Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a Gram-negative plant-pathogenic bacterium that causes crown gall disease by transferring and integrating its transferred DNA (T-DNA) into the host genome. We characterized the chromosomally encoded alpha-crystallin-type small heat-shock protein (α-Hsp) HspL, which was induced by the virulence (vir) gene inducer acetosyringone (AS). The transcription of hspL but not three other α-Hsp genes (hspC, hspAT1, hspAT2) was upregulated by AS. Further expression analysis in various vir mutants suggested that AS-induced hspL transcription is not directly activated by the VirG response regulator but rather depends on the expression of VirG-activated virB genes encoding components of the type IV secretion system (T4SS). Among the 11 virB genes encoded by the virB operon, HspL protein levels were reduced in strains with deletions of virB6, virB8 or virB11. VirB protein accumulation but not virB transcription levels were reduced in an hspL deletion mutant early after AS induction, implying that HspL may affect the stability of individual VirB proteins or of the T4S complex directly or indirectly. Tumorigenesis efficiency and the VirB/D4-mediated conjugal transfer of an IncQ plasmid RSF1010 derivative between A. tumefaciens strains were reduced in the absence of HspL. In conclusion, increased HspL abundance is triggered in response to certain VirB protein(s) and plays a role in optimal VirB protein accumulation, VirB/D4-mediated DNA transfer and tumorigenesis

Charakterisierung und physiologische Relevanz des Membranlipids Phosphatidylcholin für das pflanzenpathogene Bakterium Agrobacterium tumefaciens\textit {Agrobacterium tumefaciens}

In $\textit {Agrobacterium tumefaciens}$ wird Phosphatidylcholin (PC) durch eine dreifache Methylierung von Phosphatidylethanolamin mittels einer Phospholipid $\it {N}$-Methyltransferase synthetisiert oder durch direkte Kondensation von Cholin mit CDP-Diacylglycerin durch die PC-Synthase. Die Expression der PC-Biosynthesegene erfolgt konstitutiv. Eine PC-defiziente $\textit {A. tumefaciens}$-Mutante ist nicht mehr in der Lage eine Tumorbildung in Pflanzen zu induzieren. Proteom-, Transkriptom- und Reportergenstudien belegen, dass die Expression der für die Virulenz benötigten Gene in Abwesenheit von PC nicht induziert wird. Außerdem zeigen vergleichende Transkriptom- und Proteomanalysen von Wildtyp und PC-defizienter Mutante, dass diverse Membran-assoziierte Prozesse in der Mutante verändert sind. Eine phänotypische Charakterisierung der Mutante weist auf eine veränderte Biofilmbildung und eine stark reduzierte Motilität hin

Expression and Physiological Relevance of Agrobacterium tumefaciens Phosphatidylcholine Biosynthesis Genes▿

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens

Narberhaus F: Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Summary Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of hostmicrobe interactions