Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103–Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103–Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.     

Mono-ADP-ribosylation of the G protein betagamma dimer is modulated by hormones and inhibited by Arf6

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation.     

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout

We have investigated the morphological effects of a genetic locus, Pgm1- t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.      

Identification and partial characterization of Rhizopus nigricans Gβ proteins and their expression in the presence of progesterone

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gβ subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gβ subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gβ from other filamentous fungi, but not from yeast. The presence of more than one Gβ subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gβ subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gβ subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.     

Ly6Clow and Not Ly6Chigh Macrophages Accumulate First in the Heart in a Model of Murine Pressure-Overload

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6C^low and Ly6C^high macrophages. Ly6C^low macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6C^low but not on Ly6C^high macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6C^low macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6C^low macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.