Post-translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis.

Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection.

Enhanced specialized transduction using recombineering in Mycobacterium tuberculosis.

UNLABELLED: G: enetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. IMPORTANCE: Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.

Absence of membrane phosphatidylcholine does not affect virulence and stress tolerance phenotypes in the opportunistic pathogen Pseudomonas aeruginosa.

During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline.

Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism.

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.

Position and blood supply of the carotid body in a Kenyan population / Posición y suministro sanguíneo del cuerpo carotídeo en una población de Kenia

Position and source of blood supply to the human carotid body displays population variations. These data are important during surgical procedures and diagnostic imaging in the neck but are only scarcely reported and altogether missing for the Kenyan population. The aim of this study was to describe the position and blood supply of the carotid body in a Kenyan population. A descriptive cross-sectional study at the Department of Human Anatomy, University of Nairobi, was designed. 136 common carotid arteries and their bifurcations were exposed by gross dissection. The carotid body was identified as a small oval structure embedded in the blood vessel adventitia. Position and source of blood supply were photographed. Data are presented by tables and macrographs. 138 carotid bodies were identified. Commonest position was carotid bifurcation (75.4 percent) followed by external carotid artery (10.2 percent), internal carotid artery (7.2 percent) and ascending pharyngeal artery (7.2 percent). Sources of arterial blood supply included the carotid bifurcation (51.4 percent), ascending pharyngeal (21.0 percent), external carotid (17.4 percent) and internal carotid (10.2 percent) arteries. Position and blood supply of the carotid body in the Kenyan population displays a different profile of variations from those described in other populations. Neck surgeons should be aware of these to avoid inadvertent injury.

La posición y la fuente de suministro sanguíneo del cuerpo carotídeo humano muestra variaciones en la población. Estos datos son importantes durante los procedimientos quirúrgicos y de diagnóstico por imagen en el cuello, pero son poco informados e inclusive faltan por completo en la población de Kenia. El objetivo de este estudio fue describir la posición y el aporte sanguíneo del cuerpo carotídeo en una población de Kenia. Se diseñó un estudio descriptivo de corte transversal en el Departamento de Anatomía Humana de la Universidad de Nairobi. 136 arterias carótidas comunes y sus bifurcaciones fueron expuestas mediante disección simple. El cuerpo carotídeo fue identificado como una pequeña estructura oval ubicada en la adventicia del vaso sanguíneo. La posición y la fuente de suministro sanguíneo fueron fotografiados. Los datos obtenidos fueron presentados en las tablas y fotomacrografías. 138 cuerpos carotídeos fueron identificados. La posición más frecuente fue la bifurcación carotídea (75,4 por ciento), seguida de la arteria carótida externa (10,2 por ciento), arteria carótida interna (7,2 por ciento) y la arteria faríngea ascendente (7,2 por ciento). Las fuentes de suministro sanguíneo arterial incluyeron la bifurcación carotídea (51,4 por ciento), arteria faríngea ascendente (21,0 por ciento), arteria carótida externa (17,4 por ciento) y arterias carótidas internas (10,2 por ciento). La posición y el suministro sanguíneo del cuerpo carotídeo en la población de Kenia muestra un perfil de variaciones diferente a las descritos en otras poblaciones. Los cirujanos de cuello deben conocer estas variaciones para así evitar lesiones accidentales.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.