Environmentally constrained mutation and adaptive evolution in Salmonella

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3], [4] and [5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7], [8], [9] and [10], or as a consequence of evolutionary processes [11], [12], [13], [14], [15] and [16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that chnages in the mutability of specific loci can be influenced by alterations in DNA topology [10] and [17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not ‘directed’ and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.     

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

Lactate as substrate for glycogen resynthesis after exercise

Muscle glycogen levels in the perfused rat hemicorpus preparation were reduced two-thirds by electrical stimulation plus exposure to epinephrine (10(-7) M) for 30 min. During the contraction period muscle lactate concentrations increased from a control level of 3.6 +/- 0.6 to a final value of 24.1 +/- 1.6 mumol/g muscle. To determine whether the lactate that had accumulated in muscle during contraction could be used to resynthesize glycogen, glycogen levels were determined after 1-3 h of recovery from the contraction period during which time the perfusion medium (flow-through system) contained low (1.3 mmol/l) or high (10.5 or 18 mmol/l) lactate concentrations but no glucose. With the low perfusate lactate concentration, muscle lactate levels declined to 7.2 +/- 0.8 mumol/g muscle by 3 h after the contraction period and muscle glycogen levels did not increase (1.28 +/- 0.07 at 3 h vs. 1.35 +/- 0.09 mg glucosyl U/g at end of exercise). Lactate disappearance from muscle was accounted for entirely by output into the venous effluent. With the high perfusate lactate concentrations, muscle lactate levels remained high (13.7 +/- 1.7 and 19.3 +/- 2.0 mumol/g) and glycogen levels increased by 1.11 and 0.86 mg glucosyl U/g, respectively, after 1 h of recovery from exercise. No more glycogen was synthesized when the recovery period was extended. Therefore, it appears that limited resynthesis of glycogen from lactate can occur after the contraction period but only when arterial lactate concentrations are high; otherwise the lactate that builds up in muscle during contraction will diffuse into the bloodstream.     

Why do anemonefishes inhabit only some host actinians?

Synopsis The 25 species ofAmphiprion and one ofPremnas (family Pomacentridae) are obligate symbionts of 10 species of facultatively symbiotic sea anemones. Throughout the tropical Indo-West Pacific range of the relationship, a fish species inhabits only certain of the hosts potentially available to it. This specificity is due to the fishes. Five fishes occupy six sea anemone species at Lizard Island, Great Barrier Reef, Australia.Entacmaea quadricolor harborsP. biaculeatus, A. melanopus andA. akindynos. Adults ofPremnas occur deeper than about 3 m in large, primarily solitary actinians; juveniles may occupy peripheral members ofEntacmaea clones in shallow water. Specimens ofA. melanopus live exclusively in clonal anemones, which are found no deeper than 3 m. Most individuals ofA. akindynos inEntacmaea are juveniles, occurring shallow and deep, in solitary anemones or at the margins of clones. Interspecific as well as intraspecific social control of growth may be responsible for keeping fish small at clone fringes. Conspicuous specimens ofE. quadricolor depend upon their anemonefish to survive. Actinians cleared of symbionts disappeared within 24 h, probably having been eaten by reef fishes.Entacmaea, the most abundant and widespread host actinian at Lizard Island and throughout the range of the association, is also arguably the most attractive to anemonefishes. I believe its vulnerability to predation was a factor in its evolving whatever makes it desirable to fishes. Experimental transfers pitted fish of one species against those of another, controlling for ecophenotype of host, and sex, size and number of fish. Competitive superiority was in the same order as abundance and over-all host specificity:P. biaculeatus, A. melanopus, A. akindynos. At least three factors are necessary to explain patterns of species specificity - innate or learned host preference, competition, and stochastic processes.     

The Tropic Effect of Intrarectal Deoxycholate On Rat Colorectum Is Unaffected By Oral Metronidazole

Abstract. Intrarectal administration of sodium deoxycholate (SDC) enhances experimental colorectal carcinogenesis, an effect that is partly vitiated by oral metronidazole. the effect of topical SDC with or without concurrent metronidazole on colorectal cell proliferation was explored in male Sprague-Dawley rats ( n = 30) allocated to five groups. Two groups received thrice weekly intrarectal instillations of 1 ml N saline or 1 ml 0.12 m SDC. A third group received SDC plus metronidazole 22.5 mg/kg/day in the drinking water. Controls had no instillations or metronidazole alone. At time of killing (10 weeks), crypt cell production rate (CCPR) was determined by the stathmokinetic technique for four large-bowel segments. Saline had no significant effect on colorectal CCPR but SDC produced increases throughout, varying from 53% in the proximal colon to 222% in the rectum ( P < 0.01). Metronidazole did not reduce this effect, although given alone it reduced colonic CCPR by 40 to 50%. the direct tropic effect of bile acids could largely explain their cocarcinogenic properties. Since metronidazole does not prevent this increase in cell proliferation, its mildly protective role against cancer may reflect the presence of fewer anaerobes capable of degrading bile acids to carcinogenic metabolites.     

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Extended models of the ventilatory response to sustained isocapnic hypoxia in humans

Liang, Pei-Ji, Daphne A. Bascom, and Peter A. Robbins.Extended models of the ventilatory response to sustained isocapnic hypoxia in humans. J. Appl. Physiol. 82(2): 667-677, 1997.The purpose of this study was to examine extensions of a modelof hypoxic ventilatory decline (HVD) in humans. In the original model (model I) devised by R. Painter, S. Khamnei, and P. Robbins(J. Appl. Physiol. 74: 2007-2015, 1993), HVD is modeledentirely by a modulation of peripheral chemoreflex sensitivity. In thefirst extension (model II), a more complicated dynamic is usedfor the change in peripheral chemoreflex sensitivity. In the secondextension (model III), HVD is modeled as a combination ofboth the mechanism of Painter et al. and a component that isindependent of peripheral chemoreflex sensitivity. In all cases, aparallel noise structure was incorporated to describe the stochasticproperties of the ventilatory behavior to remove the correlation of theresiduals. Data came from six subjects from a study by D. A. Bascom, J. J. Pandit, I. D. Clement, and P. A. Robbins (Respir. Physiol.88: 299-312, 1992). For model II, there was a significantimprovement in fit for two out of six subjects. The reasons for thiswere not entirely clear. For model III, the fit was againsignificantly improved in two subjects, but in this case the subjectswere those who had the most marked undershoot and recovery ofventilation at the relief of hypoxia. In these two subjects, thechemoreflex-independent component contributed ~50% to total HVD.In the other four subjects, the chemoreflex-independent componentcontributed ~10% to total HVD. It is concluded that in somesubjects, but not in others, there may be a component of HVD thatis independent of peripheral chemoreflex sensitivity.