Microbial Lifestyle and Genome Signatures

Microbes are known for their unique ability to adapt to varying lifestyle and environment, even to the extreme or adverse ones. The genomic architecture of a microbe may bear the signatures not only of its phylogenetic position, but also of the kind of lifestyle to which it is adapted. The present review aims to provide an account of the specific genome signatures observed in microbes acclimatized to distinct lifestyles or ecological niches. Niche-specific signatures identified at different levels of microbial genome organization like base composition, GC-skew, purine-pyrimidine ratio, dinucleotide abundance, codon bias, oligonucleotide composition etc. have been discussed. Among the specific cases highlighted in the review are the phenomena of genome shrinkage in obligatory host-restricted microbes, genome expansion in strictly intra-amoebal pathogens, strand-specific codon usage in intracellular species, acquisition of genome islands in pathogenic or symbiotic organisms, discriminatory genomic traits of marine microbes with distinct trophic strategies, and conspicuous sequence features of certain extremophiles like those adapted to high temperature or high salinity.     

Signaling from the secretory granule to the nucleus

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40-60 nm vesicles carry conventional or small molecule neurotransmitters, and 200-400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.     

An immunochemical method for detection and analysis of changes in methylome

DNA methylation is an important epigenetic modification involved in the ability of an organism to respond to stress and adaptation. It has been implicated in development, differentiation, oncogenesis, chromatin remodelling, nutrigenomics, and appears to play a pivotal role in many regulatory and adaptive functions. It is therefore important to analyze the status of DNA methylation and its changes under various developmental, carcinogenic, pharmacological, and environmental conditions. In this report we describe an immunochemical method for the detection of genome wide DNA methylation and its alterations under various conditions along with the analysis of DNA methyltransferase activity. The ability of this approach to detect and provide a map of methylomic changes in a genome facilitates assessment of various agents and conditions which can alter this important epigenetic signal. This experimental system permits rapid evaluation of potential target genes which would be modulated by DNA methylation changes and thus the gene networks that govern the processes.     

Sialoglycosylation of RBC in Visceral Leishmaniasis Leads to Enhanced Oxidative Stress, Calpain-Induced Fragmentation of Spectrin and Hemolysis

Visceral leishmaniasis (VL) caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC) of VL patients (RBC(VL)) express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) that are not detected in the RBC of healthy individuals (RBC(N)). At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBC(VL) but not RBC(N) to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBC(VL) which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBC(VL) which are involved in the hemolysis related to the anemia which develops in VL patients.     

Phosphoenolpyruvate Carboxylase Purified from Leaves of C3, C4, and C3-C4 intermediate species of Alternanthera: Properties at Limiting and Saturating Bicarbonate

Phosphoenolpyruvate carboxylase (PEPC) was purified from leaves of four species of Alternanthera differing in their photosynthetic carbon metabolism: Alternanthera sessilis (C₃), A. pungens (C₄), A. ficoides and A. tenella (C₃-C₄ intermediates or C₃-C₄). The activity and properties of PEPC were examined at limiting (0.05 mM) or saturating (10 mM) bicarbonate concentrations. The V_max as well as K_m values (for Mg²⁺ or PEP) of PEPC from A. ficoides and A. tenella (C₃-C₄ intermediates) were in between those of C₃ (A. sessilis) and C₄ species (A. pungens). Similarly, the sensitivity of PEPC to malate (an inhibitor) or G-6-P (an activator) of A. ficoides and A. tenella (C₃-C₄) was also of intermediate status between those of C₃ and C₄ species of A. sessilis and A. pungens, respectively. In all the four species, the maximal activity (V_max), affinity for PEP (K_m), and the sensitivity to malate (K_I) or G-6-P (K_A) of PEPC were higher at 10 mM bicarbonate than at 0.05 mM bicarbonate. Again, the sensitivity to bicarbonate of PEPC from C₃-C₄ intermediates was in between those of C₃- and C₄-species. Thus the characteristics of PEPC of C₃-C₄ intermediate species of Alternanthera are intermediate between C₃- and C₄-type, in both their kinetic and regulatory properties. Bicarbonate could be an important modulator of PEPC, particularly in C₄ plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily

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Cloning of a cryptochrome homologue from the holoparasitic plant Orobanche minor Sm.

Orobanche minor is a non-photosynthetic root holoparasitic plant. Although it is known that photosynthesis-related genes are inactivated or have been eliminated from the plastid genomes of holoparasites, little is known about the alterations in their genes involved in the signaling networks by which light regulates photosynthesis. Cryptochromes (crys), which are blue-light receptors, appear to control both photosynthesis-related and non-photosynthetic responses to light in higher plants. Because we are interested in to what extent a cry-mediated light signaling network remains in the holoparasites, we cloned CRY homologous cDNA from O. minor (OmCRY1) and used real-time RT-PCR to compare its expression under natural daylight and darkness. We found that the OmCRY1 has a high degree of homology with CRY1 s from photosynthetic plants. Expression of the OmCRY1 gene was higher in plants grown in the dark than that in the plants grown under natural daylight. This is the first report of the gene expression of a blue-light receptor in non-photosynthetic plants.     

Phenotypic consequences of transforming growth factor β1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis

The profound effects of transforming growth factor β1 (TGF-β1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-β1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-β1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-β1 null mice (TGF-β1^−/−). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-β1^+/+) and TGF-β1 null mice were compared by cell counting, by ³H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-α1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-β1^+/+ and TGF-β1^−/− embryonic fibroblasts. We report that TGF-β1^−/− cells proliferated at about twice the rate of TGF-β1^+/+ cells. Further, TGF-β1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-α1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-β1^+/+ and TGF-β1^−/− cells could be eliminated by treatment of TGF-β1^+/+ cells with a neutralizing antibody of TGF-β1. Thus, our results are consistent with the hypothesis that TGF-β1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts. 176:67–75, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.