Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction

125 Pages·2016·4.21 MB·English

Master’s Thesis 2016 60 ECTS
Department of Chemistry, Biotechnology and Food Science (IKBM), at the
Norwegian University of Life Sciences

Quorum sensing circuits in
Pseudomonas aeruginosa regulate
N2O reduction

Kristine Lindtveit
Microbiology
Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction

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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction

Quorum sensing circuits in Pseudomonas
aeruginosa regulate N2O reduction

Master Thesis
Kristine Lindtveit

Institute of Chemistry, Biotechnology and Food Science
Norwegian university of Life Sciences
Ås 2016

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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
Mater Thesis Supervisors
PhD Linda Liberg Bergaust (Main superviso) r
NMBU-N group, researche r
Norwegian University of Life Science s
IKBM
NMBU
Dep. of Chemistry, Biotechnology and Food Scien ce
Norwegian University of Life Science s
P.O. Box 5003, N-1432 Aas, Norway
Phone: [phone]
Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction

Acknowledgements
The experiments were conducted at the Institute of Chemistry, Biotechnology and Food
Science (IKBM) at the Norwegian University of Life Sciences, during spring and autumn
2016, in connection with completion of a Master degree in Microbiology.
One student, Kristine Lindtveit, performed the experiments at the institute´s
laboratories following the experimental set-up, designed by Scientist Linda Liberg
Bergaust.
During experimental preparations, experimental performance and collection of
results, I was frequently and whole-heartedly thankful for the assistance of Ph.D student
Daniel Aleksanteri Milligan; the clever advices from a skilful Technician Rannei Tjåland
and her technician-colleague Inga Lena Angel. Also, I am thankful to Åsa Helena
Frostegård for the possibility to make a Poster-presentation, for the 4th Norwegian
Microbiology Meeting (NoMi-16), Sarpsborg. Most of all, I am grateful for the excellent
supervision and refreshingly sharp work attitude given by my supervisor Linda L.
Bergaust.

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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
Table of Contents
Abstract ................................................................................................................ 9
Sammendrag ....................................................................................................... 10
INTRODUCTION .......................................................................................................... 11
Environmental aspects ......................................................................................... 13
N2O; the forgotten climate gas ............................................................................. 16
Denitrification ..................................................................................................... 17
Regulation of denitrification ................................................................................ 21
Pseudomonas aeruginosa .................................................................................... 23
Quorum sensing (QS) circuits ............................................................................... 24
Investigation ....................................................................................................... 26
MATERIALS AND METHODS ......................................................................................... 28
Bacteria ............................................................................................................... 28
Media and culturing conditions ............................................................................ 29
Estimating Biological Parameters ............................................................................. 31
Growth curves and optical density versus cell number/biomass............................ 31
Experimental systems ............................................................................................. 36
Robot incubation system and NOA-system .......................................................... 36
Designing primer pairs ........................................................................................... 39
DNA extraction .................................................................................................... 43
PCR and gel electrophoresis ................................................................................. 43
Experimental protocol ............................................................................................. 45
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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
1. First experimentations ..................................................................................... 46
2. AHLs systems experiments .............................................................................. 46
3. Gene expression experiments .......................................................................... 48
Sample processing .................................................................................................. 50
Quantitative PCR (qPCR) ....................................................................................... 54
Melt point curve .................................................................................................. 58
Computational tools ............................................................................................... 59
Data analyses ...................................................................................................... 59
Excel-Spreadsheet for analysis of gas data .......................................................... 60
RESULTS ..................................................................................................................... 61
Estimation of Biological Parameters ......................................................................... 61
Specific growth rate (µ) and doubling time (Td) ..................................................... 64
Testing of primer pairs for quantitative PCR and confirmation of lasI/rhlI deletions . 65
Gel pictures ......................................................................................................... 65
Physiological experiments ....................................................................................... 67
DRP and AHLs systems experiment ...................................................................... 71
Gene expression experiment ................................................................................... 76
DISCUSSION ............................................................................................................... 82
Critical parameters and troubleshooting .............................................................. 82
Estimation of Biological Parameters...................................................................... 84
Anticipated results and their implications ................................................................ 91
Initial phenotypic experiment with 0% and 7 % initial O2 ...................................... 91
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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
AHLs system experiment & Gene expressions ...................................................... 93
Humans .............................................................................................................. 98
Climate ............................................................................................................... 99
REFERENCES ..............................................................................................................102
APPENDIX ..................................................................................................................115

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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
Abstract
Many bacteria respire in the absence of oxygen throughti orned ou fncitrogen oxides
(NOx) in the process called denitrification. Denitrification is the main greenhouse gas
emitter by its release of N2O when high amounts of N-fertilizers are applied globally. pH
is known to be a regulatory factor for N2O emission, but little is known about how
quorum sensing regulates denitrification. When respiratory physiology of denitrifying
organisms is studied under a defined set of conditions, their phenotypic traits are
encompassed by the term denitrification regulatory phenotype (DRP). Pseudomonas
aeruginosa is a model organism, well studied due to its widespread denitrifying and
opportunistic pathogenic abilities. Detailed gas kinetics of this proteobacterium was
studied to characterize its DRP.
DRP of strains from P. aeruginosa (a type strain and PAO1 wild type) were
characterized with respect to their denitrification phenotype at different initial oxygen
-
concentrations (0, and 7%). This was done by monitoring O2, CO2, NO2 , NO, N2O and N2
by gas chromatography (GC) and an NO-analyzer during their transition from aerobic
respiration to denitrification. This study showed that the denser the culture, the higher
the accumulation of N2O during denitrification, and implied that quorum sensing (QS) is
mediating the N2O emission from denitrification. The question became whether this
occurred due to the regulation by one or both of the AHL systems.
Further characterization on how QS regulates the denitrification phenotype was
- -
done by monitoring denitrification gases under treatment with AHLs on PAO1 rhlI lasI
mutant and a QS-inhibitor on its PAO1 parent strain (PAO1-UW). Their transcriptional
activities of narG, nirS, norB, and nosZ during transition from aerobic respiration to
denitrification were quantified by ddPCR. The gas measurements, as well as cell
densities, cell numbers and initial biomass were measured to describe specific aerobic
-1
and anaerobic respiration rates (µoxic and µanoxic h ), cell yields per e-acceptor and
mRNA per cell.
The results showed that the AHL systems´ regulatory effect on denitrification in
PAO1 is inhibiting N2OR activity, most likely on a post-transcriptional level. This was
directly due to repression of N2O reductase by the Las quorum sensing system.

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Quorum sensing circuits in Pseudomonas aeruginosa regulate N2O reduction
Sammendrag

Mange bakterier respirerer ved fravær av oksygen gjennom reduksjon av
nitrogenoksider (Nx)O i en prosess kalt denitrifikasjon. Denitrifikasjon er hoved-
klimagass frigjøreren gjennom sitt utslipp av N2O når store mengder N-kunstgjødsel
anvendes globalt. pH er en kjent reguleringsfaktor for N2O utslipp, mens quorum
sensing (bakterielt kommunikasjonssystem) er en svært lite gjennomsøkt
reguleringsmekanisme ved denitrifikasjon. Når respirasjonsfysiologien hos
denitrifiserende organismer studeres under definerte forhold, blir de fenotypiske
karakterene omfattet av terminen en ”denitrifikasjonsregulatorisk fenotype” (DRP).
Pseudomonas aeruginosa er en modellorganisme som er velstudert på grunn av dens
utbredte denitrifiserende og opportunistiske patogene egenskaper. Detaljert
gasskinetikk av denne proteobakterien ble studert for å karakterisere dens DRP.
DRP av stammer fra P. aeruginosa (en type stamme og PAO1 villtypen) ble
karakterisert med hensyn til deres denitrifikasjonsfenotype under ulike initielle oksygen
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konsentrasjoner (0 og 7 %). Dette ble gjort ved overvåkning av O2, CO2, NO2 , NO, N2O
og N2 gjennom gasskromatografi (GC) og nitrogen okside-analyse (NOA) under deres
overgang fra aerob respirasjon til denitrifikasjon. Dette studiet viste at jo høyere
celletettheten var, desto høyere ble N2O akkumuleringen under denitrifikasjon, og
antydet at quorum sensing (QS) er medvirkende til N2O utslippet fra denitrifikasjon.
Spørsmålet ble om dette skjedde på grunn av reguleringen fra en eller flere AHL
systemer.
Videre karakterisering av hvordan quorum sensing regulerer denne
denitrifikasjonsfenotypen ble gjort ved å overvåke denitrifikasjonsgassene under
- -
behandling med ulike AHL på en PAO1 rhIl lasI mutant og en QS-hemmer på dens
PAO1 forelderstamme. Deres transkripsjons aktivitet av narG, nirS, norB, og nosZ under
overgangen fra aerob respirasjon til denitrifikasjon ble kvantifisert ved ddPCR.
Gassmålingene, såvel som celletetthet, celletall og initiell biomasse ble målt for å
beskrive spesifikk aerob og anaerob respirasjons rate (µoxic and µanoxic h-1), celleutbytte
per e- akseptor og mRNA per celle.
Resultatene viste at AHL systemenes regulerende effekt på denitrifikasjon i PAO1
hemmer N2OR aktivitet, mest sannsynlig på et post-transkripsjonelt nivå. Dette var
direkte på grunn av N2O reduktase-undertrykkelsen ved Las quorum sensing systemet.

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