase (ADPRT) activity while ExoT protein has a 76% amino
acid identity to ExoS with the same bifunctionality (same reference). This GAP activity can cause
cytoskeleton disruptions in the host while ADPRT activity causes cell death, actin cytoskeleton
disruption and inhibition of DNA synthesis, vesicular trafficking and endocytosis (1). The exoY
gene encodes for adenylyl cyclase that causes an elevation of intracellular cAMP concentration as
well as differential expression of multiple genes, some of which are known to be regulated by

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cAMP; this leads to the disruption of the actin cytoskeleton, inhibition of bacterial uptake by host
cells and increased endothelial permeability (1).
Diagram 4: Structural components of the type III secretion system and the genes that encode them.
(Adapted from Hauser, A. R. 2009. The type III secretion system of Pseudomonas aeruginosa:
infection by injection. Nat. Rev. Microbiol. 7:654-665)
Las Quorum Sensing
LasA and lasB are two of the many products regulated by the las quorum sensing, which will
be explained later (See Quorum Sensing Systems). LasA encodes for a protease while lasB encodes
for an elastase; both of which function to degrade elastin of the host cell using different

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mechanisms. A summary of the presence/absence of these two genes is listed below the pathway
(Table 2); results of other las genes are listed in table 2.
Type IV Pili
Type IV pili (Tfp) are flexible filamentous appendages that are associated to various
virulence traits in pathogens; some of these traits include adhesion, twitching motility, biofilm
formation, and competence for DNA transformation and importance for host colonization (4). PilA
encodes for the major subunit of the pilus fiber that is expressed as a prepilin upon cleaveage by the
prepilin peptidase pilD. PilB encodes for an ATPase that mediates extension of the pilus; retraction
of the pilus is mediated by ATPase pilT (not analyzed in this report). PilC encodes for a
transmembrane protein in the inner membrane. Diagram 5 shows the type IV pili structure; a
summary of the presence/absence of these four genes is listed at the end of this section (Table 2).
Diagram 5: Structural components of the type IV pili and the genes that encode them. (Adapted
from Salomonsson, E. N., A. Forslund, and A. Forsberg. 2011. Type IV pili in Fracisella- a
virulence trait in an intracellular pathogen. Front. Microbiol. 2:29)

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Pathway/System Gene
AU1314 AU11103 AU14022 AU17465
ExoS + + + +
ExoT + + + +
ExoU - - - -
ExoY + + + +
ExsA + + + +
TypA + + + +
SpcS + + + +
SpcU - - - -
PscC + + + +
PscJ + + + +
YscS + + + +
Las Quorum Sensing
LasA + + + +
LasB + + + +
Type IV Pili
PilA + - - -
PilB + + + +
PilC + + + +
PilD + + + +
Table 2: Summary of presence/absence of all virulence related genes in the unknown P. aeruginosa
specimens (+ indicates presence and - indicates absence; actual data listed in appendix in tables 12-
15)
Alginate Production
Alginate is a type of exopolysaccharide that is involved in the formation of biofilm, usually
during the post colonization stages of infection; during earlier stages of biofilm formation,
extracellular DNA acts as the main component of the biofilm. The production of alginate is
associated to the conversion of P. aeruginosa phenotype from non-mucoid to mucoid. Mucoid
strains are able to resist phagocytic activation activation and uptake in both opsonized and
nonopsonized conditions and while alginate production is one of the contributing factors, the
mechanism of how this is achieved in unclear (5) The production of hydrogen peroxide, a process
commonly found in nonopsonic phagocytosis, was inhibited by alginate (6)
The algC and algD genes are involved in directly in the alginate metabolism pathway, in the
sense that their protein products are important enzymes of the pathway; algC codes for
phosphomannomutase (PMM) that converts mannose-6-phosphate into mannose-1-phosphate while
algD codes for GDP-mannose 6-dehydrogenase (GMD) (7). AlgP, algQ and algR, on the other hand,
are involved in the regulation of the pathway; algQ and algP are also known as algR2 and algR3
respectively. AlgR and algQ function cooperatively to increase the level of algD activation. AlgP
also functions to increase transcription levels of algD but it is possible for it to be involved with
other genetic and physiological processes (8). Below is alginate production pathway (Diagram 6);
again, as algR, algQ and algP are involved in the regulation of the pathway, they do not appear in it.
A summary of the presence/absence of the set of genes mentioned above is listed below the pathway
(Table 3).

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Diagram 6: Simplified alginate production pathway (Note genes algC and algD that code for PMM
and GMD, respectively) (Adapted from Galindo, E., C. Pena, C. Nunez, D. Segura, and G. Espin.
2007. Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate
production by Azotobacter vinelandii. Microb. Cell. Fact. 6:7.)
Pathway/System Gene
AU1314 AU11103 AU14022 AU17465
Alginate Biosynthesis
AlgC + + + +
AlgD + + + +
AlgP + + + +
AlgQ + + + +
AlgR + + + +
Table 3: Summary of presence/absence of all genes related to alginate biosynthesis in the unknown
P. aeruginosa specimens (+ indicates presence and - indicates absence; actual data listed in appendix
in tables 16-19)
Quorum Sensing Systems
Quorum sensing is an important group process for bacteria as it allows for the activation of
specific genes at high densities in response to the production of common chemical signals such as an
N-acyl homoserine lactone signal. This allows for the control and regulation of group bacterial
behavior such as the coordinated release of toxin to kill a host cell or biofilm formation; without such
group behavior, bacterial colonies would not be able to kill a host or form a structurally strong
biofilm. Both Las and Rhl quorum sensing systems have transcriptional activators and autoinducer
synthases; the transcriptional activator controls the regulation of the system while autoinducer
molecules bind to transcriptional activators allowing for the activation of various other genes (9).

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Las Quorum Sensing System
In this system, PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) is the autoinducer while
lasR serves as the transcriptional activator. The Las system controls the induction of the lasB gene,
an elastase, and other virulence genes that are controlled by the products of the las system (10).
Some of these genes include lasA (lasA protease), apr (alkaline protease A), toxA (exotoxin A), and
lasI (the PAI-1 synthase). While lasR, as mentioned above, serves as the transcriptional activator,
lasI gene directs PAI-1 synthesis, which is able to bind to the protein from the lasR gene. The
binding of the two causes the expression of the mentioned genes above. The pathway of las quorum
sensing system is shown below (Diagram 7). A summary of the presence/absence of the two las
genes mentioned above is listed at the end of this section (Table 4).
Diagram 7: Summary of las quorum sensing system. (Note the lasR and lasI genes and that the
binding of both their protein products lead to the activation of other genes below) (Adapted from
https://www.bio.cmu.edu/courses/03441/TermPapers/99TermPapers/Quorum/LasSys.jpg)
Rhl Quorum Sensing System
Similar to the las quorum sensing system, the rhl system consists of the transcriptional
activator, rhlR and an autoinducer synthase, rhlI. Both rhlR and rhlI have similar functions to their
las gene counterparts; rhlI directs the synthesis of the autoinducer PAI-2 (N-butyoyl-1-homoserine
lactone) that will bind to the rhlR gene product, activating the transcription of rhlI, rhlA, and rhlB
(not analyzed in this report), both of which encode a rhamnosyltransferase. This enzyme is required

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for the production of rhamnolipids, a chemical that has hemolytic and biosurfactant properties
(10).The binding of protein products of rhlR and rhlI lead to the activation of other genes, as shown
in Diagram 8. A summary of the presence/absence of the three rhl genes mentioned above is listed at
the end of this section (Table 4).
Diagram 8: Summary of rhl quorum sensing system. (Note the rhlR and rhlI genes and that the
binding of both their protein products lead to the activation of other genes below, particularly rhlA)
(Adapted from https://www.bio.cmu.edu/courses/03441/TermPapers/99TermPapers/Quorum/rhl.jpg)
Pseudomonas Quinolone Signal (PQS) Quorum Sensing Pathway
In this particular pathway, the signaling molecule is a quinolone compound known as 2-
heptyl-3-hydroxy-4(1H)-quinolone (9). PQS controls the expression of other virulence factors usch
as the phz and hcn operon, and also follows a hierarchy with respect to the las and rhl quorum
sensing systems; this hierarchy is shown in diagram13of the appendix. PQS is thought to be a
secondary regulatory signal for a subset of QS-controlled genes as compared to the other two
systems as maximal PQS production was observed at the end of the exponential growth phase (11).
In general, the operon pqsABCDE encode enzymes that direct the synthesis of five classes of 4-
hydroxy-2-alkylquinolines (HAQs) that function as antibiotics, and cytochrome inhibitors but more
significantly, intercellular communication molecules (11). PqsR acts as the transcriptional regulator
while pqsE is the response effector (9). Gene pqsA was shown to be responsible for the catalysis of
the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA; effectively, the protein
encoded by pqsA primes anthranilate for entry into the PQS pathway (12). Specific functions for

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genes pqsB, pqsC and pqsD could not be found but it was suggested that they are involved in the
production of a long chain hydrocarbon which reacts with anthranilate in the PQS pathway (13). A
proposed pathway for PQS is shown below (Diagram 9); a summary of the presence/absence of the
six pqs genes mentioned above is listed at the end of this section (Table 4).
Diagram 9: Summary of PQS quorum sensing system. (Note the pqsR and pqsA genes in particular)
(Adapted from Coleman, J. P., L. L. Hudson, S. L. McKnight, J. M. Farroww III, M. W. Calfee,
C. A. Lindsey, and E. C. Pesci. 2008. Pseudomonas aeruginosa PqsA Is an Anthranilate-Coenzyme
A Ligase. J. Bacteriol. 190:1247-1255)
Pathway/System Gene
AU1314 AU11103 AU14022 AU17465
Las Quorum Sensing
LasI + + + +
LasR + + + +
Rhl Quorum Sensing
RhlA + + + +
RhlI + + + +
RhlR + + + +
Pseudomonas Quinolone Signalling
PqsA + + + +
PqsB + + + +
PqsC + + + +
PqsD + + + +
PqsE + + + +
PqsR + + + +
Table 4: Summary of presence/absence of sets of genes related to three types of quorum sensing in
the unknown P. aeruginosa specimens (+ indicates presence and - indicates absence; actual data
listed in appendix in tables 20-27)
Biofilm Formation

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Biofilms are sessile, slime-encased community of bacterial cells; these aggregations of cells
are relatively non-motile and tend to have higher resistances against environmental stress. A biofilm
first begins its development by the loose associate of planktonic cells to surfaces, followed by robust
adhesion. These cells begin to grow and form microcolonies; this is followed by further growth and
maturation. Once a large enough population has been achieved, quorum sensing genes are activated
allowing the control of its virulence, if in a host, or further development of the biofilm such as the
formation of exopolysaccharide maxtrix. Thus, based on this simplified development process of a
biofilm, the following systems or pathways are required: type IV pili for adhesion, quorum sensing
systems for regulation of group behavior, and exopolysaccharide production. The presence/absence
of genes related to these systems and pathways are shown in table 5.
Pathway/System Gene
AU1314 AU11103 AU14022 AU17465
Type IV Pili
PilA + - - -
PilB + + + +
PilC + + + +
PilD + + + +
Las Quorum Sensing
LasI + + + +
LasR + + + +
Rhl Quorum Sensing
RhlI + + + +
RhlR + + + +
PqsA + + + +
PqsB + + + +
PqsC + + + +
PqsD + + + +
PqsE + + + +
PqsR + + + +
AlgC + + + +
AlgD + + + +
AlgP + + + +
AlgQ + + + +
AlgR + + + +
Table 5: Summary of presence/absence of gene related to systems and pathways needed for biofilm
formation in the unknown P. aeruginosa specimens (+ indicates presence and - indicates absence)
Flagellin Biosynthesis
The flagellum is a specialized structure of the cell that allows for movement of the bacteria in
their microenvironment. These structures are long, thin appendages that are attached to the cell and
consist of many different components, each of which are encoded by different genes. For example,
genes flgH encodes for the L ring protein that provides support for the flagellum while flgE encodes
for the hook of the flagellum that is attached to the L ring (14). Genes flgK and flgL form the hook-
filament junction that connects the hook and filament, that is encoded by fliC, of the flagellum; fliD
encodes the filament cap. Diagram 10 shows the flagellar components and their respective genes that
encode them. Table 6 shows the summary of the presence/absence of the six flagellin biosynthesis
genes mentioned above.

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Diagram 10: Flagellar components and their respective genes that code them. (Note the genes fliC,
fliD, flgE, flgH, flgK and flgL that are shown in the rectangular boxes.) (Adapted from Chevance, F.
F. V., and K. T. Hughes. 2008. Coordinating assembly of a bacterial macromolecular machine. Nat.
Rev. 6:455-465.)
Pathway/System Gene
AU1314 AU11103 AU14022 AU17465
Flagellin Synthesis
FlgE + + + +
FlgH + + + +
FlgK + + + +
FlgL +*
+ +*
+*
FliC +*
+ +*
+*
FliD - + - -
Table 6: Summary of presence/absence of gene related to flagellin synthesis in the unknown P.
aeruginosa specimens (+ indicates presence, +*
indicates weak presence and - indicates absence;
actual data listed in appendix in tables 28-31)

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Summary of All Systems and Pathways Analyzed
Pathway/System
AU1314 AU11103 AU14022 AU17465
Lactose Utilization - - - -
Glycolysis + + + +
Type III Secretion System + + + +
Type IV Pili + - - -
Alginate Biosynthesis + + + +
Las Quorum Sensing + + + +
Rhl Quorum Sensing + + + +
Pseudomonas Quinolone Signalling + + + +
Flagellin Synthesis - + - -
Table 7: Summary of presence/absence of pathways and systems in the unknown P. aeruginosa
specimens (+ indicates absolute presence and - indicates absence)
Discussion
With respect to virulence, unknown P. aeruginosa AU1314 appears to be the most virulent
strain as it has fully functional type IV pili; the remaining three unknown strains do not appear to
have a pilA gene that encodes for the major subunits of the pilus fiber. Without this fiber, the type IV
pili is essentially absent as there is nothing for pilB to extend or for pilT to retract and thus, the
bacterial cell is unable to adhere strongly to surfaces. For planktonic cell, this feature is important as
it will eventually lead to the development of a biofilm, further increasing the bacteria’s resistance in
the host. However, it is likely that all four strains have already developed into biofilms, as they all
have alginate production genes and biofilm formation related genes. Thus, P. aeruginosa AU1314
should only be considered slightly more virulent than the rest, based on the data obtained from local
blastp results.
Another difference observed is that only P. aeruginosa AU11103 has a fully functional
flagellin synthesis process; the remaining three strains lack the gene fliD, which encodes for the
filament cap. The genes flgL and fliC are also weakly present in these three strains, based on the
results obtained. Assuming that the filament structures (flgL and fliC) are still present, the lack of the
filament cap still prevents the proper assembly of flagellar filament; the cap exerts a significant
stabilization effect on the filament assembly that consists of thousands of flagellin molecules (15).
Thus, P. aeruginosa strains AU11103, 14022, 17465 are essentially lacking the entire filament
section of the flagella; without it, motility will be severely hampered. However, it is likely that all
four strains have already developed into biofilms, as they all have alginate production genes and
biofilm formation related genes, and even the lack of these three genes would not completely
compromise the bacteria’s survivability. It would pose a challenge, however, should the biofilm
release planktonic cells in order to colonize a different part of the lung.
All of the unknown strains lacked genes for lactose utilization and thus are unable to
metabolize lactose. They lacked a method (both lacS and lacY were absent) to allow entry of lactose
into the cell as well as a way of cleaving lactose (absence of lacZ) should it enter the cell. They are
probably obtaining some of their energy from glucose through glycolysis as they have a functional
pathway, based on the genes and enzymes analyzed. In oxygen-limiting conditions, P. aeruginosa is
able to grow in the presence of nitrate or nitrite using denitrification or through fermentation.

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For the remaining pathways and systems (T3SS, alginate biosynthesis, and all three quorum
sensing systems), all relevant genes were present in each of the bacterial strains indicating they have
fully functional pathways and systems. It is very likely that they have successfully formed some
biofilm in the patients as they are able to produce alginate for the exopolysaccharide matrix and are
able to use quorum sensing to activate other relevant genes. While the type III secretion system is
also functional, the coordinated release of the exotoxins would not be enough to kill the patients but
might be able to do some damage to lung cells.
Overall, P. aeruginosa AU1314 can be considered the most virulent among the four
unknowns as it has type IV pili that aids in adhesion and biofilm formation. Planktonic cells of P.
aeruginosa AU11103 are the most motile as they are capable of synthesizing stable flagellar
structures. All of the strains are unable to utilize lactose for energy metabolism but are able to
conduct glycolysis if needed. The strains are capable of biofilm formation and producing exotoxins
in the host; however, as the patient is very much larger than the biofilm, the bacteria is unable to kill
the host in a short period of time, even with quorum sensing systems causing coordinated expression
of virulent genes.
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Bacteriol. 174:824-831.
9. Kievit, T. R. 2009. Quorum sensing in Pseudomonas aeruginosa biofilms. Environ. Microbiol.
11:279-288.
10. Pearson, J. P., E. C. Pesci, and B. H. Iglewski. 1997. Roles of Pseudomonas aeruginosa las
and rhl Quorum-Sensing Systems in Control of Elastase and Rhamnolipid Biosynthesis Genes.
J. Bacteriol. 179:5756-5767.

19
11. Deziel, E., F. Lepine, S. Milot, J. He, M. N. Mindrinos, R. G. Tompkins, and L. G. Rahme.
2004. Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role
for 4-hydroxy-2-heptylquinoline in cell-to-cell communication . Proc. Natl. Acad. Sci.
101:1339-1344.
12. Coleman, J. P., L. L. Hudson, S. L. McKnight, J. M. Farroww III, M. W. Calfee, C. A.
Lindsey, and E. C. Pesci. 2008. Pseudomonas aeruginosa PqsA Is an Anthranilate-Coenzyme
A Ligase. J. Bacteriol. 190:1247-1255.
13. Gallagher, L. A., S. L. McKnight, M. S. Kuznetsova, E. C. Pesci, and C. Manoil. Functions
Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa. 2002. J. Bacteriol.
184:6472-6480.
14. Chevance, F. F. V., and K. T. Hughes. 2008. Coordinating assembly of a bacterial
macromolecular machine. Nat. Rev. 6:455-465.
15. Diozeghy, Z., P. Zavodszky, K. Namba, and F. Vonderviszt. 2004. Stabilization of flagellar
filaments by HAP2 capping. Febs. Lett. 568:105-109.

20
Appendix
Diagram 11: Simplified glycolysis pathway (Adapted from Pessione, A., C. Lamberti, and E.
Pessione. 2010. Proteomics as a tool for studying energy metabolism in lactic acid bacteri. Mol.
BioSyst. 6:1419-1430.)

21
Diagram 12: Structure components of the needle complex of type III secretion system. (Note the
components labeled yscS and yscJ that were not in 4 (Adapted from
http://www.cell.com/cms/attachment/580770/4369022/gr1.jpg)

22
Diagram 13: Hierarchy of the three different signaling systems found in P. aeruginosa. (Adapted
from Wade, D. S., M. W. Calfee, E. R. Rocha, E. A. Ling, E. Engstrom, J. P. Coleman, and E.
C. Pesci. 2005. Regulation of Pseudomonas Quinolone Signal Synthesis in Pseudomonas
aeruginosa. J. Bacteriol. 187:4372-4380.)
Lactose Utilization
Score Query
value
E value Identity (%)
Lactose Utilization
LacS 238 17 4.00e-20 43.81
LacY 86 34 0.033 26.76
LacZ - - - -
Glycolysis
PFK-1 1580 100 0 99.68
Fructose-
bisphosphate
aldolase
1895 100 0 100
Gllucose-6-
phosphate
isomerase
2966 100 0 100
Pyruvate
Kinase
1273 49
3.00e-
1740
100
L-Lactate
Dehydrogenase
1971 100 0 100
Table 8: Blastp results of lactose utilization and glycolysis genes of P. aeruginosa AU1314 using P.
aeruginosa PAO1 as the main query for most of the genes/protein sequences.
Score Query
value
Lactose Utilization
LacS 239 17 4.00e-20 43.81
LacY 86 34 0.033 26.76
LacZ - - - -
Glycolysis
PFK-1 1583 100 0 100
Fructose-
bisphosphate
aldolase
1895 100 0 100
Gllucose-6-
phosphate
isomerase
2966 100 0 100
Pyruvate
Kinase
2539 100 0 100
L-Lactate
Dehydrogenase
1971 100 0 100
Table 9: Blastp results of lactose utilization and glycolysis genes of P. aeruginosa AU11103 using P.
aeruginosa PAO1 as the main query for most of the genes/protein sequences.
Note: For the lacS and lacZ genes, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was
used as the query as these genes was absent in P. aeruginosa PAO1; for the lacY gene, Escherichia
coli K12was used as the query for the same reason as above.

23
Score Query
value
Lactose Utilization
LacS 238 17 4.00e-20 43.81
LacY 86 34 0.033 26.76
LacZ - - - -
Glycolysis
PFK-1 1564 100 0 99.04
Fructose-
bisphosphate
aldolase
1895 100 0 100
Gllucose-6-
phosphate
isomerase
2966 100 0 100
Pyruvate
Kinase
2539 100 0 100
L-Lactate
Dehydrogenase
1971 100 0 100
Table 10: Blastp results of lactose utilization and glycolysis genes of P. aeruginosa AU14022 using
P. aeruginosa PAO1 as the main query for most of the genes/protein sequences.
Score Query
value
Lactose Utilization
LacS 214 15 4.00e-17 45.65
LacY 86 34 0.033 26.76
LacZ - - - -
Glycolysis
PFK-1 1566 100 0 99.04
Fructose-
bisphosphate
aldolase
1895 100 0 100
Gllucose-6-
phosphate
isomerase
2966 100 0 100
Pyruvate
Kinase
2539 100 0 100
L-Lactate
Dehydrogenase
1971 100 0 100
Table 11: Blastp results of lactose utilization and glycolysis genes of P. aeruginosa AU17465 using
P. aeruginosa PAO1 as the main query for most of the genes/protein sequences.
Note: For the lacS and lacZ genes, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was
used as the query as these genes was absent in P. aeruginosa PAO1; for the lacY gene, Escherichia
coli K12was used as the query for the same reason as above.

24
Virulence Gene Data
Pathway/System Gene Score Query value E value Identity (%)
ExoS 2323 100 0 100
ExoT 1646 100 0 74.4
ExoU 128 37 1.00e-06 25.79
ExoY 2012 100 0 99.74
ExsA 1475 100 0 99.64
Las Quorum Sensing
LasA 2177 100 0 98.8
LasB 2672 100 0 100
PilA 553 99 7.00e-72 81.76
PilB 3009 100 0 100
PilC 1960 100 0 100
PilD 1481 100 0 99.66
PscC 3117 100 0 99.67
PscJ 1252 98 7.00e-175 99.59
SpcS 1236 100 2.00e-172 100
SpcU - - - -
TypA 3189 100 0 100
YscS 428 100 7.00e-55 100
Table 12: Blastp results of virulence genes of P. aeruginosa AU1314 using P. aeruginosa PAO1 as
the main query for most of the genes/protein sequences.
ExoS 2317 100 0 99.78
ExoT 2315 100 0 99.34
ExoU 128 37 9.00e-07 25.79
ExoY 1878 93 0 99.72
ExsA 1484 100 0 100
Las Quorum Sensing
LasA 2179 100 0 99.04
LasB 2664 100 0 99.8
PilA 177 99 9.00e-16 42.5
PilB 2680 100 0 88.34
PilC 1629 99 0 84.41
PilD 1475 100 0 99.31
PscC 3116 100 0 99.67
PscJ 1252 98 7.00e-175 99.59
SpcS 1236 100 2.00e-172 100
SpcU - - - -
TypA 3189 100 0 100
YscS 424 100 3.00e-54 98.86
Note: For the ExoU and SpcU genes, P. aeruginosa UCBPP-PA14 was used as the query as this
gene was absent in P. aeruginosa PAO1; for the YscS gene, P. aeruginosa PA1 was used as the
query for the same reason as above.

25
ExoS 2317 100 0 99.78
ExoT 2320 100 0 99.78
ExoU 128 37 9.00e-07 25.79
ExoY 1260 64 7.00e-174 99.59
ExsA 1484 100 0 100
Las Quorum Sensing
LasA 2184 100 0 99.04
LasB 2672 100 0 100
PilA 181 99 1.00e-16 41.67
PilB 2676 100 0 88.16
PilC 1629 99 0 84.41
PilD 1479 100 0 99.66
PscC 3125 100 0 100
PscJ 1252 98 7.00e-175 99.59
SpcS 1234 100 4.00e-172 99.59
SpcU - - - -
TypA 3184 100 0 99.83
YscS 420 100 1.00e-53 98.86
ExoS 2130 91 0 100
ExoT 2315 100 0 99.34
ExoU 128 37 9.00e-07 25.79
ExoY 2000 100 0 99.47
ExsA 1484 100 0 100
Las Quorum Sensing
LasA 2188 100 0 99.28
LasB 2510 94 0 100
PilA 183 99 1.00e-16 41.67
PilB 2676 100 0 88.16
PilC 1629 99 0 84.41
PilD 1476 100 0 99.31
PscC 3119 100 0 99.83
PscJ 1244 98 1.00e-173 99.18
SpcS 1236 100 2.00e-172 100
SpcU - - - -
TypA 3183 100 0 99.83
YscS 428 100 7.00e-55 100
Note: For the ExoU and SpcU genes, P. aeruginosa UCBPP-PA14 was used as the query as this
gene was absent in P. aeruginosa PAO1; for the YscS gene, P. aeruginosa PA1 was used as the
query for the same reason as above.

26
Alginate Biosynthesis Gene Data
AlgC 2469 100 0 100
AlgD 2156 94 0 99.75
AlgP 730 44 5.00e-93 100
AlgQ 755 91 2.00e-102 99.32
AlgR 1271 100 1.00e-177 100
Table 16: Blastp results of alginate biosynthesis genes of P. aeruginosa AU1314 using P. aeruginosa
PAO1 as query for all genes/protein sequences.
AlgC 2471 100 0 100
AlgD 2156 94 0 99.75
AlgP 1494 100 0 99.43
AlgQ 755 91 2.00e-102 99.32
AlgR 1271 100 1.00e-177 100
Table 17: Blastp results of alginate biosynthesis genes of P. aeruginosa AU11103 using P.
aeruginosa PAO1 as query for all genes/protein sequences.
AlgC 2469 100 0 100
AlgD 2309 100 0 100
AlgP 739 45 5.00e-95 100
AlgQ 755 91 2.00e-102 99.32
AlgR 1271 100 1.00e-177 100
AlgC 2471 100 0 100
AlgD 2309 100 0 100
AlgP 728 44 3.00e-92 99.35
AlgQ 755 91 2.00e-102 99.32
AlgR 1271 100 1.00e-177 100

27
Quorum Sensing Gene Data
Las Quorum Sensing
LasI 1074 100 2.00e-149 100
LasR 991 78 3.00e-136 100
Rhl Quorum Sensing
RhlA 1565 100 0 99.66
RhlI 1057 100 9.00e-147 99
RhlR 1255 97 1.00e-175 99.57
Table 20: Blastp results of two types of quorum sensing genes of P. aeruginosa AU1314 using P.
Las Quorum Sensing
LasI 1074 100 2.00e-149 100
LasR 1279 100 3.00e-179 100
Rhl Quorum Sensing
RhlA 1565 100 0 99.66
RhlI 1063 100 1.00e-147 99.5
RhlR 1255 97 1.00e-175 99.57
Las Quorum Sensing
LasI 1067 100 3.00e-148 99.5
LasR 1279 100 3.00e-179 100
Rhl Quorum Sensing
RhlA 1565 100 0 99.66
RhlI 1057 100 9.00e-147 99
RhlR 1255 97 1.00e-175 99.57
Las Quorum Sensing
LasI 1074 100 2.00e-149 100
LasR 1269 100 1.00e-177 99.58
Rhl Quorum Sensing
RhlA 1565 100 0 99.66
RhlI 1057 100 9.00e-147 99
RhlR 1255 97 1.00e-175 99.57

28
Pseudomonas Quinolone Signalling Gene Data
PqsA 2710 100 0 100
PqsB 1448 100 0 99.65
PqsC 1848 100 0 100
PqsD 1756 100 0 99.7
PqsE 1536 100 0 100
PqsR 1751 100 0 99.4
Table 24: Blastp results of pseudomonas quinolone signalling genes of P. aeruginosa AU1314 using
P. aeruginosa PAO1 as query for all genes/protein sequences.
PqsA 2710 100 0 100
PqsB 1452 100 0 100
PqsC 1848 100 0 100
PqsD 1756 100 0 99.7
PqsE 1536 100 0 100
PqsR 1764 100 0 100
Table 25: Blastp results of pseudomonas quinolone signalling genes of P. aeruginosa AU11103
using P. aeruginosa PAO1 as query for all genes/protein sequences.
PqsA 2710 100 0 100
PqsB 1452 100 0 100
PqsC 1848 100 0 100
PqsD 1756 100 0 99.7
PqsE 1166 75 1.00e-160 100
PqsR 1764 100 0 100
PqsA 2710 100 0 100
PqsB 1452 100 0 100
PqsC 1848 100 0 99.71
PqsD 1752 100 0 99.41
PqsE 1529 100 0 99.67
PqsR 1764 100 0 100

29
Flagellin Synthesis Gene Data
Flagellin Synthesis
FlgE 2391 100 0 100
FlgH 1207 100 1.00e-168 100
FlgK 3108 100 0 87.55
FlgL 1517 99 0 67.58
FliC 662 72 3.00e-80 68.12
FliD 827 96 8.00e-104 43.01
Table 28: Blastp results of flagellin synthesis genes of P. aeruginosa AU1314 using P. aeruginosa
Flagellin Synthesis
FlgE 2385 100 0 99.78
FlgH 1207 100 1.00e-168 100
FlgK 3482 100 0 98.98
FlgL 2271 100 0 99.77
FliC 2415 100 0 100
FliD 2414 100 0 100
Flagellin Synthesis
FlgE 2387 100 0 99.78
FlgH 1207 100 1.00e-168 100
FlgK 3111 100 0 87.8
FlgL 1517 99 0 67.58
FliC 662 72 3.00e-80 68.12
FliD 827 96 8.00e-104 43.01
Flagellin Synthesis
FlgE 2391 100 0 100
FlgH 1207 100 1.00e-168 100
FlgK 3107 100 0 87.55
FlgL 1517 99 0 67.58
FliC 662 72 3.00e-80 68.12
FliD 798 92 2.00e-99 43.56

30
Query Sequence Information
Regulatory protein TypA [Pseudomonas
aeruginosa PAO1]
GenBank: NP_253804.1
605 aa
RecName: Full=Exoenzyme S synthesis regulatory
protein ExsA [Pseudomonas aeruginosa PAO1]
GenBank: P26993.2
278 aa
Type III secretion inner membrane protein (YscS,
flagellar export components) [Pseudomonas
aeruginosa PA1]
GenBank: AHA21178.1
88 aa
ExoU [Pseudomonas aeruginosa UCBPP-PA14]
GenBank: YP_792285.1
687 aa
SpcU [Pseudomonas aeruginosa UCBPP-PA14]
137 aa
exoenzyme S [Pseudomonas aeruginosa PAO1]
453 aa
ORF1 [Pseudomonas aeruginosa PAO1]
GenBank: BAA33551.1
246 aa
Exoenzyme T [Pseudomonas aeruginosa PAO1]
457 aa
Exoenzyme Y [Pseudomonas aeruginosa PAO1]
GenBank: AAG05579.1
378 aa
Type III export protein PscJ [Pseudomonas
aeruginosa PAO1]
248 aa
Type III secretion outer membrane
protein PscC [Pseudomonas aeruginosa PAO1]
600 aa
LasA protease [Pseudomonas aeruginosa PAO1]
418 aa
Elastase LasB [Pseudomonas aeruginosa PAO1]
498 aa
Type 4 fimbrial PilA [Pseudomonas aeruginosa
PAO1]
149 aa
RecName: Full=Type 4 fimbrial assembly
protein PilB[Pseudomonas aeruginosa PAO1]
GenBank: P22608.2
566 aa
RecName: Full=Type 4 fimbrial assembly
protein PilC[Pseudomonas aeruginosa PAO1]
GenBank: P22609.2
374 aa
RecName: Full=Type 4 prepilin-like proteins
leader peptide-processing enzyme; AltName:
Full=Protein PilD; AltName: Full=Protein
secretion protein XCPA; Includes: RecName:
Full=Leader peptidase; AltName: Full=Prepilin
peptidase; Includes: RecName: Full=N-
methyltransferase [Pseudomonas aeruginosa
PAO1]
GenBank: P22610.3
290 aa
Phosphomannomutase AlgC [Pseudomonas
aeruginosa PAO1]

31
GenBank: AAG08707.1
463 aa
GDP-mannose 6-
dehydrogenase AlgD [Pseudomonas aeruginosa
PAO1]
436 aa
RecName: Full=Transcriptional regulatory
protein AlgP; AltName: Full=Alginate regulatory
protein AlgR3 [Pseudomonas aeruginosa PAO1]
GenBank: P15276.2
352 aa
Alginate regulatory protein AlgQ [Pseudomonas
aeruginosa PAO1]
GenBank: AAG08640.1
160 aa
Alginate biosynthesis regulatory
protein AlgR [Pseudomonas aeruginosa PAO1]
248 aa
RecName: Full=Transcriptional activator
protein LasR[Pseudomonas aeruginosa PAO1]
GenBank: P25084.1
239 aa
RecName: Full=Acyl-homoserine-lactone
synthase; AltName: Full=Autoinducer synthesis
protein LasI [Pseudomonas aeruginosa PAO1]
GenBank: P33883.1
201 aa
RhlR [Pseudomonas aeruginosa PAO1]
GenBank: AAC44036.1
241 aa
RecName: Full=Acyl-homoserine-lactone
synthase; AltName: Full=Autoinducer synthesis
protein RhlI [Pseudomonas aeruginosa PAO1]
GenBank: P54291.2
201 aa
RecName: Full=Rhamnosyltransferase 1 subunit A
[Pseudomonas aeruginosa PAO1]
GenBank: Q51559.2
295 aa
RecName: Full=B-type flagellin [Pseudomonas
aeruginosa PAO1]
GenBank: P72151.2
488 aa
Flagellar cap protein FliD [Pseudomonas
aeruginosa PAO1]
GenBank: AAF35976.1
474 aa
Flagellar hook protein FlgE [Pseudomonas
aeruginosa PAO1]
462 aa
Flagellar L-ring protein
precursor FlgH [Pseudomonas aeruginosa PAO1]
GenBank: AAG04472.1
231 aa
Flagellar hook-associated
protein FlgK [Pseudomonas aeruginosa PAO1]
683 aa
Flagellar hook-associated
protein FlgL [Pseudomonas aeruginosa PAO1]
439 aa
RecName: Full=Anthranilate--CoA ligase
GenBank: Q9I4X3.1
517 aa
PqsB [Pseudomonas aeruginosa PAO1]
GenBank: AAG04386.1
283 aa

32
PqsB [Pseudomonas aeruginosa PAO1]
GenBank: AAG04386.1
283 aa
PqsC [Pseudomonas aeruginosa PAO1]
GenBank: AAG04387.1
348 aa
RecName: Full=2-heptyl-4(1H)-quinolone
synthase PqsD; Short=PqsD [Pseudomonas
aeruginosa PAO1]
GenBank: P20582.2
337 aa
Quinolone signal response protein [Pseudomonas
aeruginosa PAO1]
301 aa
Transcriptional regulator MvfR
332 aa
lactose permease [Lactobacillus delbrueckii subsp.
bulgaricus ATCC 11842 = JCM 1002]
627 aa
lactose permease [Escherichia coli str. K-12 substr.
MG1655]
417 aa
beta-galactosidase [Lactobacillus delbrueckii
subsp. bulgaricus ATCC 11842 = JCM 1002]
1008 aa
L-lactate dehydrogenase [Pseudomonas aeruginosa
PAO1]
381 aa
pyruvate kinase [Pseudomonas aeruginosa PAO1]
483 aa
RecName: Full=Glucose-6-phosphate isomerase;
Short=GPI; AltName: Full=Phosphoglucose
isomerase; Short=PGI; AltName:
Full=Phosphohexose isomerase; Short=PHI
GenBank: Q9HV67.1
554 aa
RecName: Full=Fructose-bisphosphate aldolase;
Short=FBP aldolase; Short=FBPA; AltName:
Full=Fructose-1,6-bisphosphate aldolase
GenBank: Q9I5Y1.1
354 aa
1-phosphofructokinase [Pseudomonas aeruginosa
PAO1]
314 aa

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