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 ContentsAcknowledgements..........................................................................................................4Figures, Tables and Abbreviations.................................................................................5

List of Figures...............................................................................................................5List of Tables.................................................................................................................6Abbreviations................................................................................................................6

1.0 Abstract......................................................................................................................72.0 Introduction...............................................................................................................8

2.1 Klebsiella................................................................................................................82.2 K. pneumoniae Infection.........................................................................................92.3 β –Lactams - Mechanism of Action......................................................................102.4 β –Lactam Resistance............................................................................................112.5 Efflux Mediated β-lactam Resistance...................................................................132.6 Porin Mediated β-Lactam Resistance...................................................................142.7 Acquiring New Antimicrobial Targets..................................................................152.8 Aim........................................................................................................................16

3.0 Materials and Methods...........................................................................................173.1 Bacterial Strain and Cultures................................................................................173.2 Antibiotic Sensitivity Testing................................................................................183.3 Transposon Transformation...................................................................................193.4 Exposure of Mutants to β-lactams........................................................................203.5 Extraction and Fragmentation of DNA.................................................................203.6 Next Generation Sequencing Preparation.............................................................213.7 Next Generation Sequencing................................................................................233.8 Data Analysis........................................................................................................24

4.0 Results......................................................................................................................274.1 Kanamycin Susceptibility.....................................................................................274.2 Mutant Harvesting.................................................................................................284.3 Sequencing Data...................................................................................................294.4 Breakdown of Gene Essentiality...........................................................................314.5 Expected Essential and Non-Essential Genes.......................................................334.6 Essential Hypothetical Genes................................................................................364.7 Exposure of Mutants to β-Lactams.......................................................................384.8 Cefotaxime-Resistance Conferring Insertions......................................................394.9 Meropenem-Resistance Conferring Insertions......................................................40

5.0 Discussion.................................................................................................................43

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5.1 Essential Genes – lipA and lipB............................................................................435.2 Essential Genes - BN373_19381..........................................................................445.3 ramR......................................................................................................................455.4 bamB.....................................................................................................................465.5 hupA......................................................................................................................475.6 Future Investigations.............................................................................................485.7 Summary...............................................................................................................49

6.0 References................................................................................................................517.0 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 AbstractKlebsiella 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.,

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2006). Sub-species pneumoniae 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

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(Tumbarello et al., 2006). Continual circulation of K. pneumoniae amongst

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

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lysis (Cho et al., 2014). Each β–lactam antibiotic differs in side chain composition

which alter bioavailability, 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

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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 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.

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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 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

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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; 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

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(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 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

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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 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 yielding 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 inactiva-

tion. The β-lactams investigated will be cefotaxime (3rd generation ceph-

alosporin) and meropenem (carbapenem), both of which are used clinically.

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3.0 Materials and Methods

3.1 Bacterial Strain and 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.

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Medium CompositionLuria-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

3.2 Antibiotic Sensitivity Testing

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).

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Reagent MethodsGlycerol 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

Powder added to de-ionised H2O to a final concentration of 0.5 mg/ml, SF and stored at -80oC for 3 months

Meropenem Tihydrate

Powder added to de-ionised H2O to a final concentration of 1 mg/ml, SF and stored at -20oC for 3 months

3.3 Transposon Transformation

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 colonies from plates into LB broth. All colonies were mixed yielding a

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mutant library. 100% glycerol was added to a concentrations of 10% before storing at -

80oC until needed.

3.4 Exposure of 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 -80oC

storage. The following protocols were completed separately but identically for the

initial mutant library and the antibiotic exposed mutants.

3.5 Extraction and Fragmentation of 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

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Biorupter ® (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 Generation Sequencing 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

11As 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 table1.3B 1-11

12As 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

1045 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.

22

Table 4│PCR parameters. Used for NEBNext® Ultra™ DNA Library Prep Kit for

Illumina® protocol for creating NGS compatible DNA.

3.7 Next Generation Sequencing

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).

3.8 Data Analysis

23

PCR Run

Description Temperature Cycles Time (Seconds)

First Initial Denaturation

98oC 1 45

Denaturation 98 oC }1015

Annealing 65 oC 30Extension 72 oC 30Final Extension 72 oC 1 60Hold 4 oC ∞ ∞

Second Initial Denaturation

98oC 1 45

Denaturation 98 oC }2015

Annealing 65 oC 30Extension 72 oC 30Final Extension 72 oC 1 60Hold 4 oC ∞ ∞

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.

24

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

present. Only one Tn insertion per bacterium. B) Sonicated DNA extracted from Tn

25

Insertions

Gene A Gene B Gene C

Tn5 Gene A

PCR with forward (P5) and reverse (P7/index) primers

Tn5 Gene A

AA

PCR with forward (Tn5) and reverse primers (adapter)

Size selection for 200 bp

Add adapterA

A A

A A

AA

AA

A

AAA

AA

P5 Tn5 Gene A Index P7

KanR

KanRTn5 Transposon

A.

B.C

.

K. pneumoniae

↑ = Direction of sequencing

Fragmented DNA

Transform

1 random insertion per cell

Mutants grow on kanamycin agar

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.

26

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.

27

BA

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.

28

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

29

Genes Across Genome

Insertion per Gene

in which several large operons of 50s ribosomal subunits reside, all of which are

essential.

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

30

Frequency

encompasses genes which have a greater number of insertions and thus a greater

insertion index.

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 Breakdown of 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).

31

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).

32

50 E. coli MG1655

K. pneumoniae ECL-8

66 258 14

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 GeneacnB lipAybeD lipBruvA pdxJcedA ppiBcysE rplAddlB rpmGfdx rpsTfolP ruvCglnD secBhscA tonBiscU tusBtusD

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 Essential and Non-Essential Genes

33

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

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

34

Region of K. pneumoniae ECL-8 genome

have insertions. This may suggest that this region of the gene is non-essential. Log2-LR

for gyrA = -16.90.

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).

35

B

A

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.

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 Essential Hypothetical 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.

36

.

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).

37

A B

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 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 Exposure of 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).

38

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.

39

A B

A B

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.

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 Conferring Insertions

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.

40

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 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 Conferring Insertions

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.

41

Figure 14│K. pneumoniae ECL-8 BN373_34991 (identified as bamB) showing

insertions across the gene. Reads = 126; UIPs = 65.

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 bamB gene, 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.

42

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 Essential Genes – lipA and lipB

43

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 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 Essential Genes - BN373_19381

44

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

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

45

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, 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 bamB gene 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

46

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 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.

47

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 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 OMP density. 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.

48

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

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.

49

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

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.

50

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57

7.0 Supplementary MaterialSupplementary table 1 – 374 Essential genes in K. pneumoniae ECL-8 chromosome.

accA cca glnD ispG mukBaccB fdx glnS ispH mukEaccC ffh gltX kdsA mukFaccD fldA glyA kdsB murAacnB fmt glyQ lepB murBacpP folA glyS leuS murCacpS folB gmk lexA murDadk folC groL lgt murEalaS folD groS ligA murFargS folE grpE lipA murGaroQ folP gyrA lipB murIasd frr gyrB lnt mviNasnS ftsA hda lolA nadDaspS ftsI hemA lolB nadEbirA ftsL hemC lolC nrdAcdsA ftsQ hemD lolD nrdBcedA ftsW hemE lolE nusAcmk ftsY hemG lpdA nusBcoaA ftsZ hemH lptA nusG

58

coaBC fusA_1 hemL lptB oxaAcoaD gapA hflB lpxA parCcoaE gcp hipB lpxB parEcsrA glmM hisS lpxC pdxHcysE glmS holA lpxD pdxJcysS dut holB lpxH pgkdapA dxr hscA lpxK pgsAdapB dxs icd lspA pheSdapD engA ileS lysS pheTdapE eno imp metG plsBddlB era infA metK plsCdnaA fabA infB minE ppadnaB fabB infC mraW ppiBdnaC fabD iscS mraY ppnKdnaE fabG iscU mrdA prfAdnaG fabH ispA mrdB prfBdnaN fabI ispB mreB priAdnaQ fabZ ispD mreC proSdnaT fbaA ispE mreD prsdnaX glmU ispF msbA psd

pssA rpmA secY erpA BN373_00791pth rpmB serS tsaE BN373_01521

purB rpmC ssb priB BN373_09361pyrG rpmD sucA lptF BN373_09861pyrH rpmG sucB lptG BN373_09901recA rpmH suhB lapB BN373_09941rep rpmI thiL ybeD BN373_10361rho rpoA thrS lptE BN373_05261ribA rpoB thyA ybeY BN373_14481ribB rpoC tilS rplY BN373_14531ribD rpoD tmk ftsK BN373_14631ribE rpoH tonB tsaB BN373_18781ribF rpsA topA yfiO (bamD) BN373_19011ribH rpsB trmD BN373_25751 BN373_05251rimM rpsC trmU BN373_26571 BN373_19381rnc rpsD tsf BN373_28461 BN373_21521

rnpA rpsE tusA BN373_28471 BN373_22201rpiA rpsF tusB BN373_28481 BN373_22461rplA rpsG tusC BN373_28491 BN373_23191rplB rpsH tusD BN373_28501 BN373_23221rplC rpsI tusE BN373_16841 BN373_23821rplD rpsJ tyrS BN373_30371 BN373_23831rplE rpsL ubiA BN373_30411 BN373_24151

59

rplF rpsM ubiB BN373_30581 BN373_24431rplJ rpsN ubiD BN373_16151rplK rpsP ubiE BN373_32301rplL rpsQ ubiF BN373_33841rplM rpsR ubiG BN373_15601rplN rpsS ubiH BN373_35981rplO rpsT uppS BN373_35991rplP rseP valS BN373_37261rplQ rsgA waaA BN373_37291rplR ruvB yaeT(bamA) BN373_40351rplS ruvC ygfZ BN373_41171rplT secA yjaC BN373_17741rplU secB zipA BN373_17751rplV secD lptC BN373_43341rplW secE tsaC BN373_17931rplX secF engB BN373_17661

Supplementary table 1 continued

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.

60

BN373_p00491BN373_p01311BN373_p01651BN373_p01721BN373_p01741BN373_p01751 BN373_p01951BN373_p01961BN373_p01971BN373_p01991BN373_p02091BN373_p02101BN373_p02111

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

fabH rpsF lptB ubiEfdx rpsT lysS ubiFfolB rsgA mraW ubiGfolP 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.

61

Supplementary table 5 – 14 essential genes in E. coli MG1655 which are non-essential in K. pneumoniae ECL-8.

62

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

BN373_19011 BN373_37261

BN373_19381 BN373_37291

BN373_21521 BN373_41171

63

bcsBcydAcydCdef

degSftsEftsNftsXmapminDpolAspoTtrpSwzyE