Genetic analysis and phenotypic characterization of three novel genes of Rhizobium tropici CIAT899 involved in symbiotic interactions with Phaseolus vulgaris plants [Elektronische Ressource] / vorgelegt von Keilor Rojas Jiménez

Genetic analysis and phenotypic characterization of three novel genes of
Rhizobium tropici CIAT899 involved in the symbiotic interactions with
Phaseolus vulgaris plants






Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)




dem
Fachbereich Biologie
Der Philipps-Universität Marburg
vorgelegt von


Keilor Rojas Jiménez
aus San Carlos, Costa Rica




Marburg/Lahn 2005














Vom Fachbereich Biologie der Philipps-Universität Marburg
Als Dissertation angenommen am ....................................2005

Erstgutachter Prof. Dr. Dietrich Werner
Zweitgutachter Prof. Dr. Michael Bölker

Tag der mündlichen Prüfung am ......................................2005

2

External Advisor Dr. Pablo Vinuesa
Centro de Ciencias Genómicas-UNAM, México




















3Part of the work performed during the development of this thesis was or will be published
in:

Keilor Rojas-Jiménez, Christian Sohlenkamp, Otto Geiger, Esperanza Martínez-Romero,
Dietrich Werner and Pablo Vinuesa. 2005. A putative chloride channel and
ornithine-containing membrane lipids of Rhizobium tropici CIAT899 are involved
in symbiotic efficiency and acid tolerance. Molecular Plant-Microbe Interactions
(accepted).

Keilor Rojas-Jiménez, Miguel A. Ramírez-Romero, Esperanza Martínez-Romero,
Dietrich Werner and Pablo Vinuesa. 2005. Rhizobium tropici CIAT899 requires a
Eputative σ factor to establish an effective symbiosis with Phaseolus vulgaris
plants. FEMS Microbiology Letters (submitted).

Ismael Hernández-Lucas, Marco Antonio Rogel-Hernández, Lorenzo Segovia, Keilor
Rojas-Jiménez, and Esperanza Martínez-Romero. 2004. Phylogenetic
Relationships of Rhizobia Based on Citrate Synthase Gene Sequences. Systematic
and Applied Microbiology. 27:703-706.







4Table of contents

Abstract.............................................................................................................................. 7
Zusammenfassung............................................................................................................. 8
General introduction ........................................................................................................ 9
LEGUME-RHIZOBIA INTERACTIONS AND BIOLOGICAL NITROGEN FIXATION ................................................ 9
STRESS TOLERANCE IN RHIZOBIA.............................................................................................................. 11
RHIZOBIUM TROPICI CIAT899 ................................................................................................................. 12
Objectives......................................................................................................................... 14
Materials and Methods................................................................................................... 15
BACTERIAL STRAINS AND PLASMIDS......................................................................................................... 15
RANDOM TRANSPOSON MUTAGENESIS OF RHIZOBIUM TROPICI CIAT899, SELECTION OF ACID-SENSITIVE
MUTANTS AND COSMID COMPLEMENTATION ............................................................................................ 16
STANDARD DNA MANIPULATIONS........................................................................................................... 17
DNA SEQUENCE ANALYSES. .................................................................................................................... 18
DETERMINATION OF OPERON STRUCTURE BY RT-PCR............................................................................. 18
CONSTRUCTION OF NON-POLAR MUTANTS................................................................................................ 19
COMPLEMENTATION................................................................................................................................. 20
PLANT TESTS............. 21
14 14IN VIVO LABELING OF RHIZOBIAL STRAINS WITH [ C]ACETATE OR [ C]ORNITHINE AND ANALYSIS OF
LIPID EXTRACTS........................................................................................................................................ 22
DETERMINATION OF SIGE TRANSCRIPTIONAL REGULATION ..................................................................... 22
IDENTIFICATION OF THE SIGE TIONAL START SITES................................................................. 23
Results .............................................................................................................................. 24
Chapter 1 ......................................................................................................................... 24
A putative ClC chloride channel from Rhizobium tropici is required for symbiosome
invasion of Phaseolus vulgaris nodules.......................................................................... 24
INTRODUCTION......................................................................................................................................... 24
RESULTS OF CHAPTER 1............................................................................................................................ 26
Complementation of mutant 899-PV9 from Rhizobium tropici CIAT899 ........................................... 26
Sequence analyses and genetic characterization of syc1.................................................................... 27
Construction of a non-polar deletion in syc1 and phenotypic characterization of the mutant strain. 29
DISCUSSION OF CHATER 1......................................................................................................................... 32
Chapter 2 ......................................................................................................................... 34
A putative β-hydroxylase from Rhizobium tropici is involved in the modification of
two ornithine-containing membrane lipids which are required for symbiosis and
acid tolerance................................................................................................................... 34
INTRODUCTION......................................................................................................................................... 34
RESULTS OF CHAPTER 2............................................................................................................................ 37
Genetic analyses of olsC..................................................................................................................... 37
RT-PCR of the intergenic spacer between syc1 and olsC confirms that both genes are independently
transcribed.......................................................................................................................................... 39
Construction of a non-polar deletion in olsC and phenotypic characterization of mutant strain....... 40
Some membrane lipids of R. tropici CIAT899 are absent in mutant 899-olsC ∆1............................... 43
5DISCUSSION OF CHAPTER 2....................................................................................................................... 48
Chapter 3 ......................................................................................................................... 50
ERhizobium tropici CIAT899 requires a putative σ -factor to establish an effective
symbiosis with Phaseolus vulgaris plants...................................................................... 50
INTRODUCTION......................................................................................................................................... 50
RESULTS OF CHAPTER 3............................................................................................................................ 52
Sequence analyses of sigE and alf1 .................................................................................................... 52
Construction of a non-polar deletion in sigE and symbiotic performance of the mutant strain ......... 54
Complementation of mutant 899- ∆sig1 with pBBRsigE...................................................................... 56
Partial deletion of sigE in mutant 899- ∆sig1 was not reflect in an increased sensibility to some
environmental stresses. ....................................................................................................................... 57
ETranscriptional regulation of the putative σ factor in R. tropici ....................................................... 58
Identification of the transcriptional start sites of sigE........................................................................ 59
DISCUSSION OF CHAPTER 3....................................................................................................................... 60
Literature Cited .............................................................................................................. 62
PROTOC