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     

In vitro TRH release from hypothalamus slices varies during the diurnal cycle

We have previously described a daily rhythm in thyrotropin releasing hormone (TRH) and TRH mRNA in the rat hypothalamus. To determine whether TRH release fluctuates in a diurnal manner, we have measured basal and potassium stimulated release from hypothalamic slices, and compared it to release from olfactory bulb slices, during the diurnal cycle. Basal TRH release was higher at 7:00 h than at any other time (1:00, 13:00 or 19:00 h) in either hypothalamus or olfactory bulb. The ratio of stimulated over basal release was higher in the hypothalamus at 19:00 h, when TRH content was highest. Potassium stimulated TRH release from olfactory bulb was not different from basal release at any time. TRH release fluctuations were not due to a rhythm of extracellular inactivation: the activity of pyroglutamyl aminopeptidase II, an ectoenzyme responsible for TRH inactivation, was constant throughout the cycle. Our data demonstrate that diurnal variations of TRH release occur in vitro and that the enhanced responsiveness to potassium stimulation in hypothalamus is correlated with increased levels of peptide.     

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.     

Early stages of vocal ontogeny in the magpie (Pica pica)

Summary The vocal repertoire of magpie (Pica pica) chicks consists of six calls: Begging Trill (BT), Soft Whistle (SW), Begging Scream (BS), Alarm Call (AC), Distress Call (DC) and Brief Contact Note (BCN). Both BT and SW have a tonal structure and their occurrence is restricted to the nestling period. At fledging, there is a gradual change from BT into BS and a sudden appearance of harsh calls similar to those of adult birds (AC, DC, BCN), without evident transitional forms with preceding tonal calls. Both the existence and the structural design of calls seem to be adapted for providing nestlings with immediate benefits linked to the two major chapters of allocation of parental care. Emission rates of BT increase with hunger motivation under laboratory conditions. Their structure suggests that they are easily located but liable to suffer from environmental degradation. BS of fledglings may be more resistant to degradation, a trait which may facilitate the identification by parents of their own offspring. Both AC and DC attract parents to defend the nest against potential predators, and their structure make them to be easily located and detectable at long distances. BCN are given by fledglings during bouts of locomotory activity (exploration and play) and they probably help in maintaining the cohesion of the group under conditions of poor visibility. In accordance, this call may be fairly located at short distances. The function of SW was unclear. It is given during periods of nestling inactivity between begging bouts, and could be easily elicited by tactile and auditory stimuli. After laboratory experiments, it is concluded that SW serve to indicate parents that nestlings are in good condition, hence to benefit from the parental willingness to invest in a brood with high prospects of survival. Since (i) there is a widespread lack of continuity in the development of adult vocalizations starting from nestling calls, and (ii) nestling calls seem to have evolved to provide birds with benefits in the short-term, these facts argue against the prevailing idea that the main function of calls early in ontogeny is to act as precursors of adult vocalizations.

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Zusammenfassung Das Lautrepertoire von Elsternestlingen besteht aus 6 Lautäußerungen: Betteltrillern (BT), Sanftes Pfeifen (SP), Bettelkreischen (BK), Alarmruf (AR), Angstschreien (AS) und kurzer Kontaktruf (KR). BT und SP sind tonal und treten nur während der Nestlingsperiode auf. Zum Zeitpunkt des Ausfliegens geht BT graduell in BK über und plötzlich treten auch ohne vorausgehende tonale Übergangsformen rauhe Rufe ähnlich denen der Altvögel auf (AR, AS, KR). Sowohl die Existenz als auch die Struktur der Lautäußerungen scheinen als Anpassungen im Zusammenhang mit einer Optimierung der Brutpflege zu interpretieren zu sein. Die Emissionsrate von BT steigt unter Laborbedingungen mit der Hungermotivation. Die Struktur legt nahe, daß BT leicht geortet werden kann, aber auch leicht von der Umgebung verschluckt wird. BS der flüggen Jungen scheint davon weniger betroffen zu sein, so daß die Eltern leichter ihre Jungen identifizieren können. AR und AS veranlassen die Altvögel, ihr Nest gegenüber potentiellen Prädatoren zu verteidigen; die Struktur der Laute begünstigt ihre Ortung und Wahrnehmung über größere Entfernungen. KR werden von den flüggen Jungen bei lebhafter lokomotorischer Aktivität (Exploration, Spiel) geäußert; sie tragen vielleicht dazu bei, die Gruppe auch unter erschwerten optischen Kontaktmöglichkeiten zusammenzuhalten. Die Funktion von SP blieb unklar. SP war während inaktiver Perioden zwischen Bettelverhalten zu hören und konnte leicht durch taktile und akustische Reize ausgelöst werden. Nach Laborexperimenten ist zu schließen, daß SP den Eltern Wohlbefinden der Jungen anzeigt und so Bereitschaft zur Investition in eine Brut mit hoher Überlebenswahrscheinlichkeit fördert. Daß (1) zwischen den Rufen der Altvögel und dem Repertoire der Nestlingen weitgehend keine kontinuierlichen Übergänge festzustellen und (2) Nestlingsrufe offensichtlich im Hinblick auf kuzfristige Gewinne zu interpretieren sind, spricht gegen die allgemein vorherrschende Ansicht, das Lautrepertoire in frühen Stadien der Ontogenie sei hauptsächlich ein Vorläufer der Adultlaute.

     

Flavodoxin overexpression confers tolerance to oxidative stress in beneficial soil bacteria and improves survival in the presence of the herbicides paraquat and atrazine

Aim

To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine.

Methods and Results

The model bacterium Escherichia coli, the symbiotic nitrogen‐fixing bacterium Ensifer meliloti and the plant growth‐promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species‐inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents.

Conclusions

Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro‐organisms.

Significance and Impact of the study

The use of plant growth‐promoting rhizobacteria or nitrogen‐fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin‐expressing bacteria to atrazine and paraquat points to potential applications in herbicide‐treated soils.     

Community dynamics of nematodes after Larsen ice‐shelf collapse in the eastern Antarctic Peninsula

Free‐living marine nematode communities of the Larsen B embayment at the eastern Antarctic Peninsula were investigated to provide insights on their response and colonization rate after large‐scale ice‐shelf collapse. This study compares published data on the post‐collapse situation from 2007 with new material from 2011, focusing on two locations in the embayment that showed highly divergent communities in 2007 and that are characterized by a difference in timing of ice‐shelf breakup. Data from 2007 exposed a more diverse community at outer station B.South, dominated by the genus Microlaimus. On the contrary, station B.West in the inner part of Larsen B was poor in both numbers of individuals and genera, with dominance of a single Halomonhystera species. Re‐assessment of the situation in 2011 showed that communities at both stations diverged even more, due to a drastic increase in Halomonhystera at B.West compared to relatively little change at B.South. On a broader geographical scale, it seems that B.South gradually starts resembling other Antarctic shelf communities, although the absence of the genus Sabatieria and the high abundance of Microlaimus still set it apart nine years after the main Larsen B collapse. In contrast, thriving of Halomonhystera at B.West further separates its community from other Antarctic shelf areas.