The mitochondrial dimeric phospholipid cardiolipin is characterized by a high degree of unsaturation of its acyl chains, which is important for its functional interaction with mitochondrial enzymes. The unusual fatty acid composition of cardiolipin molecular species emerges from a de novo synthesized "premature" species by extensive acyl chain remodeling that involves as yet only partially identified acyltransferases and phospholipases. Recently, the yeast protein Taz1p was shown to function as a transacylase, which catalyzes the reacylation of monolysocardiolipin to mature cardiolipin. A defect in the orthologous human TAZ gene is associated with Barth syndrome, a severe genetic disorder, which may lead to cardiac failure and death in childhood. We now identified the protein encoded by reading frame YGR110W as a mitochondrial phospholipase, which deacylates de novo synthesized cardiolipin. Ygr110wp has a strong substrate preference for palmitic acid residues and functions upstream of Taz1p, to generate monolysocardiolipin for Taz1p-dependent reacylation with unsaturated fatty acids. We therefore rename the Ygr110wp as Cld1p (cardiolipin-specific deacylase 1). Cardiolipin (CL)2 is a dimeric phospholipid specifically enriched in mitochondrial membranes (1). It plays an important role in mitochondrial structure and function and stabilizes respiratory chain super complexes and individual electron transport complexes (2-4). The CL biosynthetic pathway is well characterized in the yeast Saccharomyces cerevisiae, and the enzymes catalyzing the three sequential reactions involved are all associated with mitochondrial membranes (4, 5). CL synthesis shares the same precursor, CDP-diacylglycerol, with the main cellular phospholipids, yet it differs significantly from other phospholipids in its acyl-chain composition that is characterized by a high degree of unsaturated fatty acids. Although cardiolipin was believed to be essential to support mitochondrial function, mutants defective in cardiolipin synthase (Crd1p) are viable and display only moderate defects of mitochondrial function (6, 7). More severe is a defect in the first committed step of CL synthesis, catalyzed by phosphatidylglycerolphosphate synthase Pgs1p/Pel1p (8). pgs1 mutants are temperature-sensitive, unable to grow on non-fermentable carbon sources for growth, and petite lethal, i.e. dependent on intact mitochondrial DNA for survival (8). More subtle mitochondrial phenotypes emerge from alterations of cardiolipin acyl-chain remodeling. "Premature" cardiolipin synthesized by Crd1p undergoes significant remodeling of its acyl-chain composition (9). Mature CL in yeast contains mainly palmitoleic acid (C16:1) and oleic acid (C18:1), which distinguishes CL from most other phospholipids by its high degree of unsaturation (4, 5, 10). In humans, a severe genetic disorder, the Barth syndrome, is associated with defective CL acyl-chain remodeling (11,12). This disease is caused by mutations in the TAZ gene encoding Tafazzin, for which a functional ortholog also exis...

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (⌬N155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 ؋ 10 7 liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.

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