We find that genome-wide DNA transfer by conjugation in mycobacteria affords bacteria that reproduce by binary fission the same advantages of sexual reproduction, and may explain the genomic evolution of Mycobacterium tuberculosis.

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gβγ as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl (C) terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Conserved residues in the GRK2 large lobe whose side-chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of twenty, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate activated GPCRs, rhodopsin and the β 2 -adrenergic receptor (β 2 AR). The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non-receptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had 12-fold lower k cat , but no changes in K M , suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated β 2 AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.G protein-coupled receptors (GPCRs), distinguished by their seven transmembrane helices and their ability to couple to heterotrimeric G proteins (Gαβγ), are activated by hormones, neurotransmitters and sensory signals, and regulate many diverse processes. Receptor stimulation catalyzes the binding of GTP to the Gα subunit, allowing Gα and Gβγ to directly † This work was funded by the National Science Foundation grants MCB0315888 and MCB0744739 (to RSM), National Institutes of Health grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N (to JJGT), and a summer undergraduate research fellowship from the Northeast Affiliate of the American Heart Association (to EDB). *To whom correspondence should be addressed. Biology Department, Siena College, Morrell Science Center, 515 Loudon Road, Loudonville, NY 12211, Tel: (518) 783-2462; Fax: (518) ; email: sternemarr@siena.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 May 26. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript modulate effectors such as adenylyl cyclase, phospholipase Cβ, cGMP phosphodiesterase, ion channels and ...

Summary The ESX-1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX-1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX-1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX-1 activity consistently localize to the cell pole, identified by time-lapse fluoro-microscopy as the non-septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein-protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX-1 apparatus in M. smegmatis. This observation illustrates the cross-species conservation of protein interactions governing assembly of ESX-1, as well as polar localization. Importantly, we describe novel non-esx1 encoded proteins that affect ESX-1 activity, that co-localize with ESX-1, and that are required for ESX-1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.