Studies on the regulation of genes related to nitrogen fixation and N-assimilation in Azoarcus sp. strain BH72 [Elektronische Ressource] : the role of NtrBC / Abhijit Sarkar

Prof. Barbara Reinhold-Hurek

Description

STUDIES ON THE REGULATION OF GENES RELATED TO NITROGEN FIXATION AND N-ASSIMILATION IN Azoarcus sp. strain BH72: THE ROLE OF NtrBC Abhijit Sarkar 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN (NATURAL) SCIENCE (Dr. rer. nat.) THE FACULTY OF BIOLOGY AND CHEMISTRY UNIVERSITY OF BREMEN GERMANY Untersuchungen zur Regulation von Genen, die in der N -2fixierung und N-Assimilation von Azoarcus sp. Stamm BH72 involviert sind: Die Rolle von NtrBC DISSERTATIONZur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Dem Fachbereich Biologie/Chemie der Universität Bremen vorgelegt von Abhijit Sarkar aus Indien Bremen 2003 The studies of the presented work have been carried out from February 2000 till March 2003 at the department of Biology/Chemistry of Bremen University, Germany, under the guidance of Prof. Dr. Barbara Reinhold-Hurek. Die Untersuchungen zur folgenden Arbeit wurden von Februar 2000 bis März 2003 am Fachbereich Biologie/Chemie der Universität Bremen unter der Leitung von Prof. Dr. Barbara Reinhold-Hurek durchgeführt. Vom Fachbereich Biologie/Chemie der Universität Bremen als Dissertation angenommen am: Datum der Disputation: 1. Erstgutachterin: Prof. Dr. Barbara Reinhold-Hurek 2. Zweitgutachterin: Prof. Dr. Friederike Koenig Dedicated to My Parents …….

Sujets

• Biologie

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2003
• Nombre de lectures: 63
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

STUDIES ON THE REGULATION OF GENES RELATED

TO NITROGEN FIXATION AND N-ASSIMILATION

sp. strain BH72: rcusAzoaIN

THE ROLE OF NtrBC

Abhijit Sarkar

2003

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

(NATURAL) SCIENCE NPHILOSOPHY I DOCTOR OF

(Dr. rer. nat.)

CHEMISTRY THE FACULTY OF BIOLOGY AND

OF BREMEN UNIVERSITY

NY GERMA

-Untersuchungen zur Regulation von Genen, die in der N2

sp. Stamm BH72 Azoarcusfixierung und N-Assimilation von

Bremen

involviert sind: Die Rolle von NtrBC

DISSERTATION

orgrades Zur Erlangung des Dokt

der Naturwissenschaften

(Dr. rer. nat.)

ereich Biologie/Chemie der Dem Fachb

Universität Bremen

n vorgelegt vo

Abhijit Sarkar

aus Indien

3 200

The studies of the

presented work

have been carried out

from February 2000 till

March 2003 at the department of Biology/Chemistry of Bremen University, Germany,

of. Dr. Barbara Reinhold-Hurek. runder the guidance of P

Die Untersuchungen zur folgenden

Arbeit wurden von Februar 2000 bis März 2003

er Universität Bremen uam Fachbereich Biologie/Chemie d

Dr. Barbara Reinhold-Hurek durchgef

Vom Fach

ührt.

nter der Leitung von Pro

bereich Biologie/Chemie der Universität Bremen als Dissertation

n am: angenomme

Datum der Disputation:

1. Erstgutachterin: Prof. Dr. Barbara Reinhold-Hurek

. Dr. Friederike Koenig 2. Zweitgutachterin: Prof

f.

…….Their

Dedicated to My Parents

strength my are dreams

results presented in the Parts of the in: following work have been published

Egener, T., Martin, D.E., Sarkar, A., and Reinhold-Hurek, B. (2001). Role of a

Ferredoxin Gene Cotranscribed with the nifHDK Operon in N2Fixation and

Nitrogenase “Switch-Off” of Azoarcussp. Strain BH72. J. Bacteriol. 3752 – 3760 183,

Egener, T., Sarkar, A., Martin, D.E., and Reinhold-Hurek, B. (2002). Identification

,Proteobacteria-subgroup of the ßof a NifL-like protein in a diazotroph of the

Azoarcus148Microbiolsp. strain BH72.

, 3202 – 3212. Sarkar, A. and Reinhold-Hurek, B. (2002). Characterization of ntrBC of Azoarcus

Book of Abstracts, 5In72. Hsp. strain B

Norwich, Great Britain.

thpean Nitrogen Fixation Conference, Euro

Contents

Abbreviations y 1 Summar 2 Introduction Material and methods 3 3.1 Material3.1.1 Chemicals Gases 3.1.2 plasmids and Strains 3.1.3 ons th conditidia and growCulture me3.2 17 E. colifor 3.2.1 Media 3.2.2 Media for Azoarcussp. BH72
Antibiotic and other supplements 3.2.3 20 E. colifor Cultures 3.2,4 3.2.5 Cultures for Azoarcus sp.
3.2.6 Set up of N2 fixing cultures of Azoarcus sp. BH72
edium semisolid mCultures in 3.2.6.1 3.2.6.2 Batch cultures for N2 fixation in liquid medium
3.2.6.3 Cultures in Laboratory fermenter
y atographchrom3.3 Gas ncentration Estimation of oxygen co3.3.1 Estimation of ethylene concentration 3.3.2 h nucleic acids witorking Standard methods for w3.4 3.4.1 Sterilisation precipitation acid Nucleic 3.4.2 d RNA) Estimation of nucleic acids (DNA an3.4.3 3.4.4 Restriction digestion
esis electrophorgel Agarose 3.4.5 ds Isolation of nucleic aci3.5 sp. BH72 Azoarcuschromosomal DNA from Isolation of 3.5.1 isolation DNA Plasmid 3.5.2 3.5.3 Isolation of DNA from agarose gel and solutions
RNA of Isolation 3.5.4 thod for RNA isolation eHot phenol m3.5.4.1 3.5.4.2 Isolation of RNA using kit (peqGOLD Trifast)

124141414141517181920202020212121212222222323232424 2425252525

3.5.4.3 DNase treatment of RNA 26
26 3.6 Cloning 26vectors cloning 3.6.1 The 27Construction of recombinant plasmid 3.6.2 3.6.2.1 Preparation of vector and insert (along with its modification if
27necessary) 3.6.2.2 Set up of ligation 27
28Transfer of foreign DNA into bacterial cells 3.7 3.7.1 Transfer of DNA in E.coli cells 28
3.7.1.1 Transformation by CaCl2 and heat shock 28
3.7.1.2 Transformation by electroporation 29
29 AzoarcusDNA in Transfer of 3.7.2 3.7.1.1 Electroporation of Azoarcus 29
3.7.1.2 Conjugation of Azoarcus by triparental mating 30
3.8 DNA hybridisation techniques 30
3.8.1 DNA transfer to membrane 30
3.8.2 Labelling DNA probes for hybridisation 31
31Hybridisation 3.8.3 3.8.4 Detection of the probe 32
3.9 Amplification of DNA by PCR 32
3.9.1 Standard method of amplification of plasmid or genomic DNA 32
3.9.2 PCR amplification using Proofstart polymerase 33
3.9.3 PCR amplification using RT-PCR beads 33
33RT-PCR 3.9.3.1 Semi-quantitative 35nsion extePrimer 3.10 36Sequencing of DNA 3.11 37 methods Protein chemistry3.12 37SDS-PAGE 3.12.1 38staining Gel 3.12.2 3.12.3 Western blot and immunodetection 38
39rophoresis elect2D-gel 3.12.4 39extraction Protein 3.12.4.1 3.12.4.2 Isoelectric focussing (I dimension) 39
3.12.4.3 SDS-PAGE (II dimension) 40
3.13 Estimation of E–gucuronidase activity (GUS assay) 40
41y Microscop 3.14 41s used for data evaluation Computer programme3.15

42 4 Results 4.1 Transcript analyses of genes (nifH and nifLA) related to
42 fixation in strain BH72 N24.1.1nifHDK is cotranscribed with fdxN from its upstream V54
43 promoter 4.1.2 nifAis cotranscribed with nifL utilizing the V54 promoter 44
ferentially according to in strain BH72 is expressed dif nifA 4.1.3 45N-availability. 46 genes ntrBCIdentification and genetic organization of the4.2 46 region. ntrBCCloning and sequencing of the 4.2.1 o acid sequences of Alignment of the NtrB and NtrC amin4.2.2 48 sp. BH72 with known sequences from the datadases Azoarcus 4.2.3 Predicted functional motifs of the NtrB and NtrC from
51 sp. BH72 Azoarcus 4.2.4 ntrB and ntrC in strain BH72 are transcriptionally linked. 52
4.3 Generation of a marker exchange ntrBC mutant of Azoarcus sp.
53 BH72 strain 4.3.1 Construction of a marker exchange deletion mutant of the ntrBC 53
4.3.2 Construction of a nonpolar ntrB mutant of strain BH72 54
4.3.3 Validation of constructs by Southern hybridisation and
55amplification PCR genomic 4.4 Phenotypes of the ntrBC mutants 56
4.4.1 Growth characteristics of wild type and the ntrBC
56 mutants wild type with the Comparison of colony/cell morphologies of the 4.4.2 ce: phenotypes sole N-sournt growing on nitrate as mutantrBC of impairment exhibited by BntrBsp 57
4.4.3 Twitching motility is upregulated in BntrBsp 59
60 operon ntrBCTranscriptional regulation of the 4.5 4.5.1 Mapping the 5’ end of the ntrBC transcript (primer extension) 60
4.5.2 Undetectable expression of ntrB::gusA in strain BH72
grown on different nitrogen sources. 62
4.5.3 Effect of nitrogen sources on ntrBtranscription: an RT PCR
63 approach. 4.5.4 ntrBC of strain BH72 is not likely to be autoregulated 64

4.6 Putative targets of NtrBC of strain BH72 65
4.6.1 Transcriptional regulation of N2fixation genes by NtrBC 65
4.6.1.1 The nifA expression in strain BH72 is NtrBC regulated in
a nitrogen dependent manner. 65
4.6.1.2 Effect of nitrate on the derepression of nitrogenase genes
66BntrBsp in 4.6.2 Transcription regulation of the gln genes of
68 sp. BH72: role of NtrBC Azoarcus 4.6.2.1 glnK regulation and effect of N2on its expression: role of
68NtrBC 4.6.2.1.1 Cotranscription of glnK and ugk 69
4.6.2.1.2 Primer extension studies to map the 5’ end of the
ugk and glnK transcript 70
4.6.2.1.3 Western blots to study the effect of nitrogen on GlnK
expression in strain BH72 72
4.6.2.1.4 Nitrogen-dependent differential glnK expression in
strain BH72 and its down-regulation in the ntrBC
mutant: RT-PCR approach 72
4.6.2.1.5 Confirmation of the nitrogen dependent glnK
expression by GUS reporter gene and its down
regulation in the ntrBC mutant 74
4.6.2.1.6 Retention of ammonium “switch off” response and
nitrogenase modification in BntrBsp under low glnK
expression levels 76
4.6.2.2 Analyses of the glnBexpression and role of NtrBC77
4.6.2.2.1 The glnB::gusA expression in strain BH72 is affected
by nitrogen 77
4.6.2.2.2 RT-PCR to analyse glnB expression in strain BH72
and its downregulation in BntrBsp 78
4.6.2.2.3 Comparable protein levels of GlnB in strain BH72
independent of nitrogen 79
4.6.2.3 Detection and analyses of the glnY in the ntrBC mutant 80
4.6.2.3.1 Detection of the glnY transcript in BntrBsp by RT-PCR 80
4.6.2.3.2 2D-gel and Western blot analyses confirming GlnY
expression along with the PIIproteins 81
4.6.2.3.3 Low but detectable glnY::gusA expression in BntrBsp 82
4.6.3 Study of genes for N-assimilation in Azoarcus: role of NtrBC 83
4.6.3.1 Identification of a putative glutamine synthetase III in
Azoarcus sp. BH72 84
4.6.3.2 GSIII transcription in strain BH72 is nitrogen dependent
but free from NtrBC regulation 85

4.6.3.3 Identification of the genetic region in Azoarcus BH72

ne-2-oxoglutarate amino utamiputatively encoding gl

tr