FINAL Thesis

1
University of Birmingham
School of Biosciences
Genome Wide Assay of Essential Genes and β-Lactam Resistance Associated Mutations
in Klebsiella pneumoniae
A research project report submitted by
Christy Collins
as part of the requirement for the
Degree of MSc in Microbiology and Infection
This project was carried out at:
Under the supervision of: Professor Ian Henderson and Karl Dunne
Date 15/08/2016
Word Count: 8,394

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Table of Contents
Acknowledgements .........................................................................................................4
Figures, Tables andAbbreviations.................................................................................5
List of Figures ...............................................................................................................5
List of Tables.................................................................................................................6
Abbreviations................................................................................................................6
1.0 Abstract......................................................................................................................7
2.0 Introduction...............................................................................................................8
2.1 Klebsiella ................................................................................................................8
2.2 K. pneumoniae Infection.........................................................................................9
2.3 β –Lactams - Mechanism of Action......................................................................10
2.4 β –Lactam Resistance ...........................................................................................11
2.5 Efflux Mediated β-lactam Resistance ...................................................................13
2.6 Porin Mediated β-Lactam Resistance ...................................................................14
2.7 Acquiring New Antimicrobial Targets ..................................................................15
2.8 Aim........................................................................................................................16
3.0 Materials and Methods...........................................................................................17
3.1 Bacterial Strain and Cultures ................................................................................17
3.2 Antibiotic Sensitivity Testing................................................................................18
3.3 Transposon Transformation ..................................................................................19
3.4 Exposure of Mutants to β-lactams ........................................................................20
3.5 Extraction and Fragmentation of DNA.................................................................20
3.6 Next Generation Sequencing Preparation.............................................................21
3.7 Next Generation Sequencing ................................................................................23
3.8 Data Analysis ........................................................................................................24
4.0 Results ......................................................................................................................27
4.1 Kanamycin Susceptibility .....................................................................................27
4.2 Mutant Harvesting.................................................................................................28
4.3 Sequencing Data ...................................................................................................29
4.4 Breakdown of Gene Essentiality...........................................................................31
4.5 Expected Essential and Non-Essential Genes.......................................................33
4.6 Essential Hypothetical Genes................................................................................36
4.7 Exposure of Mutants to β-Lactams .......................................................................38
4.8 Cefotaxime-Resistance Conferring Insertions ......................................................40
4.9 Meropenem-Resistance Conferring Insertions .....................................................41
5.0 Discussion.................................................................................................................43

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5.1 Essential Genes – lipA and lipB............................................................................43
5.2 Essential Genes - BN373_19381 ..........................................................................44
5.3 ramR......................................................................................................................45
5.4 bamB.....................................................................................................................46
5.5 hupA......................................................................................................................47
5.6 Future Investigations.............................................................................................48
5.7 Summary...............................................................................................................49
6.0 References................................................................................................................51
7.0 Supplementary Material ........................................................................................58

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Acknowledgements
Thanks to everyone in T101 who made myself and my course mates feel welcome from
the very beginning.
Thanks to Danny, Sam and Laura for being hilarious lab partners.
Thanks to Ian for letting us make a mess of his lab and destroy his cheque book. Despite
all this he has continued to be a great support.
Huge thanks to Karl Dunne for his idiosyncrasies and for giving up 3 months of his life
to babysit us. We really appreciate it.
Finally thanks to my wonderful family for their relentless support. I am unbelievably
lucky to have them.

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Figures, Tables and Abbreviations
List of Figures
Figure 1: TraDIS workflow in chronological order
Figure 2: Kanamycin susceptibility of K. pneumoniae ECL-8
Figure 3: Tn mutants cultured on 32 µg/ml (MIC) kanamycin agar
Figure 4: Genome wide representation of Tn insertions
Figure 5: Frequency of insertion indexes
Figure 6: Essential K. pneumoniae genes vs E. coli
Figure 7: Annotated Artemis 16.0.0 screenshot
Figure 8: Mutual redundancy of genes
Figure 9: K. pneumoniae ECL-8 gene BN373_19381
Figure 10: BN373_19381 predicted structure
Figure 11: Mutant exposure to cefotaxime
Figure 12: Mutant exposure to meropenem
Figure 13: K. pneumoniae ECL-8 gene ramR
Figure 14: K. pneumoniae ECL-8 gene bamB
Figure 15: K. pneumoniae ECL-8 gene hupA

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List of Tables
Table 1: Composition of media
Table 2: Reagents used, their preparation and storage
Table 3: DNA extraction protocol
Table 4: PCR parameters
Table 5: K. pneumoniae-only essential genes
Abbreviations
PBP: Penicillin binding protein
WHO: World Health Organisation
NAM: N-acetyl-glucosamine
DAP: Diamino-pimelic acid
ESBL: Extended spectrum β-lactamase
KPC: Klebsiella pneumoniae carbapenemase
PMF: Proton motive force
Omp: Outer membrane protein
LB: Luria-Bertani
MIC: Minimum inhibitory concentration
RT: Room temperature
Tn: Transposon
PCR: Polymerase chain reaction
UIP: Unique insertion point
LR: Likelihood ratio
Supp.: Supplementary
ATP: Adenosine triphosphate
MFS: Major facilitator superfamily
SMR: Small multidrug resistance family
RND: Resistance-nodulation- cell division superfamily
MATE: Multi antimicrobial extrusion protein family

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1.0 Abstract
Klebsiella pneumoniae is an opportunistic pathogen increasingly associated with multi-
drug resistance. Resistance to one class of antibiotics, the β-lactams, is particularly
troublesome. Different families of β-lactams are used empirically and as a last resort to
treat Klebsiella infections. An increasing number of isolates are showing resistance to all
β-lactams in association with ever increasing mortality rates. To curb this problem two
lines of enquiry are important: 1) Understanding the fundamental gene set required for
K. pneumoniae viability and 2) Understanding resistance mechanisms. Both tactics are
important in assessing potential targets for novel therapies. An efficient way to do this is
to assay the whole genome in one experiment. This can be done by transposon directed
insertion-site sequencing (TraDIS) which couples transposon mutagenesis with next
generation sequencing. Here an essential gene set for K. pneumoniae was found under
laboratory conditions. 374 of 5,006 genes were found to be essential; 50 of which are
hypothetical genes which have no homologues in S. Typhi or E. coli. Of these genes a
putative inner membrane receptor with an SH3-like domain is described and may be a
good antimicrobial target. Additionally, transposon insertions into the marR, bamB and
hupA genes were associated with resistance to clinically relevant β-lactams. Of these
genes, hupA has not before been associated with β-lactam resistance and warrants further
investigation. Thus, elucidated here are several genes which warrant further study in the
quest for novel antimicrobials against K. pneumoniae.

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2.0 Introduction
2.1 Klebsiella
The Gram negative bacterial family Enterobacteriaceae encompasses an array of human
commensals and clinically important pathogens including the genera Escherichia,
Salmonella, Yersinia and Klebsiella (Guentzel, 1996). The genus Klebsiella represents a
ubiquitous taxon which can be isolated globally from soil, water sources, plants and
animals (Brisse et al., 2006). All Klebsiellae are facultative anaerobic, 0.6 to 6 µm long
straight rods (Grimont and Grimont, 2015). Immobility (bar K. mobilis) and a thick
hydrophobic polysaccharide capsule are also defining characteristics (Grimont and
Grimont, 2015). Taxonomically the general consensus is that the genus is most closely
related to the genera Enterobacter and Raoultella and comprises 5 species belonging to
three polyphyletic groups: K. pneumoniae, K. granulomatis, K. oxytoca, K. mobilis and
K. variicola (Brisse et al., 2006). The type species of the genus, K. pneumoniae, is the
most clinically relevant and has historically been sub-classified into 3 sub-species
(subsp.): K. pneumoniae subsp. pneumoniae, ozaenae and rhinoscleromatis (Brisse et al.,
2006). Distinction between sub-species is based on the clinical conditions they cause and
by phenotypic differences (Brisse et al., 2006). Further classification after investigations
including DNA sequencing of the gyrA and parC genes has phylogenetically separated
K. pneumoniae into three clusters: KpI, KpII and KpIII (Brisse and Verhoef, 2001). Most
clinical infections are caused by cluster KpI which include all 3 K. pneumoniae sub-
species, with KpII and KpIII implicated to a lesser extent (Brisse and Verhoef, 2001).
Sub-species rhinoscleromatis and ozaenae are responsible for the rare diseases
rhinosclererma and ozena respectively (Brisse et al., 2006). Sub-species pneumoniae

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manifests as multiple types of infection and is overwhelmingly the commonest cause of
disease amongst the three (Podschun and Ullmann, 1998). Hereafter K. pneumoniae will
be used in reference to the subspecies clinically classified as K. pneumoniae subsp.
pneumoniae which is represented in phylogenetic cluster group KpI.
2.2 K. pneumoniae Infection
K. pneumoniae is usually a non-pathogenic human commensal of the large intestine and
nasopharynx in an estimated 30-43% and 3-4% of the population respectively (Davis and
Matsen, 1974). Active infection is opportunistic and seen almost exclusively in
immunosuppressed individuals with a significant association between K. pneumoniae
community-acquired pneumonia in chronic alcoholics in the mid to late 20th century
(Carpenter, 1990). Throughout the 21st century however the majority of infections have
occurred in healthcare settings, particularly hospitals (Brisse et al., 2006). Opportunistic
infections caused by K. pneumoniae are predicted to represent up to 8% of all healthcare
associated infections in the USA and Europe, manifesting mainly as urinary tract
infections, pneumonia and sepsis with wound infections and meningitis representing
more rare pathologies (Brisse et al., 2006; Podschun and Ullmann, 1998). Common
predisposing immunosuppressive conditions include diabetes mellitus, neoplastic
disease, renal failure and chronic alcoholism (Podschun and Ullmann, 1998). Mortality
rates for individuals with K. pneumoniae sepsis and pneumonia have been described as
high as 52% (Tumbarello et al., 2006) and >50% (Podschun and Ullmann, 1998)
respectively. These high mortality rates are associated with protracted infections due to
the ineffectiveness of antibiotics to which K. pneumoniae has become resistant
(Tumbarello et al., 2006). Continual circulation of K. pneumoniae amongst

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immunosuppressed individuals in healthcare environments has allowed for the selection
of resistance-determining factors rendering many important treatments ineffective
(Garbati and Godhair, 2013). One important class of antibiotics to which important
resistance is seen are the β-lactams. Of the β-lactams the penicillins and cephalosporins
are 2 families which are used empirically for many K. pneumoniae infections, with drug
of last resort status claimed by the carbapenem family (Brisse et al., 2006). Resistance of
K. pneumoniae is now seen to all families of β-lactams including the carbapenems leading
the WHO to declare the current situation as a serious cause of international concern
(WHO, 2014).
2.3 β –Lactams - Mechanism of Action
For penicillins, cephalosporins, carbapenems and all other β-lactams, the bacterial target
is the same: the PBPs. PBP transpeptidase catalytic sites form peptide cross-links in
peptidoglycan by removing the terminal D-alanine from the pentapeptide side chain
attached to NAM. The energy released allows the transpeptidase to link the position 4 D-
alanine from one NAM-peptide molecule to the position 3 DAP of another in Gram
negative bacteria (Kohanski et al., 2010a). This crosslinking of peptidoglycan allows the
structure to resist lysis due to the intense turgor pressure from the cytoplasm (Sobhanifar
et al., 2013). Constant autolysis of the peptidoglycan by hydrolases occurs naturally and
is balanced by the activity of transpeptidases so as to not lead to lysis (Sobhanifar et al.,
2013). β–lactams possess a 4-membered lactam ring which is hydrolysed by
transpeptidases to form an irreversible acyl-enzyme complex at the active site leading to
malfunctioning of peptidoglycan synthesis and eventually cell lysis (Cho et al., 2014).
Each β–lactam antibiotic differs in side chain composition which alter bioavailability,

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ability to cross the bacterial membrane in Gram negatives and/or susceptibility to β–
lactamases (Hamilton-Miller, 1999). Steric and electrostatic alterations can alter
penetration through outer membrane channels as well as interactions with the active sites
of β–lactamases. For example, the presence of an oxyimino side chain on some
cephalosporins allows for activity against K. pneumoniae which express certain β–
lactamases, as the side chain is sterically incompatible with the active site (Jacoby, 1997).
2.4 β –Lactam Resistance
An important mechanism of resistance to β-lactams is the production of β-lactamases
which hydrolyse the β-lactam ring rendering them unable to act on PBPs (Majiduddin et
al., 2002). β-lactamases can be broadly classified into 4 groups based on their molecular
configuration. Groups A, C and D possess a serine residue in their active site which is
used to hydrolyse the beta lactam ring whereas group B β-lactamases are metalloenzymes
which utilise zinc as a co-factor to hydrolyse the β-lactam (Bush et al., 1995). More
complex classifications can be made based on the substrates they can hydrolyse and the
susceptibility to β-lactamase inhibitors (Bush and Jacoby, 2010). β-lactamase inhibitors
(clavulanate and tazobactam) contain a β-lactam ring which is used to competitively
compete with β-lactams for the active site of β-lactamases, thus increasing β-lactam
efficacy (Maiti et al., 1998).
All K. pneumoniae strains constitutively express at a low level at least one of three related
chromosomal class β-lactamases encoded by the blaSHV, blaOKP or blaLEN genes (Siebor
et al., 2005). The β-lactamases produced are SHV-1, OKP, and LEN which correspond
with the K. pneumoniae phylogenetic cluster groups with KpI associated only with SHV-
1 (Hæggman et al., 2004). All three are active against the penicillins and 1st and 2nd

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generation cephalosporins but susceptible to β-lactamase inhibitors (Arnold et al., 2011).
For this reason two commonly used penicillins in treatment, pipericillin and amoxicillin,
are combined with β-lactamase inhibitors tazobactam and clavulanate for use as a clinical
treatment option against susceptible strains (Brisse et al., 2006). SHV-1 can also be
plasmid encoded and passed from strain to strain by horizontal gene transfer (Tärnberg
et al., 2009). The plasmid-based β-lactamase TEM-1 (blaTEM gene) is related to SHV-1
and is also found in some K. pneumoniae isolates giving resistance to a similar range of
β-lactams (Brisse et al., 2006). Nearly 200 variants of SHV-1 and >200 TEM-1 variants
which differ by at least one amino acid substitution have been categorised, many of which
in K. pneumoniae (Bush et al., 2015). Mutations which alter the orientation of the
hydrolysing serine and other residues in the enzyme’s active site alter the β-lactams to
which the enzyme confers resistance to (Hæggman, 2010). Mutations leading to increased
activity against 3rd generation cephalosporins with an oxyimino side-chain allow the β-
lactamase to sterically complement the bulky β-lactam resulting in an ability to hydrolyse
a new substrate and yield ESBLs (Jacoby and Munoz-Price, 2005). ESBLs have been
identified as many SHV-1 and TEM-1 variants in K. pneumoniae (Bush et al., 2015).
Examples include TEM-3, TEM-50, SHV-2 and SHV-10; all of which are able to
hydrolyse 3rd generation cephalosporins and penicillins (Bush and Jacoby, 2010). The
circulation of ESBL plasmids housing fluoroquinolone and aminoglycoside resistance
genes is becoming more common (Filippa et al., 2013). For multi-drug resistant infections
caused by K. pneumoniae carbapenems are the antibiotic of choice (Morrill et al., 2015).
Now however, there is an increasing occurrence of K. pneumoniae which produce β-
lactamases termed carbapenemases. These include the plasmid-based KPC (blaKPC gene)
which confers resistance to all β-lactams and β-lactamase inhibitors, and are often found
in multi-drug resistance plasmids (Jiang et al., 2010). This leads to using drugs such as

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polymyxin B and colistin which are less effective and associated with detrimental side
effects (Morrill et al., 2015). Furthermore resistance is now also seen to these drugs in K.
pneumoniae (Gu et al., 2016a).
Alongside β-lactamases, mechanisms including a decrease in permeability of the
bacterial cell envelope and active efflux of antibiotics post internalisation often work in
synergy to confer resistance (Tenover, 2006).
2.5 Efflux Mediated β-lactam Resistance
Gram negative bacteria possess 5 major families of membrane-spanning efflux pumps
which actively transport waste and toxic material from within the cell using PMF or ATP
hydrolysis (Webber and Piddock, 2003) . Members from four (RND, MATE, MFS and
SMR) of the five families have been documented as increasing drug resistance to multiple
compounds including β-lactams in K. pneumoniae (Srinivasan and Rajamohan, 2013).
The major efflux pump associated with antibiotic resistance is the AcrAB, RND family
pump (Webber and Piddock, 2003). Increase in AcrAB expression allows for greater
efflux of antibiotics out of the cell and thus increases resistance. AcrB is an inner
membrane-integrated ATPase and AcrA an accessory complex which links AcrB to the
outer membrane channel protein TolC in Enterobacteriaceae (Du et al., 2014). Using PMF
to drive conformational changes, AcrB forces substrate through the TolC channel out of
the bacterium (Pos, 2009). In K. pneumoniae, acrB gene deleted strains are more
susceptible to β-lactams, with gene regulation the defining factor (Padilla et al., 2010a).
acrAB and tolC expression is induced by several transcriptional regulators, including
MarA, RamA and SoxS, which stimulate expression of stress-associated genes in
response to stressful conditions (including oxidative and antibiotic stress) (Grove, 2013;

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Nikaido, 2003). Mutations in the genes encoding their repressors (marR, ramR and soxR)
which diminishes or stop repressor activity can lead to upregulated expression of MarA,
RamA and SoxS and ergo acrAB and tolC as well (Webber and Piddock, 2003).
Inactivating mutations in SoxR were shown to increase resistance to cephalosporins and
the carbapenem, ertapenem, due to an increase in AcrAB-TolC expression in K.
pneumoniae (Bialek-Davenet et al., 2011). Furthermore ramR and marR mutants are
associated with an increase in antibiotic resistance amongst members of the
Enterobacteriaceae (Abouzeed et al., 2008). A similar mechanism of regulation is seen in
the local transcription repressor of acrAB, AcrR (Ruiz and Levy, 2014). Gene knock-outs
of acrR have also yielded K. pneumoniae significantly more resistant to cephalosporins
(Padilla et al., 2010b).
2.6 Porin Mediated β-Lactam Resistance
Outer membrane porins are hollow hydrous transmembrane proteins which allow the
non-specific diffusion of small hydrophilic molecules (including β-lactams) from outside
to inside the cell (Galdiero et al., 2012). Alteration to membrane permeability by porin
structural modification, absence of expression and change in representation between
types are all associated with β-lactam resistance in the Enterobacteriaceae (Nordmann et
al., 2012). These alterations decrease antibiotic entrance into the bacterium. K.
pneumoniae expresses three major porins: OmpK34, OmpK35 and OmpK36 which are
homologous to OmpA, OmpF and OmpC in E. coli respectively (Findlay, 2011). An
increase in resistance of K. pneumoniae isolates to cephalosporins is associated with a
decrease in the presence of OmpK35 and OmpK36 (Ananthan and Subha, 2005).
Furthermore insertional mutations into the OkpK36 gene have been shown to inhibit

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expression, increasing resistance to cephalosporins in vitro (Hernández-Allés et al.,
1999). Transformation of Ompk36-deficient carbapenem and cephalosporin resisant K.
pneumoniae with a plasmid containing the Ompk36 gene has been shown to restore a
susceptible phenotype (Arnold et al., 2011; Martínez-Martínez et al., 1999). β-lactam
resistant strains have also been demonstrated with a joint absence of Opk35 and Opk36
(Doménech-Sánchez et al., 1999). As well as direct mutation to porin genes,
transcriptional regulator mutations also occur. MarA regulates porin expression by
inducing the synthesis of micF. The non-coding RNA, micF, binds to OmpF (Ompk35
homologue) mRNA in E. coli and prevents translation at the ribosomes (Chubiz and Rao,
2011). Thus mutations to marR also decrease the porin density in the outer membrane
which has been associated with multi-drug resistance in E. coli (Chubiz and Rao, 2011).
2.7 Acquiring New Antimicrobial Targets
Many elements can contribute towards β-lactam resistance in K. pneumoniae, all of which
are under highly complex and integrated genetic control much of which is yet to be
elucidated. To counter the ever worsening paradigm of antibiotic resistance in general it
is key to elucidate these elements to give new direction for the development of therapies.
Using the TraDIS (transposon directed insertion-site sequencing) method which couples
high density transposon mutagenesis with next generation sequencing (NGS) it is
possible to efficiently assay: 1. A required gene set for a bacterium under a given
condition and 2. Inactivating mutations which confer resistance to clinically relevant
conditions (Langridge et al., 2009). Firstly, as all antibiotics target processes essential to
life this method allows the identification of essential genes, proteins and processes which
may be identified as novel drug targets. Secondly, due to the highly complex nature of

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genetic regulation and our incomplete knowledge of it, TraDIS offers an unbiased method
of assaying every gene under antibiotic stress and may yield novel genes, proteins or
pathways involved in resistance, increasing our knowledge base and potentially yield ing
new targets for therapy.
2.8 Aims
The aims of this investigation are as follows:
1. Acquire an essential gene list for K. pneumoniae under laboratory conditions.
2. Elucidate which genes confer resistance to β-lactams after transposon
inactivation. The β-lactams investigated will be cefotaxime (3rd generation
cephalosporin) and meropenem (carbapenem), both of which are used clinically.

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3.0 Materials and Methods
3.1 Bacterial Strainand Cultures
The human associated strain K. pneumoniae ECL-8 (Forage and Lin, 1982) was acquired
streaked out into single colonies on LB agar (media composition in table 1). Overnight
cultures were prepared when required for each experimental session by removing part of
a single colony from the agar plate followed by inoculation into a 50 ml Falcon centrifuge
tube (Fisher Scientific, Loughborough, UK) containing 15 ml of LB broth. Overnight
incubation occurred at 37oC with shaking at 180 rpm.
Table 1│Composition of media. All media was prepared by adding dried powder to
deionised H2O followed by autoclaving at 121oC for 15 minutes.
Medium Composition
Luria-Bertani Broth
(Melford Laboratories,
Ipswich, UK)
10 g/L peptone, 5 g/L yeast extract, 5
g/L NaCl
Luria-Bertani Agar
(Melford Laboratories,
Ipswich, UK)
10 g/L peptone, 5 g/L yeast extract, 5
g/L NaCl, 10 g/L agar
2XTY Broth 16 g/L Tryptone (BD, Oxford, UK),
10 g/L (Merck, New Jersey, USA), 5
g/L NaCl (Sigma-Aldrich, Dorset,
UK)
Brain-Heart Infusion
Broth
(Oxoid, Hampshire,
UK)
12.5 g/L brain infusion solids, 5 g/L
heart infusion solids, 10 g/L peptone,
2 g/L glucose, 5 g/L NaCl, 2.5 g/L
Na2HPO4

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3.2 AntibioticSensitivityTesting
For use in selecting transposon mutants the MIC of kanamycin monosulphate (Melford
Laboratories, Ipswich, UK) was determined to be 32 µg/ml. For exposure of transposon
mutants to β-lactams the MICs of cefotaxime sodium salt and Meropenem tihydrate (both
Sigma-Aldrich, Dorset, UK) were determined at 0.125 µg/ml and 0.0625 µg/ml
respectively. All MICs were found by inoculating 50 µl of overnight culture on to LB
agar containing varying concentrations of antibiotic. Growth was examined after
overnight incubation at 37oC. The lowest concentration at which there was no visible
growth was determined as the MIC. Table 2 shows antibiotic preparation information.
For use in agar, sterile antibiotics in solution were added to the desired concentration to
sterile LB agar at ~55oC.
Table 2│Reagents used, their preparation and storage. AC = autoclaved at 121oC for 15
minutes; SF = sterile filtered using a 0.22 µm Millex® syringe filter (Merck, Nottingham,
UK).
Reagent Methods
Glycerol 100% glycerol used as is or added to de-ionised
H2O to a concentration of 10%, AC and stored at
-20oC
Ethanol 100% ethanol added to de-ionised H2O to the
desired concentration, SF and stored at RT
Kanamycin
Monosulphate
Powder added to de-ionised H2O to a final
concentration of 50 mg/ml, SF and stored at -
20oC for 3 months.
Cefotaxime
Disodium Salt
concentration of 0.5 mg/ml, SF and stored at -
80oC for 3 months
Meropenem
Tihydrate
concentration of 1 mg/ml, SF and stored at -20oC
for 3 months

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3.3 TransposonTransformation
Overnight K. pneumoniae ECL-8 culture was added to 2XTY broth in conical flasks.
EDTA at a final concentration of 0.7 mM was added to increase transformation efficiency
(Fayard et al 1995). Flasks were incubated at 37oC with shaking at 180 rpm. Once the
optical density at 600 nm (OD600) reached circa 0.4 the culture was aliquoted into 50 ml
Falcon tubes at 4oC and submerge in ice. After 30 minutes on ice samples were
centrifuged in an Eppendorf 5810R model (Eppendorf, Hamburg, Germany) at 4,000 g
and 4oC for 15 minutes before returning to ice. The supernatant was decanted and the
pellet re-suspended in 50 ml -20oC 10% glycerol. Centrifugation and re-suspension was
repeated with combination of two pellets into one 50 ml sample. Centrifugation and re-
suspension without recombination occurred two more times with a final re-suspension in
circa 100 µl glycerol. 100 µl of sample was added to sterile 1.5 ml micro-centrifuge tubes
(Eppendorf, Hamburg, Germany) and left on ice. After 15 minutes samples were
centrifuged at 5,000 g and 4oC for 5 minutes. 0.2 µl of EZ-Tn5™ <KAN-2> Tnp
Transposoome™ (Epicentre Biotechnologies, Madison, USA) was then added to each
sample. After 1 hour on ice each sample was added to a separate 0.2 cm BioRad®
GenePulser™ electroporation cuvette (BioRad, Hertfordshire, UK) followed by
electroporation (23 kV, 600 Ω, 10 µF) using an Eppendorf Eporator® electroporator
(Eppendorf, Hamburg, Germany). After electroporation 900 µl of brain-heart infusion
broth at 37oC was rapidly added to the cuvette, mixed and transferred to a 50 ml Falcon
tube before incubation at 37oC with shaking at 180 rpm for 2 hours. 100 µl of culture was
then inoculated and evenly spread onto LB agar enriched with 32 µg/ml kanamycin.
Incubation for at least 12 hours before harvesting followed by removing individual

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colonies from plates into LB broth. All colonies were mixed yielding a mutant library.
100% glycerol was added to a concentrations of 10% before storing at -80oCuntil needed.
3.4 Exposureof Mutants to β-lactams
Transposon mutants obtained in section 2.3 were diluted 1/3 using LB broth and 150 µl
added to LB agar plates supplemented with MIC concentrations cefotaxime and
meropenem (see section 2.2). Non-mutant K. pneumoniae ECL-8 overnight culture was
also cultured on MIC plates as controls. After 12 hours incubation at 37oC individual
colonies were harvested as in section 2.3 if the control plate showed an absence of growth.
100% glycerol was added to a final concentration of 10% followed by -80oCstorage. The
following protocols were completed separately but identically for the initial mutant
library and the antibiotic exposed mutants.
3.5 Extractionand Fragmentationof DNA
500 µl of transformed K. pneumoniae ECL-8 was defrosted and made to an OD600 of 1
by diluting with LB broth to a final volume of 5 ml. Bacterial DNA was then extracted
using the QIAamp® DNA Blood Mini Kit (Qiagen, California, USA) as outlined by the
manufacturer (Qiagen, 2015). DNA concentration was then deduced using the Qubit®
Fluorometer 2.0 (Invitrogen, Massachusetts, USA) as per the manufacturer’s instructions
(Life Technologies, 2015). DNA was then added to a nuclease free 15 ml Falcon tube to
yield a final DNA mass of 1 μg and made up to 500 µl using nuclease free H2O. The
sample was then fragmented to ~150 bp fragments using a Diagenode Biorupter ®

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(Diagenode, Seraing, Belgium) using the parameters: 30 second on time, 90 seconds off
time, low power, 13 cycles. Concentration followed using the Eppendorf Concentratrator
5301 (Eppendorf, Hamburg, Germany) to a final volume of 55 µl.
3.6 Next GenerationSequencing Preparation
The 55 µl sample was prepared for Illumina® MiSeq® NGS using the NEBNext®
Ultra™ DNA Library Prep Kit for Illumina® (New England BioLabs, Massachusetts,
USA). Table 3 outlines the chronological order of the extraction with reference to any
deviations from the manufacturer’s protocol (New England BioLabs, 2016).
Concentration of Illumina® compatible DNA was determined by Stratagene Mx3005P
qPCR (Agilent Technologies, California, USA). Preparation of samples was completed
using the KAPA Library Quantification Kit for Illumina® Platforms (KAPA Biosystems,
Massachusetts, USA) as per manufacturer’s instructions (KAPA Biosystems, 2014).

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Table 3│DNA extraction protocol. Outline of adherence to and deviance from the
NEBNext® Ultra™ DNA Library Prep Kit for Illumina® manufacturer’s protocol in
chronological order.
Section in
Protocol
Step in
Section
Description
1.1 1 - 3 As per manufacturer’s instruction.
1.2 1 - 5 As per manufacturer’s instruction.
1.3A 1 – 10, 12
11
As per manufacturer’s instruction (Selected for 200 bp).
17.5 µl of DNA solution was added to a PCR tube.
1.4A 1 2.5 ul of two custom primers (Eurogentech, Seraing,
Belgium) were added to select for the Tn5 transposon
(forward primer) and the Illumina® adapter sequence
(reverse primer) rather than the primers stated. All other
steps completed as per manufacturer’s instruction.
1.4B 2 The first PCR step parameters are presented in table
1.3B 1-11
12
As per manufacturer’s instruction. The addition of 17.5 µl
of DNA solution to a PCR tube for amplification.
1.4A 1 2.5 µl of two custom primers (Eurogentech, Seraing,
Belgium) were added to select for: 1. Transposon (forward
primer – with added P5 and index sequence); 2. Illumina®
adapter sequence (reverse primer) rather than the primers
stated.
1.4A 2 The first PCR step parameters are presented in table.
1.5 1, 3 - 8 As per manufacturer’s instruction.
1.5 2, 9 and 10 45 µl of AMPure® XP beads were used and 20 µl of buffer
EB (Qiagen, California, USA) to elute DNA. Size
distribution was not completed.
1.4B 1 1 µl of one index primer from NEBNext® Oligos for
Illumina® Index Primers Set 1, 1 µl of custom primer, 25 µl
of NEBNext® HiGi PCR Master Mix, 6.5 µl nuclease free
H2O and 17.5 µl of DNA from section 1.5 were added
together.
1.4B 2 The second PCR step parameters are presented in table.
1.5 - Repeated as directed in this table.

23
Table 4│PCR parameters. Used for NEBNext® Ultra™ DNA Library Prep Kit for
Illumina® protocol for creating NGS compatible DNA.
3.7 Next GenerationSequencing
For NGS using the Illumina MiSeq® platform, the reagent kit v3 (Illumina, California,
USA) was used. DNA was denatured and diluted following manufacturer’s protocol
(Illumina, 2016). The v3 cartridge was then prepared and loaded with the DNA sample
and sequencing initiated as per the manufacturer’s instruction (Illumina, 2013).
PCR
Run
Description Temperature Cycles Time (Seconds)
First Initial
Denaturation
98oC 1 45
Denaturation 98 oC
}10
15
Annealing 65 oC 30
Extension 72 oC 30
Final Extension 72 oC 1 60
Hold 4 oC ∞ ∞
Second Initial
Denaturation
98oC 1 45
Denaturation 98 oC
}20
15
Annealing 65 oC 30
Extension 72 oC 30
Final Extension 72 oC 1 60
Hold 4 oC ∞ ∞

24
3.8 Data Analysis
Sequence data from the MiSeq® sequence run were processed using a series of in-house
scripts to discard any read without transposon sequence present, discard transposon and
index sequences before alignment to the K. pneumoniae ECL-8 reference genome and
segregate reads based on indexes. Data were then analysed as numbers of insertions
across the genome using the Artemis 16.0.0 software package (Rutherford et al., 2000).
For the initial investigation of essential genes in K. pneumoniae, data were normalised
by deducing insertion indexes calculated by dividing the number of insertions per gene
by the length of the gene. Plotting the insertion index (x-axis) against frequency of genes
(y-axis) shows a bimodal distribution (results section) with a peak at an insertion index
of 0 (genes with no insertions) and another peak representing genes with insertions. Using
the modes from each peak, gamma distributions were fitted from which Log2-LRs were
deduced to assess the likelihood of a gene belonging to the essential distribution. A Log2-
LR cut-off of -3.6 was used to assign a gene the title of essential. A LR of -3.6 or below
was considered essential, 3.6 or above non-essential and an LR between 3.6 and -3.6
indeterminable. Single or minimal insertion peaks in genes classified as essential may be
due to incorrect sequence data or a mark of the sensitivity of TraDIS (DNA may be
sequenced from lethal insertions and therefore appear as a peak). Genes were compared
to essentiality data in E. coli MG1655 from the KEIO collection using the EcoCyc
database (Keseler et al., 2013; Yamamoto et al., 2009). Unnamed genes were searched
using NCBI’s BLAST function.
An outline of the TraDIS workflow as completed here is shown in figure 1.

25
Figure 1│TraDIS workflow in chronological order. A) Kanamycin sensitive K.
pneumoniae ECL-8 are transformed with a Tn containing a kanamycin resistance gene
(KanR). The Tn randomly inserts into the genome allowing growth with kanamycin
Tn5 Transposon
KanR
KanR
P5 Tn5 Gene A Index P7
A
A
A
A A
A
A
A
A A
AA
AA
A
Add adapter
Size selectionfor 200 bp
PCR with forward (Tn5)
and reverse primers
(adapter)
A A
Tn5 Gene A
PCR with forward (P5)
and reverse (P7/index)
primers
Tn5 Gene A
Gene A Gene B Gene C
Insertions
A
.
B
.
C
.
K. pneumoniae
↑ = Direction
of sequencing
Fragmented
DNA
Transform
1 random
insertion
per cell
Mutants grow
on kanamycin
agar

26
present. Only one Tn insertion per bacterium. B) Sonicated DNA extracted from Tn
mutants is prepared for Illumina® sequencing by adaptor ligation at the 3 and 5 prime
ends of all DNA fragments. Size selection for 200 bp fragments occurs followed by PCR
enrichment for fragments containing the Tn, gene segment and adaptor using adaptor-
and Tn-complementary primers. Fragments are then made Illumina® compatible by PCR
amplification using adaptor- and Tn-complementary primers ligated to P5 and P7
adaptors which allow amplified DNA fragments to bind to Illumina® flow cell oligos.
Index sequences ligated to the P7 primer allow for multiplexing. C) The Illumina®
compatible DNA binds to flow cell oligos and is sequenced by NGS. Raw sequence data
is processed and aligned to a reference genome yielding the identity of the gene fragment
into which the transposon is inserted. Sequence data is then pooled allowing visualisation
of insertion points throughout the genome with an absence of insertions (Gene B)
associated with gene essentiality.

27
4.0 Results
4.1 Kanamycin Susceptibility
The Tn of the EZ-Tn5™ <KAN-2> Tnp Transposoome™ kit used contains a kanamycin
resistance cassette that confers kanamycin resistance to bacteria which become
transformed. It was therefore essential to demonstrate the sensitivity of K. pneumoniae
ECL-8 to kanamycin is shown in figure 2.
Figure 2│Kanamycin susceptibility of K. pneumoniae ECL-8. A) Confluent growth on
LB agar without kanamycin. B) Absence of growth on LB agar supplemented with 32
µg/ml kanamycin. 16 µg/ml and 64 µg/ml plates showed growth and no growth
respectively thus the MIC = 32 µg/ml.
This confirmed sensitivity means that only transposon mutants can be selected for after
transformation with the transposon by culturing on kanamycin MIC agar plates.
A B

28
4.2 Mutant Harvesting
Following transformation with the Tn by electroporation, incubation of K. pneumoniae
ECL-8 on kanamycin agar at MIC allowed for only the Tn mutants to grow (figure 3).
Figure 3│ Tn mutants cultured on 32 µg/ml (MIC) kanamycin agar. Growth is seen after
Tn due acquisition of kanamycin resistance.
Mutant colonies from 1,704 kanamycin MIC agar plates were harvested (figure 3 shows
1 example plate). A representative sample of plates were photographed plates using a
G:BOX F3 imager followed by colony counting using GeneTools 4.3.5 software (both
Syngene, Cambridge, UK). This yielded a total estimated number of colonies (ergo
mutants) harvested as ~1.2 million. Dense TraDIS mutant libraries have been created with
circa one million mutants (Langridge et al., 2009) thus this collection of mutants was
expected to be sufficient to attain the resolution required to assess the essentiality of all
the genes within the genome.

29
4.3 Sequencing Data
The mutant library was sequenced using an Illumine® MiSeq® to yield ~3.5 million
sequence reads with ~310,000 UIPs mapped to the chromosome. This results in an
insertion on average every 17.18 bases when taking into account the 5,324,709 bp length
of the K. pneumoniae ECL-8 chromosome. Though the consensus that ~8 million reads
are ideal for optimal density (Langridge et al., 2009), here the density is sufficient (figure
4) to assay the essentiality of genes with good resolution across the entire chromosome.
Figure 4│Genome wide representation of Tn insertions. Insertions per gene across all
5,006 genes in the K. pneumoniae ECL-8 chromosome and its 206,102 bp plasmid shows
sufficient density to assay every gene in the genome. Any gaps may be associated with
sections of essential genes. The black triangle highlights such a section in which several
large operons of 50s ribosomal subunits reside, all of which are essential.
InsertionperGene
Genes AcrossGenome

30
As longer genes will generally have more insertions than shorter genes it is necessary to
normalise the data in figure 4 by deducing the insertion index (number of insertions
divided by the length of the gene) per gene. Plotting insertion indexes against frequency
gives a bimodal distribution (figure 5).
Figure 5│Frequency of insertion indexes. The left peak represents genes into which there
are very few or no insertions with the mode residing at 0 insertions. The right peak
encompasses genes which have a greater number of insertions and thus a greater insertion
index.
Frequency
Insertion Index

31
In the left-most peak of figure 5 resides genes which have no of few insertions. This is
because the genes were not sequence as transposon insertion into them inhibited growth
ergo these genes are essential for life under laboratory conditions. In the right peak, genes
which have many insertions represent non-essential genes as growth leading to
sequencing has occurred despite transposon insertion. Using the modes from each peak,
gamma distributions were fitted from which Log2-LRs were deduced to assess the
likelihood of a gene belonging to the essential distribution. A Log2-LR cut-off of below -
3.6 was used to assign a gene the title of essential.
4.4 Breakdownof Gene Essentiality
Using the parameters in section 3.3 374 genes from the K. pneumoniae ECL-8
chromosome (supp. table 1) and 13 plasmid genes (supp. table 2) were defined as
essential for growth under laboratory conditions. 4,390 chromosomal genes were classed
as non-essential for growth. 228 genes from the chromosome and plasmid had log2-LRs
of between 3.6 and -3.6 and were thus unassigned to either category. Comparisons to the
essential gene requirement in E. coli MG1655 using data derived from the Keio collection
are shown in figure 6 (Yamamoto et al., 2009).

32
Figure 6│Essential K. pneumoniae genes vs E. coli. 66 essential genes in K. pneumoniae
ECL-8 are non-essential in E. coli MG1655 and 50 extra (red) essential K. pneumoniae
genes have no significant homologues in E. coli (supp. tables 3 and 4). 14 genes are non-
essential in K. pneumoniae but essential in E. coli (supp. table 5) and 258 essential genes
are shared. E. coli MG1655 essentiality data from the Keio collection data (Yamamoto et
al., 2009).
Of the 66 K. pneumoniae ECL-8 essential genes which were non-essential in E. coli
MG1655, 23 were also non-essential in Salmonella enterica serovar Typhi Ty2
(Langridge et al., 2009) (table 5).
66 258 14
K. pneumoniae
ECL-8
E. coli
MG1655
50

33
Table 5│K. pneumoniae-only essential genes. Essential K. pneumoniae ECL-8 genes
which are non-essential in S. Typhi Ty2 and E. coli MG1655.
K. pneumoniae ECL-8 Gene
acnB lipA
ybeD lipB
ruvA pdxJ
cedA ppiB
cysE rplA
ddlB rpmG
fdx rpsT
folP ruvC
glnD secB
hscA tonB
iscU tusB
tusD
Of these genes, lipA and lipB are involved in lipoic acid metabolism. Both form part of
the lipoic acid synthesis pathway in S. Typhi, E. coli and K. pnuemoniae. In the former
two, these genes are redundant due to the presence of another pathway of lipoic acid
acquisition. In K. pneumoniae, this second pathway is non-essential also, thus the
essentiality of lipA and lipB is intruiging.
4.5 Expected Essentialand Non-EssentialGenes
The entire genome of K. pneumoniae ECL-8 was viewed using Artemis 16.0.0 software
which presents the insertion frequency across the entire genome. Figure 7 shows an
example screenshot which includes the gyrA gene region with annotation (Rutherford et

34
al., 2000). By assessing the sequence data yielded for genes for which are well known
to be essential then accuracy of other genes in the data set can be inferred.
Figure 7│Annotated Artemis 16.0.0 screenshot. Red and blue arrows represent the 5’ to
3’ direction of the forward and reverse complimentary DNA strands respectively. Black
boxes represent gene names (BN373 genes refer to genes without a common name).
White rectangles above gene names represent the coverage of the gene across that portion
of the genome. Red dashed lines represent X and Y axes where X = position within the
genome (corresponds to white rectangles) and Y = frequency of sequence reads (lines
added for demonstrative purposes). Black spikes represent sequence reads and therefore
indirectly transposon insertions. Purple triangle represents 10 insertions on the Y-axis.
The red triangle is above a small region at the end of the gene which does have insertions.
This may suggest that this region of the gene is non-essential. Log2-LR for gyrA = -16.90.
Region of K. pneumoniae ECL-8 genome

35
The gyrA gene is amongst the 375 genes classified as essential in K. pneumoniae ECL-8
Essentiality was also seen to other topoisomerase genes parE, parC, gyrB and topA in K.
pneumoniae ECL-8 and E. coli MG1655.
The peptidoglycan synthesis genes murA murB murC murD murE murF murG murI
mraY murJ and ddlB are also essential in both K. pneumoniae ECL-8 and E. coli
MG1655. The alr gene involved in peptidoglycan synthesis encodes alanine rasmase
which converts L-alanine to D-alanine for use in peptidoglycan crosslinking. K.
pneumoniae ECL-8 has 2 alr genes and ergo both are mutually redundancy. The data here
confirms this, showing both alr genes as non-essential (figure 8).
Figure 8│Mutual redundancy of genes. Demonstrated by A) alr_1 (log2-LR = 5.82)
and B) alr_2 (log2-LR = 13.44) in K. pneumoniae ECL-8.
A
B

36
Topoisomerases are essential for cellular replication as demonstrated by the lethal
action of fluoroquinolones. Peptidoglycan synthesis is also an essential bacterial
process as demonstrated by the action of the β-lactams. Taken together these data
confirm the ability of TraDIS to identify known essentiality patterns in genes and can be
accepted as controls when assessing the essentiality of other genes.
4.6 EssentialHypothetical Genes
50 genes were found to be essential in K. pneumoniae ECL-8 with no significant matches
to E. coli MG1655 after BLAST searches. Further investigation showed no significant
matches to another member of the Enterobacteriaceae, S. Typhi Ty2, either (Langridge et
al., 2009). Such hypothetical genes offer a rich environment for investigation into
potential novel drug targets.
One such essential (figure 9) hypothetical protein of note is BN373_19381.
BN373_19381 encodes a 377 amino acid long protein. 35 amino acid residues were
modelled with 56% confidence of possessing an SH3-like barrel fold. 7 transmembrane
α helices were also predicted as presented in figure 10.
.

37
Figure 9│ K. pneumoniae ECL-8 gene BN373_19381 (log2-LR = -11.36).
Figure 10│ BN373_19381 predicted structure. A) Predicted tertiary structure of
BN373_19381 SH3-like barrel domain from K. pneumoniae ECL-8. B) Predicted
topology of 7 transmembrane α helices of BN373_16151. Images from Phyre (Kelley et
al., 2015).
SH3 domains are important constituents of Eukaryotic signal transduction proteins such
as kinases. SH3-like domains in prokaryotes have been associated with signal
transduction and intracellular pathogenicity (bacterial proteins which interfere with host
cell signal transduction) (Whisstock and James, 1999). SH3-like domains have also been
implicated in iron sequestration in K. pneumoniae (Hung et al., 2012). Thus it is possible
A B

38
that BN373_19381 may be involved in the activation, inactivation or alteration of a
protein which plays a role in a pathway essential for viability.
Further investigations into the structure and function of BN373_19381 will allow for
identification of any potential target for novel antimicrobial therapy.
4.7 Exposureof Mutants to β-Lactams
Exposure of the mutant library to β-lactams to which the non-transformed K. pneumoniae
ECL-8 cells are sensitive was completed on antibiotic enriched LB agar. Growth of the
mutant library at MIC concentrations of cefotaxime and meropenem suggested that some
transposon insertions might confer resistance (figures11 and 12).

39
Figure 11│Mutant exposure to cefotaxime. A) Exposure of non-transformed K.
pneumoniae ECL-8 to 0.125 µg/ml cefotaxime sodium salt (MIC) showing no growth.
B) Exposure of transposon mutants 0.125 µg/ml of cefotaxime sodium salt showing
growth.
Figure 12│ Mutant exposure to meropenem. A) Exposure of non-transformed K.
pneumoniae ECL-8 to 0.0625 µg/ml meropenem tihydrate (MIC) showing no growth. B)
Exposure of transposon mutants 0.0625 µg/ml meropenem tihydrate showing growth.
A B
A B

40
The mutants which grew as single colonies at MIC values of cefotaxime or meropenem
were harvested. Sequencing was undertaken to identify which genes had a transposon
insertions.
4.8 Cefotaxime-Resistance ConferringInsertions
After exposure to an MIC concentration of cefotaxime any isolated mutant colonies
which grew were sequenced using the same parameters as the initial mutant library
sequence run. Insertions identified after sequencing represent those genes into which a
transposon insertion has allowed growth at the MIC. Figure 13 shows the insertions found
in the ramR gene.
Figure 13│K. pneumoniae ECL-8 gene BN373_11261 (identified as ramR) showing
insertions across the gene. Reads = 27,495; UIPs = 124.
ramR is a global transcriptional regulator of bacterial stress response genes. Mutations
inactivating ramR are associated with increased antibiotic resistance though activation of

41
multiple genes including those which include multidrug efflux pumps which actively
efflux β-lactams from within the cell. This would correspond with the ability of K.
pneumoniae ECL-8 to grow when ramR is inserted into and thus inactivated as shown in
figure 13.
4.9 Meropenem-Resistance ConferringInsertions
After exposure to an MIC concentration of meropenem any isolated mutant colonies
which grew were sequenced. Figures 14 and 15 show the two genes into which most
insertions were found: bamB and hupA.
Figure 14│K. pneumoniae ECL-8 BN373_34991 (identified as bamB) showing
insertions across the gene. Reads = 126; UIPs = 65.

42
Figure 15│K. pneumoniae ECL-8 hupA showing insertions across the gene. Reads =
492; UIPs = 51.
bamB is part of the β barrel assembly machinery (BAM) in Gram negative bacteria
(Bakelar et al., 2016). Insertions into the bamBgene, as shown in figure 11, may influence
the proportion of β barrels such as porins in the outer membrane and thus decrease the
ability of β-lactams to enter the cell.
hupA encodes the DNA binding protein, HU, α sub-unit. Together with the β sub-unit
encoded by hupB, HU protects DNA against stressors such as UV radiation by
compacting the chromosome. Its role in β-lactam resistance is unknown.

43
5.0 Discussion
This investigation has yielded important information regarding the essential gene set
required under laboratory conditions for K. pneumoniae ECL-8. Furthermore the
identification of a selection of genes which confer resistance to β-lactams has been
inferred. The essential gene set of a bacterium encompasses the very fundamental genes
and therefore proteins and processes required for life. Antibiotics interfere with these
essential processes to either halt growth or kill the bacterium altogether (Kohanski et al.,
2010b). Although essential processes are well known amongst bacteria (for example fatty
acid metabolism, DNA replication and peptidoglycan synthesis) the essentiality of genes
responsible for individual proteins involved can vary. This can be exploited clinically as
exemplified by different classes of antibiotics that target different molecules of the same
process to ultimately cause a detrimental effect to an essential pathway (e.g.
glycopeptides and β-lactams).
5.1 EssentialGenes – lipAand lipB
lipA and lipB genes encode the proteins LipA and LipB respectively. Both catalyse a step
in the de novo synthesis of lipoic acid. Lipoic acid is an essential 8-carbon long fatty acid
well known to be a co-factor to several enzymes in the Enterobacteriaceae (Zhang et al.,
2015). Lipoic acid can also be acquired through a separate system in E. coli through the
action of the LplA protein encoded by the gene, lplA. LplA converts the precursor lipolyl-
adenylate from the environment into lipoic acid (Zhang et al., 2015). In E. coli the actions
of lipA/lipB and lplA have been shown to be mutually redundant, with mutants in

44
lipA/lipB or lplA showing wild-type growth characteristics but mutants in lplA/lipA or
lplA/lipB showing growth defects (Morris et al., 1995). Though lipoic acid metabolism
in prokaryotes has been investigated using mainly E. coli, this redundancy appears to be
the case for S. Typhi Ty2 also (Langridge et al., 2009). Here, in K. pneumoniae ECL-8,
transposon insertions into lplA were shown to be non-essential (log2-LR= 14.37- data not
shown) but insertions into both lipA and lipB were essential suggesting a non-redundant
relationship between genes. It may therefore be feasible to develop an antibiotic molecule
which inhibits the action of LipA or LipB resulting in a lethal effect for the bacterium.
Such a target would have the added positive of being non-lethal to commensals such as
E. coli which have redundant genes. This would decrease the chance of developing
further opportunistic infections with bacteria such as Clostridium difficile (Leffler and
Lamont, 2015). Based on these data the role of lipoic acid metabolism in K. pneumoniae
appears different to is close relatives and thus warrants further investigation.
5.2 EssentialGenes - BN373_19381
BN373_19381 was found to possess a domain reasonably homologous to SH3-like
domains. Mainly found in eukaryotic kinases, SH3 domains modulate protein-protein
interactions through preferentially binding to proline rich sequences (Kurochkina and
Guha, 2013). SH3 domains play a key role in regulation of kinase and GTPase activity;
influencing cellular phosphorylation states and therefore signalling pathways
(Kurochkina and Guha, 2013). A regulatory function in bacteria is known but less well
understood (Bakal and Davies, 2000). One example of an SH3-like domain in
Enterobacteriaceae family members is the Feo system. The Feo system is an iron
acquisition system which includes a membrane spanning iron permease (FeoB) and a

45
transcriptional regulator (FeoC) (Lau et al., 2013). An additional protein, FeoA, contains
a SH3-like domain which was thought to act as a GTPase activating protein leading to
repression of Feo system genes in iron rich environments (Hung et al., 2012). This has
since been dismissed, rendering the role of FeoA and its SH3-like domain unknown (Lau
et al., 2013). BN373_19381 is predicted to have 7 membrane spanning α-helices with the
SH3-like domain residing in the cytoplasm. As multiple transmembrane helices rarely
appear in the outer membrane it can be predicted that BN373_19381 is an inner
membrane protein (Silhavy et al., 2010). Thus given the role of SH3 domains in signal
transduction and link to a potential transmembrane domain it can be speculated that
BN373_19381 may be a putative receptor. The SH3 domain may transduce signals from
the periplasmic binging of a ligand to the cytoplasm and have a downstream effect. In
FeoA and many other proteins the SH3-like domain has an unknown function thus further
elucidation studies are essential. If BN373_19381 is a receptor then its substrate may be
small enough enter the cell through porins. If this is the case it may be possible to develop
a molecule to irreversibly bind to the periplasmic side of the receptor leading to its
constitutive action which may yield lethal effects. If BN373_19381 is a receptor then it
could be a favourable target as there is no need to enter the cytoplasm which can prove
difficult.
5.3 ramR
Transposon insertions throughout the ramR gene were associated with resistance to the
cephalosporin β-lactam cefotaxime. This corresponds with the finding in clinical isolates
of K. pneumoniae which have shown ramR mutations (including deletions) increase
resistance to antibiotics such as tigecycline, fluorquinilones and the cephalosporin,

46
cefotoxin (Bialek-Davenet et al., 2011; Wang et al., 2015). RamR represses the
expression of ramA. RamA induces transcription of the acrA and acrB efflux pump genes
thus inactivating mutations in ramR lead to constitutive AcrAB efflux pump synthesis
through RamA induction (Rosenblum et al., 2011). This has been demonstrated by a non-
resistant fluoroquinolone phenotype seen in ramA overexpressed acrA/acrB deleted
mutants (Schneiders et al., 2003). Thus the findings here in K. pneumoniae ECL-8 further
support a role for ramA overexpression in β-lactam resistant mutants.
5.4 bamB
The bamBgene showed multiple insertions upon NGS of the meropenem-exposed mutant
library. BamB is the protein encoded by bamB which is part of the beta-barrel assembly
machinery (BAM) in Gram negative bacteria (Gu et al., 2016b). BAM receives
cytoplasmic-derived proteins from periplasmic chaperones and folds them into beta-
barrel outer membrane proteins (OMPs) before releasing them into the outer membrane
(Gu et al., 2016b). BAM is composed of 5 subunits (BamA, B, C, D and E) of which
BamA is the central component within which proteins are folded. BamB to D are BamA-
associated lipoproteins (Gu et al., 2016b). Whilst BamA and BamD are essential in E.
coli and K. pneumoniae (supp. table 1), mutations in bamB are associated with inefficient
but non-lethal OMP production (Bakelar et al., 2016). OMPs provide essential pathways
for nutrients to enter the bacterium thus their absolute absence (in bamA and bamD
mutants which yield a non-functional BAM) is associated with cell death (Bakelar et al.,
2016; Bialek-Davenet et al., 2011). In fact, the BAM complex has been proposed as a
potential target for novel therapies due to its overall essentiality (Gu et al., 2016b). bamB
mutants however only limit the efficiency of OMP production thus a lower density will

47
be present in the outer membrane (Bakelar et al., 2016). As stated in the introduction, the
K. pneumoniae OMPs OmpK35 and OmpK36 have been associated with increased
resistance to antibiotics. As the BAM is responsible for the assembly of such OMPs in
the outer membrane it is here proposed that a bamB mutation limits the number of OMPs
in the OM to such a level that decreases meropenem diffusion into cell but does not
lethally block entrance of essential nutrients. Mutations in bamB have also been
demonstrated to increase resistance to some antimicrobials in S. enterica serovar
enteritidis (Namdari et al., 2012). Here it is demonstrated that K. pneumoniae can
decrease its OMP profile not only by mutations to porin genes directly (Hernández-Allés
et al., 1999). If these mutations are present in clinical isolates then this mechanism further
demonstrates the versatility of bacteria in taking different routes to attain the same.
5.5 hupA
HupA and HupB (genes hupA and hupB) form a heterodimer which comprise the histone-
like protein (HU) in E. coli (Bi et al., 2009). HU is predicted to be involved with the
compaction of bacterial DNA and gene regulation (Bi et al., 2009; Dri et al., 1991). Whilst
the exact significance in gene regulation is unknown, HU mutants (hupA/B deleted) have
showed decreased survivability in acidic environments as well as inhibition of cell
division in E. coli (Bi et al., 2009; Dri et al., 1991). HU has further been implicated in the
binding of non-coding RNAs, tRNAs and mRNAs (Macvanin et al., 2012). Though the
function of RNA binding remains unknown, a regulatory role is presumed (Macvanin et
al., 2012). One such gene regulatory role was demonstrated in hupA/B-deleted mutants
which were shown to decrease micF transcription and OMP expression in E. coli leading
to an increased sensitivity to antibiotics (Painbeni et al., 1997). Deletion of hupB did not

48
alter antimicrobial sensitivity to the macrolide, chloramphenicol (Painbeni et al., 1997).
Thus the result of hupA Tn-insertion here does not correlate with the existing evidence in
regards to increasing OMPdensity. If this were the case, then an increase in susceptibility
to meropenem would be predicted. It is clear that regulation played by HU is complex,
much of which is yet to be elucidated. It is possible that hupA mutation here alters the
expression one or multiple genes, with the resulting gene product responsible for a
resistance phenotype. hupA mutations have not been associated with antibiotic resistance,
thus investigation into role of hupA mutations may elucidate a novel resistant mechanism
and therapeutic targets.
5.6 Future Investigations
Further sequencing of the mutant library is required to provide an optimal amount of
sequence reads to yield sufficient density of transposon insertions. This will allow for a
more reliable identification of essential and non-essential genes. For any essential gene
which warrants further investigation after this targeted gene knockouts should be used to
confirm essentiality (Datsenko and Wanner, 2000). This should be followed with
investigations elucidating the role of the gene in K. pneumoniae. For hypothetical genes
further investigation is required to assign a definitive structure and function of the
encoded protein. Such investigations will reveal whether there is an opportunity to target
these gene, gene products or processed in the hope of developing novel antimicrobial
therapies.
Greater density is also needed to derive definitive conclusions regarding β-lactam-
resistance conferring mutations. Many genes assayed contained low amounts of
insertions (data not shown) whilst cultured under meropenem and cefotaxime MICs

49
which were too ambiguous to draw conclusions from. This was due to an insufficient
number of sequence reads. Further sequencing of these exposed bacterium should
increase reads in those genes which confer-resistance, diminish any ambiguity and thus
yield more insertion-associated mutations.
The identification of genes advantageous to growth under β-lactam stress using TraDIS
may yield useful insights. By passaging K. pneumoniae under conditions of non-lethal
but stressful levels of β-lactams and then mapping which genes disappear from the mutant
population over each passage advantageous genes for growth under β-lactam stress can
be elicited (Langridge et al., 2009). Those genes which disappear from the population
may offer further insight into the mechanisms of antibiotic resistance.
Finally, as plasmids are associated with multidrug resistance genes it would be interesting
to assess the essentiality of genes to a clinically isolated multidrug resistant plasmid. This
may allow for the targeting of plasmid genes or proteins which are essential for its own
survival rather than the bacterium itself. In rendering plasmids inactive it may be possible
to increase sensitivity of the whole bacterium to existing antimicrobials.
5.7 Summary
The first aim of this investigation was to acquire an essential gene list in K. pneumoniae
ECL-8 with the hope of identifying unique essential genes which may be feasible drug
targets. Here 374 chromosomal genes have been classified as essential under laboratory
conditions, several of which are also non-essential in E. coli and S. Typhi. Further
research is needed to assess their feasibility as therapy targets. The second aim was to
discover gene which give resistance to clinically relevant β-lactams. Of the three noted,
hupA mutations have not been previously associated with an increase in β-lactam

50
resistance. Thus overall this investigation has demonstrated the essentiality of known and
hypothetical genes to K.pneumoniae and demonstrated known and unknown mechanism
of β-lactam resistance. These data should be investigated further in order to find new
ways of tackling the increasingly problematic opportunistic pathogen, K. pneumoniae.

51
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58
7.0 Supplementary Material
Supplementary table 1 – 374 Essential genes in K. pneumoniae ECL-8 chromosome.
accA cca glnD ispG mukB
accB fdx glnS ispH mukE
accC ffh gltX kdsA mukF
accD fldA glyA kdsB murA
acnB fmt glyQ lepB murB
acpP folA glyS leuS murC
acpS folB gmk lexA murD
adk folC groL lgt murE
alaS folD groS ligA murF
argS folE grpE lipA murG
aroQ folP gyrA lipB murI
asd frr gyrB lnt mviN
asnS ftsA hda lolA nadD
aspS ftsI hemA lolB nadE
birA ftsL hemC lolC nrdA
cdsA ftsQ hemD lolD nrdB
cedA ftsW hemE lolE nusA
cmk ftsY hemG lpdA nusB
coaA ftsZ hemH lptA nusG
coaBC fusA_1 hemL lptB oxaA
coaD gapA hflB lpxA parC
coaE gcp hipB lpxB parE
csrA glmM hisS lpxC pdxH
cysE glmS holA lpxD pdxJ
cysS dut holB lpxH pgk
dapA dxr hscA lpxK pgsA
dapB dxs icd lspA pheS
dapD engA ileS lysS pheT
dapE eno imp metG plsB
ddlB era infA metK plsC
dnaA fabA infB minE ppa
dnaB fabB infC mraW ppiB
dnaC fabD iscS mraY ppnK
dnaE fabG iscU mrdA prfA
dnaG fabH ispA mrdB prfB
dnaN fabI ispB mreB priA

59
dnaQ fabZ ispD mreC proS
dnaT fbaA ispE mreD prs
dnaX glmU ispF msbA psd
Supplementary table 1 continued
pssA rpmA secY erpA BN373_00791
pth rpmB serS tsaE BN373_01521
purB rpmC ssb priB BN373_09361
pyrG rpmD sucA lptF BN373_09861
pyrH rpmG sucB lptG BN373_09901
recA rpmH suhB lapB BN373_09941
rep rpmI thiL ybeD BN373_10361
rho rpoA thrS lptE BN373_05261
ribA rpoB thyA ybeY BN373_14481
ribB rpoC tilS rplY BN373_14531
ribD rpoD tmk ftsK BN373_14631
ribE rpoH tonB tsaB BN373_18781
ribF rpsA topA yfiO (bamD) BN373_19011
ribH rpsB trmD BN373_25751 BN373_05251
rimM rpsC trmU BN373_26571 BN373_19381
rnc rpsD tsf BN373_28461 BN373_21521
rnpA rpsE tusA BN373_28471 BN373_22201
rpiA rpsF tusB BN373_28481 BN373_22461
rplA rpsG tusC BN373_28491 BN373_23191
rplB rpsH tusD BN373_28501 BN373_23221
rplC rpsI tusE BN373_16841 BN373_23821
rplD rpsJ tyrS BN373_30371 BN373_23831
rplE rpsL ubiA BN373_30411 BN373_24151
rplF rpsM ubiB BN373_30581 BN373_24431
rplJ rpsN ubiD BN373_16151
rplK rpsP ubiE BN373_32301
rplL rpsQ ubiF BN373_33841
rplM rpsR ubiG BN373_15601
rplN rpsS ubiH BN373_35981
rplO rpsT uppS BN373_35991
rplP rseP valS BN373_37261
rplQ rsgA waaA BN373_37291
rplR ruvB yaeT(bamA) BN373_40351
rplS ruvC ygfZ BN373_41171
rplT secA yjaC BN373_17741
rplU secB zipA BN373_17751
rplV secD lptC BN373_43341

60
Supplementary table 2 – 13 Essential genes in K. pneumoniae ECL-8 plasid.
Supplementary table 3 – 66 K. pneumoniae ECL-8 genes which are non-essential in E.
coli MG1655.
acnB pdxH glyA secB
priB pdxJ hda sucA
ybeD ppiB hemE sucB
lapB priA hipB thyA
rplY recA hscA tonB
cedA rep icd trmU
cmk rpiA iscS tusA
cysE rplA iscU tusB
ddlB rplK lipA tusC
dnaQ rpmG lipB tusD
dnaT rpmI lpdA tusE
rplW secE tsaC BN373_17931
rplX secF engB BN373_17661
BN373_p00491
BN373_p01311
BN373_p01651
BN373_p01721
BN373_p01741
BN373_p01751
BN373_p01951
BN373_p01961
BN373_p01971
BN373_p01991
BN373_p02091
BN373_p02101
BN373_p02111

61
fabH rpsF lptB ubiE
fdx rpsT lysS ubiF
folB rsgA mraW ubiG
folP ruvB nusB ubiH
glnD ruvC rimM ygfZ
ruvA yjaC
Supplementary table 4 – conserved, putative or hypothetical K. pneumoniae ECL-8
essential genes which have no significant homologous genes in E. coli MG1655.
BN373_00791 BN373_22201
BN373_01521 BN373_22461
BN373_05251 BN373_23191
BN373_05261 BN373_23221
BN373_09361 BN373_23821
BN373_09861 BN373_23831
BN373_09901 BN373_24151
BN373_09941 BN373_24431
BN373_10361 BN373_25751
BN373_12531 BN373_26571
BN373_12641 BN373_28461
BN373_14481 BN373_28471
BN373_14531 BN373_28481
BN373_14631 BN373_28491
BN373_15601 BN373_28501
BN373_16151 BN373_30371
BN373_16841 BN373_30411
BN373_17661 BN373_30581
BN373_17741 BN373_32301
BN373_17751 BN373_33841
BN373_17931 BN373_35981
BN373_18781 BN373_35991

62
Supplementary table 5 – 14 essential genes in E. coli MG1655 which are non-essential
in K. pneumoniae ECL-8.
BN373_19011 BN373_37261
BN373_19381 BN373_37291
BN373_21521 BN373_41171
bcsB
cydA
cydC
def
degS
ftsE
ftsN
ftsX
map
minD
polA
spoT
trpS
wzyE

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