Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.     

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.     

The Radical SAM Superfamily

ABSTRACT

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]^{1 +} –SAM complex to [4Fe–4S]^{2 +}–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.