Preliminary Content

A thesis submitted to the board of the Faculty of Biological Sciences at the University of Oxford in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Abstract

The nirS gene, which encodes the enzyme cytochrome cd₁ nitrite reductase in the denitrifying bacterium Thiosphaera pantotropha, was cloned and sequenced. The sequence of this cytochrome cd₁ was found to be very similar to those of the proteins not only from the closely-related organism P. denitrificans but also several other species of bacteria. However, the N-terminal region of these proteins shows less similarity than the overall protein sequence. Furthermore, sequence analysis in combination with spectroscopic and structural data indicates that the ligands to the c- and d₁-haem groups are not identical in cytochromes cd₁ from different sources. Implications for the mechanism of the enzyme are discussed.

Three different approaches were taken to the problem of expressing recombinant cytochrome cd₁ from the cloned nirS gene. First, the gene was expressed in Escherichia coli, yielding a semi-apo enzyme that contained only the c-type haem. The protein could be reconstituted to some degree using purified haem d₁, producing an enzyme with some of the spectroscopic features of the native holoenzyme and around 50% of the native nitrite reductase activity. This approach should be of use in the future expression of site-directed mutants of cytochrome cd₁, which would be structurally unstable or enzymically inactive if expressed as the holoenzyme in a heterologous denitrifying host.

Second, the T. pantotropha nirS gene was expressed in the denitrifying bacterium Ps. aeruginosa which synthesises an endogenous cytochrome cd₁. Under the growth conditions employed, a mixture of holo- and semi-apo recombinant cytochrome cd₁ was produced. Differences in the visible spectrum of the holoenzyme and attenuated nitrite reductase and cytochrome c oxidase activities suggested that it was not fully assembled in this system. However, the system may be of use in distinguishing site-directed mutants of the enzyme that are enzymically inactive from those that retain some activity.

Third, an unmarked deletion was constructed in the genomic copy of the T. pantotropha nirS gene which abolished the synthesis of cytochrome cd₁. It then proved possible to restore denitrifying growth to the mutant by complementation with the cloned wild-type nirS gene. However, complementation using a site-directed mutant of cytochrome cd₁, in which the d₁ haem ligand Tyr-25 was replaced with phenylalanine failed to restore growth and the cells contained greatly attenuated levels of the mutant cytochrome cd₁. Possible reasons for this result are discussed. These data illustrate the problem of finding an expression system that can synthesise the holoenzyme but does not rely upon its activity to maintain the growth of the host organism.

The transcription start site of the nirS gene was determined using 5’-RACE and primer extension. The nirS gene was found to be transcribed as a monocistronic mRNA. Comparison of the upstream region of the nirS gene with other denitrification genes indicates a common mechanism of transcriptional regulation and suggests that such genes may be transcribed using a specific sigma factor.

Acknowledgements

My thanks first to Stuart, for taking me on in the first place, organising the funding and for his advice and encouragement during the project, particularly in the writing of this thesis. Thanks are also due to the Wellcome Trust, for their absurdly generous studentship, to Graham Pettigrew, who got me into bacterial c-type cytochromes in the first place, and to Thon de Boer, for his cytochrome cd₁ antibody and for the gift of pTNIR3, the plasmid that saved the project! Thanks too, to Val for a huge number of perfect primers and to Colin for excellent photography.

Lab 5-9 has been graced by a number of wonderful postdocs; my thanks especially to Simon, for his support throughout the project and for not laughing (too much) during my transformation (ho ho) to a molecular biologist. And a big thank-you to Dudley, for advice on almost everything and conversation during the late nights, long weekends and even the more normal times of day. Variety has certainly been the spice of life in 5-9 thanks to Dave, Ben, Yoshi, Fiona, Alrik, Neeti, Isobel, our latest arrivals and all our visiting students. Outside of 5-9, thanks especially to Pam, Vilmos and Janos, Andy, the 6th floor crowd and of course Gerry and Rachel for much-needed caffeine and sugar, with a smile.

On the brief occasions that I escaped to the world outside, the Oxford “1st wave”, John, Hannah, Jacqui, Caroline and Sara had a lot to answer for. Thanks too to the Oxford “2nd wave”, especially Mat and Lucy, Jem and Lisa, Ralph, Charlie, Craig, Mark and Amanda and of course Marian, for rescuing me and making me realise it wasn’t all bad! Most of all, thanks to the folks in Edinburgh, for great holidays, time spent amongst mountains, accommodation, beers, entertainment and empathy: Mark, Leo, Jo, Anita and Lorna, and to my fellow “refugees in England” or further abroad; Mary Jane, James and Nim, Marc and Karen, and Colin and Michelle. I’d never have made it without all of you. I must also thank Mark E. Smith and The Fall, for twenty years of brilliant records.

Last, but definitely not least, a big thank-you to my parents, for all their support over the last eight years. I guess this is for you.

Publications

Some of the work in this thesis has been published.

Baker, S.C., Saunders, N.F.W., Willis, A.C., Ferguson, S.J., Hajdu, J. and Fülöp, V. (1997). Cytochrome cd₁ structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to β-propeller folds. J. Mol. Biol. 269: 440-455.
Williams, P.A., Fülöp, V., Garman, E.F., Saunders, N.F.W., Ferguson, S.J. and Hajdu, J. (1997). Haem-ligand switching during catalysis in crystals of a nitrogen cycle enzyme. Nature 389: 406-412.

Conference abstract

Baker, S.C., Saunders, N., Fülöp, V., Hajdu, J. and Ferguson, S.J. (1995). The structure of cd₁ nitrite reductase and analysis of the nirS gene of the denitrifying bacterium Thiosphaera pantotropha. Poster presented at the Beijerinck Centennial Conference on Microbial Physiology and Gene Regulation: Emerging Principles and Applications. 10-14 December 1995, The Hague, The Netherlands.

Publications not directly related to the present work

Goodhew, C.F., Pettigrew, G.W., Devreese, B., Beeumen, J.V., van Spanning, R.J.M., Baker, S.C., Saunders, N.F.W., Ferguson, S.J. and Thompson, I.P. (1996). The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures. FEMS Microbiol. Letts. 137: 95-101.
Page, M.D., Saunders, N.F.W. and Ferguson, S.J. (1997). Disruption of the Pseudomonas aeruginosa dipZ gene, encoding a putative protein-disulfide reductase, leads to partial pleiotropic deficiency in c-type cytochrome biogenesis. Microbiol. 143: 3111-3122.
Hu, W., Van Driessche, G., Devreese, B., Goodhew, C.F., McGinnity, D.F., Saunders, N., Fülöp, V., Pettigrew, G.W. and Van Beeumen, J.J. (1997). Structural characterization of Paracoccus denitrificans cytochrome c peroxidase and assignment of the low and high potential heme sites. Biochemistry 36: 7958-7966.

List of Figures

Chapter	Figure	Title
Chapter 1	1.1	The Nitrogen Cycle
	1.2	The denitrifying electron transport chain of Paracoccus denitrificans
	1.3	Structure of (a) d\(_1\) haem and (b) of protoporphyrin IX
	1.4	Crystal structure of cytochrome cd\(_1\) in the oxidised form from T. pantotropha
	1.5	Proposed mechanism for nitrite reduction by cytochrome cd\(_1\) from Thiosphaera pantotropha
	1.6	Denitrification gene clusters in Ps. stutzeri Zobell, Ps. aeruginosa and P. denitrificans PD1222
	1.7	Unrooted phylogenetic tree of the FNR family of transcriptional activator proteins
Chapter 3	3.1	PCR products used to sequence the nirS gene of T. pantotropha
	3.2	Sequence of the nirS gene and its product, cytochrome cd\(_1\), from T. pantotropha
	3.3	Predicted cleavage site of the periplasmic targeting sequence in cytochrome cd\(_1\) from T. pantotropha
	3.4	Multiple alignment of cytochrome cd\(_1\) sequences
	3.5	Phylogenetic tree of cytochrome cd\(_1\) sequences compared with a phylogenetic tree of 16S rRNA sequences from the same organisms
	3.6	Putative NNR binding sites in the upstream regions of the nirS gene aligned with the FNR consensus binding site
	3.7	Early region of the nirS gene from Ps. stutzeri ATCC 14405, showing primers used for re-sequencing and the sequence obtained
	3.8	Visible spectra of cytochrome cd\(_1\) (a) in the oxidised and (b) in the reduced forms from Ps. stutzeri Zobell
	3.9	Visible spectra of (a) holocytochrome cd\(_1\) and (b) semi-apo cytochrome cd\(_1\) in the reduced forms from T. pantotropha
	3.10	Alternative reaction schemes for the reduction of nitrite by cytochrome cd\(_1\) starting from the fully oxidised enzyme
Chapter 4	4.1	Physical map of the plasmid pTNIR3 produced using six restriction enzymes
	4.2	Location of denitrification genes of T. pantotropha on the plasmid pTNIR3 as determined by physical mapping and limited sequencing.
	4.3	Diagram of the broad host range expression vector pMMB67EH, showing relevant features
	4.4	Restriction digests illustrating the problem of vector carryover during cloning of the T. pantotropha nirS gene into pMMB67EH
	4.5	Restriction digest analysis of the plasmids pMMBSE and pMMBSEK
	4.6	Comparison of the aerobic growth of E. coli, E. coli [pMMB67EH], E. coli [pMMBSEK] and E. coli [pMMBSE]
	4.7	Comparison of the anaerobic growth of E. coli JM83 with that of E. coli [pMMB67EH]
	4.8	Comparison of the protein content of E. coli JM83 with that of E. coli [pMMB67EH], E. coli [pMMBSEK] and E. coli [pMMBSE].
	4.9	Western blot of protein samples from E. coli JM83, E. coli [pMMB67EH], E. coli [pMMBSEK] and E. coli [pMMBSE], probed using antibody to cytochrome cd\(_1\)
	4.10	Comparison of the c-type cytochrome content of E. coli [pMMBSE] grown under aerobic or anaerobic conditions
	4.11	Visible spectrum of soluble extract from E. coli [pMMBSE] grown anaerobically and induced with 1 mM IPTG
	4.12	Elution profile during ion exchange chromatography of soluble extract from anaerobically-grown E. coli [pMMBSE] induced using IPTG
	4.13	UV-visible spectrum of recombinant semi-apo cytochrome cd\(_1\) following ion-exchange chromatography
	4.14	Visible spectra of recombinant cytochrome cd\(_1\) following reconstitution with purified haem d\(_1\) (a) as prepared, (b) following reduction using sodium dithionite, compared with (c) native cytochrome cd\(_1\) from T. pantotropha
	4.15	Assays for nitrite reductase activity using reduced methyl viologen as an electron donor: (a) recombinant semi-apo cytochrome cd\(_1\), (b) recombinant cytochrome cd\(_1\) following reconstitution with d\(_1\) haem, (c) native cytochrome cd\(_1\) purified from T. pantotropha
Chapter 5	5.1	Re-isolation of intact plasmid pMMBSE from Ps. aeruginosa PAO1
	5.2	Comparison of the growth curves of Ps. aeruginosa and Ps. aeruginosa [pMMBSE] under different growth conditions
	5.3	Comparison of (a) the growth curve and (b) nitrite accumulation of Ps. aeruginosa with that of Ps. aeruginosa [pMMBSE]
	5.4	Comparison of the protein content of Ps. aeruginosa with that of Ps. aeruginosa [pMMBSE] under different growth conditions
	5.5	Western blot of protein samples from Ps. aeruginosa or Ps. aeruginosa [pMMBSE] grown under different growth conditions, probed using antibody to cytochrome cd\(_1\)
	5.6	Comparison of the c-type cytochrome content of Ps. aeruginosa with that of Ps. aeruginosa [pMMBSE] under different growth conditions
	5.7	Elution profile during ion-exchange chromatography of soluble extract from Ps. aeruginosa [pMMBSE] grown semi-aerobically with nitrate and induced using IPTG
	5.8	Analysis of the c-type cytochrome content in the elution profile of soluble extract from Ps. aeruginosa [pMMBSE] following ion exchange chromatography
	5.9	UV-visible spectrum of concentrated fractions eluting under peak 2 following DEAE-Sepharose chromatography
	5.10	a) UV-visible spectrum of concentrated fractions eluting under peak 3 after DEAE-Sepharose chromatography, as isolated and (b) after the addition of excess sodium dithionite
	5.11	Difference spectra of (a) dithionite-reduced minus as-isolated spectra from peak 3 and (b) dithionite-reduced peak 3 minus peak 2
	5.12	Cytochrome c oxidase and (b) nitrite reductase activities of the concentrated fractions from peak 3 following ion exchange chromatography
Chapter 6	6.1	Construction of a kanamycin insertional mutant in the nir region of the T. pantotropha genome
	6.2	Construction of a nirS deletion mutant strain in T. pantotropha
	6.3	Construction of the suicide plasmid pGRPN3K
	6.4	Restriction digest analysis of plasmid pGRPN3K
	6.5	Southern blot of genomic DNA from T. pantotropha K7 using the kanamycin resistance gene as a probe
	6.6	Construction of the suicide plasmid pRVS\(\Delta\)nir
	6.7	Restriction digest analysis of plasmids pLN3N and pLN3N\(\Delta\)nir
	6.8	Restriction digest analysis of plasmids pRVS1 and pRVS\(\Delta\)nir
	6.9	PCR analysis of plasmid pRVS\(\Delta\)nir showing a 382 bp deletion in the cloned nirS gene
	6.10	Southern blot of genomic DNA from wild-type T. pantotropha, T. pantotropha K7 and the nirS deletion mutants \(\Delta5\), \(\Delta7\) and \(\Delta8\) using a nirS-derived probe
	6.11	Comparison of the anaerobic growth of T. pantotropha with that of T. pantotropha K7 and T. pantotropha \(\Delta7\)
	6.12	Comparison of (a) the anaerobic growth and (b) nitrite accumulation of T. pantotropha with that of T. pantotropha K7 and T. pantotropha \(\Delta7\)
	6.13	Western blot of total soluble extracts from wild-type T. pantotropha, T. pantotropha K7, T. pantotropha \(\Delta7\), T. pantotropha \(\Delta7\) [pMMBSEK] and T. pantotropha \(\Delta7\) [pMMBY25F] using antibody raised against P. denitrificans cytochrome cd\(_1\)
	6.14	Complementation of T. pantotropha \(\Delta7\) using plasmid pMMBSEK
	6.15	(a) Anaerobic growth of T. pantotropha \(\Delta7\) [pMMBSEK] and (b) nitrite concentration in the medium during growth
	6.16	Construction of the plasmid pVA191-Y25F containing the Y25F mutation in the nirS gene of T. pantotropha
	6.17	Construction of the expression plasmid pMMBY25F containing the Y25F mutation in the nirS gene of T. pantotropha
	6.18	Restriction digest analysis of plasmid pBY25FK
	6.19	Restriction digest analysis of plasmid pMMBY25F using MluI
	6.20	Comparison of (a) anaerobic growth and (b) nitrite accumulation in T. pantotropha \(\Delta7\) [pMMBY25F] compared with that of T. pantotropha, T. pantotropha \(\Delta7\) and T. pantotropha \(\Delta7\) [pMMBSEK]
	6.21	Elution profile during ion exchange chromatography of soluble extract from anaerobically grown T. pantotropha \(\Delta7\) [pMMBY25F]
	6.22	SDS-PAGE gel of fractions eluting from the DEAE-Sepharose column during chromatography of soluble extract from T. pantotropha \(\Delta7\) [pMMBY25F], stained for c-type cytochromes
	6.23	Visible spectra of an unidentified c-type cytochrome from T. pantotropha [pMMBY25F]
	6.24	Visible spectra of (a) cytochrome c\(_{550}\) and (b) cytochrome c’ from T. pantotropha [pMMBY25F]
	6.25	Visible spectra of cytochrome c peroxidase from T. pantotropha [pMMBY25F]
	6.26	Assay of cytochrome c peroxidase activity in peak 4 following ion exchange chromatography
	6.27	Visible spectrum of pseudoazurin from T. pantotropha [pMMBY25F]
Chapter 7	7.1	Analysis of total RNA samples from human cells, E. coli and T. pantotropha using denaturing agarose gel electrophoresis
	7.2	Northern blot of total RNA from anaerobically grown T. pantotropha using a nirS-derived probe
	7.3	Northern blot of total RNA from anaerobically grown T. pantotropha, using a nirS-derived probe together with chemiluminescent detection
	7.4	Location of primers used in primer extension and 5’-RACE experiments to determine the transcription start site of the T. pantotropha nirS gene
	7.5	Primer extension product synthesised from nirS mRNA, compared with a sequencing ladder generated from the nirS gene using the same primer
	7.6	The upstream region of the T. pantotropha nirS gene, showing the transcription start site as determined using primer extension and 5’-RACE
	7.7	Schematic diagram illustrating the principle of the 5’-RACE method
	7.8	Analysis using agarose gel electrophoresis of the PCR product generated from nirS mRNA by 5’-RACE
	7.9	Restriction digest analysis of the cloned PCR products generated from nirS mRNA by 5’-RACE
	7.10	Sequence of the cloned PCR product synthesised from nirS mRNA by 5’-RACE
	7.11	Model for transcriptional termination of the T. pantotropha nirS gene
	7.12	Alignments of the upstream regions of NNR-regulated genes from P. denitrificans and T. pantotropha showing regions of conserved sequence
	7.13	Putative factors influencing the transcription of nitrite and nitric oxide reductases in T. pantotropha
Appendix A	A.1	Schematic diagram of the \(\beta\)-propeller d\(_1\)-haem binding domain of T. pantotropha cytochrome cd\(_1\)

List of Tables

Chapter	Table	Title
Chapter 1	1.1	Reduction potentials and thermodynamics of the reactions of denitrification
	1.2	Standard reduction potentials reported by other workers of the haem groups in cytochrome cd\(_1\)
Chapter 3	3.1	Primers used to sequence the nirS gene from T. pantotropha
	3.2	Comparison of inverted repeats in the DNA sequence of the nir region from T. pantotropha with those in the same region of P. denitrificans PD1222
	3.3	Matrix of similarities and identities between the cytochromes cd\(_1\)
	3.4	Comparison of the physicochemical properties of cytochrome cd\(_1\) from different sources
Chapter 4	4.1	Plasmids referred to in this chapter
	4.2	Different growth conditions used to compare the growth of E. coli JM83 with that of E. coli containing either pMMB67EH, pMMBSEK or pMMBSE
	4.3	Comparison of the ratios of the absorbance maxima in (left) the reduced and (right) the oxidised states of native holo- and chemically prepared semi-apo cytochrome cd\(_1\) from T. pantotropha with those of the recombinant protein from E. coli
Chapter 5	5.1	Different growth conditions used to compare the growth of Ps. aeruginosa PAO1 with that of Ps. aeruginosa [pMMBSE]
	5.2	Comparison of the ratios of the absorbance maxima in the reduced state of native holo- and chemically-prepared semi-apo cytochrome cd\(_1\) from T. pantotropha with those of the partially purified recombinant protein from Ps. aeruginosa or E. coli
Chapter 6	6.1	Bacterial strains and plasmids referred to in this chapter
Chapter 7	7.1	Proteins involved in regulating the expression of denitrification enzymes in P. denitrificans

List of Abbreviations

Abbreviation	Meaning
AMV	Avian myeloblastosis virus
ATP	Adenosine triphosphate
A\(_{xxx}\)	Absorbance at wavelength XXX nm
bp	Base pairs
BSA	Bovine serum albumin
DEAE	Diethylaminoethyl
\(\Delta\)G°’	Standard Gibbs free energy change at pH 7.0
DIG	Digoxigenin
DIG-11-dUTP	Digoxigenin-11-deoxyuridine triphosphate
DMSO	Dimethylsulphoxide
DNase	Deoxyribonuclease
dNTP	Deoxynucleotide triphosphate
DTT	Dithiothreitol
e\(^-\)	Electron
E°’	Standard redox potential at pH 7.0
EPR	Electron paramagnetic resonance
IPTG	Isopropylthio-\(\beta\)-D-galactoside
LB	Luria Bertani
MCD	Magnetic circular dichroism
MGD	Molybdopterin guanine dinucleotide
MMLV	Moloney murine leukaemia virus
Na\(_2\)EDTA/EDTA	Disodium ethylenediamine tetraacetic acid
NAD	Nicotinamide adenine dinucleotide
NMR	Nuclear magnetic resonance
O.D. (xxx)	Optical density at XXX nm
PBS	Phosphate buffered saline
PCR	Polymerase chain reaction
PEG	Polyethylene glycol
RNase	Ribonuclease
SDS-PAGE	Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SSC	0.15 M NaCl, 0.15 mM sodium citrate
TAE	40 mM Tris-acetate pH 8.0, 1 mM EDTA
TBE	45 mM Tris-borate pH 8.3, 1 mM EDTA
TE	10 mM Tris-HCl, 1 mM EDTA
TEMED	N, N, N’, N’-tetramethylethylenediamine
UQ/UQH\(_2\)	Ubiquinone/ubiquinol
X-Gal	5-bromo-4-chloro-3-indolyl-\(\beta\)-D-galactoside

Quote

If we do not succeed, then we run the risk of failure”

Former U.S. Vice-President Dan Quayle, 1990 (attrib.)

Cloning, sequence analysis and studies on the expression of the nirS gene, encoding cytochrome cd\(_1\) nitrite reductase, from Thiosphaera pantotropha