Summary The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX-5-secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG5. EspG5 represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG5-binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG5 and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG5 chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE–PPE proteins in their soluble state.
In many Lactobacillales species ( lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in and In conclusion, the elucidation of GAC biosynthesis in reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.
Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of 14 Clabeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and -hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.Methylobacterium extorquens AM1 is a facultative methylotroph that utilizes the serine cycle to assimilate formaldehyde and supply C 3 units for cell biosynthesis (1,30). While most of the reactions in this cycle have been characterized at the gene and/or enzyme level (6-12, 17), one part of the serine cycle that involves regeneration of a molecule of glyoxylate from a molecule of acetyl coenzyme A (acetyl-CoA) still remains unresolved. Earlier work in which the fate of carbon atoms originating from methanol, ethanol, or acetate was monitored indicated that this pathway is also essential for growth of M. extorquens AM1 on C 2 compounds, and chemically induced mutants in this pathway had a characteristic phenotype: they were not able to grow on C 1 or C 2 compounds and were rescued on these compounds by the addition of glyoxylate or glycolate (15,16,30). Later, a region on the chromosome of M. extorquens AM1 containing three genes mutations in which produced this characteristic phenotype was identified. These were the genes for propionyl-CoA carboxylase (pccB), a putative alcohol dehydrogenase later identified as crotonyl-CoA dehydrogenase (ccr [19]), and a mutase similar to methylmalonyl-CoA mutase (MCM) but fulfilling a different and yet unknown function (meaA [12,33]). Orthologs of meaA and crr were also found in Streptomyces species and were shown to be involved in C 2 metabolism (19, 41), suggesting that this pathway might be found outside of serine cycle methylotrophs. In our more recent studies involving genes for poly--hydroxybutyrate (PHB) biosynthesis in M. extorquens AM1, we discovered that two genes essential for the pathway for PHB synthesis from acetyl-CoA, encoding -ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB), are also involved in the glyoxylate regeneration pathway (23). These data indicated that the first step of the pathway must be the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA (catalyzed by PhaA), which is later converted into (R)--hydroxybutyryl-CoA (catalyzed by PhaB), crotonyl-CoA (catalyzed by an R-specific crotonase), and butyryl-CoA (catalyzed by Ccr). Further steps of the pathway leading to propionyl...
SummaryCarcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N-terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgVlike domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.
Coenzyme B 12 (5Ј-deoxyadenosylcobalamin) (AdoCbl)
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