Mol Genet Genomics (2008) 280:59–72 DOI 10.1007/s00438-008-0345-2
ORIGINAL PAPER
Global consequences of phosphatidylcholine reduction in Bradyrhizobium japonicum Stephanie Hacker · Julia Gödeke · Andrea Lindemann · Socorro Mesa · Gabriella Pessi · Franz Narberhaus
Received: 14 March 2008 / Accepted: 15 April 2008 / Published online: 30 April 2008 © Springer-Verlag 2008
Abstract Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a limited number of bacteria including members of the Rhizobiales. Here, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum, respectively. Two diVerent phospholipid N-methyltransferases, PmtA and PmtX1, convert phosphatidylethanolamine (PE) to PC by three consecutive methylation reactions in B. japonicum. PmtA mainly catalyzes the Wrst methylation reaction converting PE to monomethyl PE, which then serves as substrate for PmtX1 performing the last two methylation reactions. Disruption of the pmtA gene results in a signiWcantly reduced PC content causing a defect in symbiosis with the soybean host. A genome-wide survey for diVerentially expressed genes in the pmtA mutant with a custom-made AVymetrix gene chip revealed that PC reduction aVects transcription of a strictly conWned set of genes. Among the 11 up regulated genes were pmtX3 and pmtX4, which code for isoenzymes of PmtA. The expression of two typical two-component systems, a MarR-like regulator and two proteins of a RND-type (resistance nodulation cell
Communicated by D. Andersson. S. Hacker · J. Gödeke · F. Narberhaus (&) Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, NDEF 06/783, 44780 Bochum, Germany e-mail:
[email protected] A. Lindemann · S. Mesa · G. Pessi Institute of Microbiology, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland
division) eZux system were diVerentially expressed in the pmtA mutant. Our data suggests that a decrease in the PC content of B. japonicum membranes induces a rather speciWc transcriptional response involving three diVerent transcriptional regulators all involved in the regulatory Wne-tuning of a RND-type transport system. Keywords Phospholipids · Phosphatidylcholine · Phosphatidylethanolamine · Methyltransferase · Rhizobium · Nitrogen Wxation
Introduction Phosphatidylcholine (PC) is a major phospholipid in eukaryotes where it fulWls important structural and signalling functions. In contrast, only about 10% of all bacterial species are predicted to produce PC (Sohlenkamp et al. 2003). PC in bacteria can be synthesized via two alternative routes. A pathway unique to bacteria comprises the direct condensation of choline and CDP-diacylglycerol catalyzed by a phosphatidylcholine synthase (Pcs). This activity has been demonstrated in Sinorhizobium meliloti, Pseudomonas aeruginosa, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti, Legionella pneumophila, Agrobacterium tumefaciens, and Brucella abortus (Sohlenkamp et al. 2000; Wilderman et al. 2002; Martínez-Morales et al. 2003; Comerci et al. 2006; Wessel et al. 2006). In the methylation pathway, PC is produced by the threefold methylation of phosphatidylethanolamine (PE) via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE) using S-adenosyl-L-methionine (SAM) as methyl donor. This reaction is catalyzed by one or more phospholipid N-methyltransferases (Pmts). Like higher eukaryotes,
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such as humans or rats (Kent 1995), Rhodobacter sphaeroides, Zymomonas mobilis, S. meliloti or A. tumefaciens use a single Pmt enzyme to catalyze the threefold methylation of PE (Kaneshiro and Law 1964; Arondel et al. 1993; Tahara et al. 1994; de Rudder et al. 1997). The soybean symbiont Bradyrhizobium japonicum on the other hand encodes several Pmt enzymes, two of which are signiWcantly expressed under aerobic growth conditions. PmtA predominantly catalyzes the Wrst methylation step from PE to MMPE, whereas PmtX1 catalyzes the second and third methylation steps (Hacker et al. 2008). This is reminiscent of PC biosynthesis in lower eukaryotes such as yeast and Neurospora, which also possess two diVerent Pmt enzymes with distinct substrate speciWcities (Kent 1995). It is notable that many of those bacteria that produce PC are either symbionts or pathogens of animal or plant hosts. There is growing evidence that bacterial PC plays a fundamental role in the infection process. A B. japonicum pmtA mutant with a decreased PC content was unable to establish an eYcient symbiosis with its soybean host (Minder et al. 2001). PC also is required for virulence of the plant pathogen A. tumefaciens. A PC-deWcient mutant lacked the type IV secretion apparatus responsible for T-DNA transfer into the plant (Wessel et al. 2006). Some human and animal pathogens also seem to rely on PC for the proper establishment of host infections. B. abortus mutants unable to synthesize PC have lowered virulence in a mouse model (Comerci et al. 2006; Conde-Alvarez et al. 2006). Recently, Conover and coworkers showed, that the presence of PC in L. pneumophila is important for both adhesion to host cells and functioning of the Dot/Icm type IV secretion system (Conover et al. 2008). Generally, phospholipids are of critical importance for membrane integrity and cell growth (Raetz and Dowhan 1990). Therefore, a tight control of membrane lipid homeostasis is crucial for survival. The composition of biological membranes is adapted in response to changes in environmental conditions, like temperature, oxygen tension, salinity or nutrient supply (Tang and Hollingsworth 1998; Medeot et al. 2007). However, the underlying regulatory mechanisms controlling membrane lipid biosynthesis are largely unknown (Schujman and de Mendoza 2005). Apart from the more generalized function of maintaining cellular integrity, phopholipids are also known to exert stabilizing and/or activating functions on certain membrane proteins. Anionic phospholipids such as phosphatidylserine or phosphatidylglycerol (PG) are known to interact with components of the Sec protein secretion machinery (Wang et al. 2003). PG is required for the assembly of a functional photosynthetic apparatus in Synechocystis sp. PCC6803 (Hagio et al. 2000; Sato et al. 2000). Ornithine lipids are crucial for normal amounts of c-type cytochromes in
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Mol Genet Genomics (2008) 280:59–72
Rhodobacter capsulatus (Aygun-Sunar et al., 2006). PE is involved in the correct assembly of the lactose permease and gamma-aminobutyric acid permease in E. coli (Bogdanov et al. 1996; Zhang et al. 2005). Furthermore, PE was recently described to be important for secretion of E. coli alkaline phosphatase and also aVected its transcription in a PhoB/R-dependent manner (Mikhaleva et al. 2001). Another two-component system, the Cpx system, is activated in PE-deWcient cells (Mileykovskaya and Dowhan 1997), whereas the expression of Xagellin is suppressed (Kitamura et al. 1994). The expression of the micF RNA involved in expression of the OmpF porin is up regulated in both PE- and PG-deWcient cells (Inoue et al. 1997). This clearly shows that phospholipids play an important role in bacterial signal transduction pathways. In this study, we set out to determine global gene expression in a B. japonicum pmtA mutant, which contains signiWcantly reduced PC levels in the membrane (Minder et al. 2001; Hacker et al. 2008). Our Wrst wholegenome analysis of a bacterial PC biosynthesis mutant demonstrates that expression of only a limited set of genes is changed in PC depleted cells. DiVerentially expressed were two so far undescribed two-component systems, a MarR-like transcriptional regulator and RND-type eZux system.
Material and methods Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli cells were routinely grown at 37°C in Luria–Bertani (LB) medium (Miller 1972) supplemented with ampicillin (200 g ml¡1), kanamycin (50 g ml¡1), chloramphenicol (50 g ml¡1) or tetracycline (10 g ml¡1), if required. B. japonicum strains were propagated aerobically at 30°C in PSY complex medium (Regensburger and Hennecke 1983) supplemented with 0.1% (w/v) L-arabinose. Aerobic cultures for microarray analysis were grown with rigorous shaking (180 rpm) in 5-l Erlenmeyer Xasks containing 200 ml of medium. For phenotypic analyses, cultures were grown in HM minimal medium (Cole and Elkan 1973) supplemented with 0.1% (w/v) L-arabinose under various pH conditions or with diVerent phosphate concentrations. If appropriate, antibiotics were added at the following concentrations (g ml¡1) to B. japonicum cultures: chloramphenicol, 20 (for counterselection against E. coli donor strains); kanamycin, 100 (solid media), 50 (liquid media); spectinomycin, 100; tetracycline, 50 (solid media), 30 (liquid media).
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Table 1 Bacterial strains and plasmids used in this study Strain or plasmid
Relevant characteristics
Source or reference
DH5
Host for plasmid ampliWcation
Hanahan (1983)
S17-1
RP4-2 (Tc::Mu) (Km::Tn7) integrated in the chromosome
Simon et al. (1983)
E. coli
B. japonicum USDA110 spc4
Spr (wild type)
Regensburger and Hennecke (1983)
5519
Spr Kmr phoB::[Km>] insertion mutant of USDA110 spc4
Minder et al. (1998)
5569
Spr Kmr pmtA::[Km>] deletion mutant of USDA110 spc4
Minder et al. (2001)
BO245
Spr Kmr blr4802-pmtX4::[Km>] deletion mutant of USDA110 spc4
This study
BO246
Spr Kmr blr4802-pmtX4::[] deletion mutant of USDA110 spc4
Hacker et al. (2008)
BO255
Spr Kmr pmtX4::[] deletion mutant of USDA110 spc4
BO263
Spr Kmr bll5263::[] m] m> [ Km WT ::Tn ]
[]
[Km>]
[
