Contracts, Reference Points, and Competition - Behavioral Effects of the Fundamental Transformation

In this paper we study the role of incomplete ex ante contracts for ex post trade. Previous experimental evidence indicates that a contract provides a reference point for entitlements when the terms are negotiated in a competitive market. We show that this finding no longer holds when the terms are determined in a non-competitive way. Our results imply that the presence of a "fundamental transformation" (i.e., the transition from a competitive market to a bilateral relationship) is important for a contract to become a reference point. To the best of our knowledge this behavioral aspect of the fundamental transformation has not been shown before.

Early Changes in Soil Metabolic Diversity and Bacterial Community Structure in Sugarcane under Two Harvest Management Systems

Preharvest burning is widely used in Brazil for sugarcane cropping. However, due to environmental restrictions, harvest without burning is becoming the predominant option. Consequently, changes in the microbial community are expected from crop residue accumulation on the soil surface, as well as alterations in soil metabolic diversity as of the first harvest. Because biological properties respond quickly and can be used to monitor environmental changes, we evaluated soil metabolic diversity and bacterial community structure after the first harvest under sugarcane management without burning compared to management with preharvest burning. Soil samples were collected under three sugarcane varieties (SP813250, SP801842 and RB72454) and two harvest management systems (without and with preharvest burning). Microbial biomass C (MBC), carbon (C) substrate utilization profiles, bacterial community structure (based on profiles of 16S rRNA gene amplicons), and soil chemical properties were determined. MBC was not different among the treatments. C-substrate utilization and metabolic diversity were lower in soil without burning, except for the evenness index of C-substrate utilization. Soil samples under the variety SP801842 showed the greatest changes in substrate utilization and metabolic diversity, but showed no differences in bacterial community structure, regardless of the harvest management system. In conclusion, combined analysis of soil chemical and microbiological data can detect early changes in microbial metabolic capacity and diversity, with lower values in management without burning. However, after the first harvest, there were no changes in the soil bacterial community structure detected by PCR-DGGE under the sugarcane variety SP801842. Therefore, the metabolic profile is a more sensitive indicator of early changes in the soil microbial community caused by the harvest management system.

A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.

An ammonium chloride procedure was used to prepare a bacterial pellet from positive blood cultures, which was used for direct inoculation of VITEK 2 cards. Correct identification reached 99% for Enterobacteriaceae and 74% for staphylococci. For antibiotic susceptibility testing, very major and major errors were 0.1 and 0.3% for Enterobacteriaceae, and 0.7 and 0.1% for staphylococci, respectively. Thus, bacterial pellets prepared with ammonium chloride allow direct inoculation of VITEK cards with excellent accuracy for Enterobacteriaceae and a lower accuracy for staphylococci.

Bacterial translocation in cirrhotic rats. Its role in the development of spontaneous bacterial peritonitis

Bacterial translocation occurs in ascitic cirrhotic rats, but its association with ascites infection is unknown. The aim of this study was to assess the relation between bacterial translocation and ascites infection in cirrhotic rats. Male Sprague-Dawley rats were induced to cirrhosis with intragastric CCl4. Ascitic fluid, portal and peripheral blood, mesenteric lymph nodes, liver and spleen samples were cultured before death in those cirrhotic rats with less (group A) or more (group B) than 250 polymorphonuclear neutrophils/mm3 in ascitic fluid, as well as in healthy control rats. Histological examination of jejunum, ileum, and caecum was also performed. Bacterial translocation occurred in 45% of ascitic rats (without differences between groups A and B), but in 0% controls (p = 0.01). Bacterial translocation was associated with positive ascitic fluid culture in 60% of the cases. In all of them the same bacterial species was isolated in both mesenteric lymph node and ascitic fluid. Submucosal caecal oedema (100%), ileal lymphangiectasia (41%), and caecal inflammatory infiltrate (41%) occurred in ascitic rats, the last being associated with ascitic fluid positive culture (p = 0.04). These results suggests that bacterial translocation occurs frequently in ascitic cirrhotic rats, and may play a permissive, but not unique, part in a number of ascites infections. Whether histological changes seen in cirrhotic ascitic rats favour bacterial translocation remains to be elucidated.

Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

[Phot. de l'hôtel du prince Roland Bonaparte avant sa transformation, don de l'Abbé Alphonse Bourzeix, le 26 juin 1896]

Donateur : Bourzeix, Alphonse (18..-19..? ; abbé)

Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii.

Penicillin resistance in Streptococcus spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal Streptococcus gordonii. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 microg/ml). The major MIC increase (> or = 35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 microg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (> or = 2 microg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.

Bacterial sensors: synthetic design and application principles

Bacterial reporters are live, genetically engineered cells with promising application in bioanalytics. They contain genetic circuitry to produce a cellular sensing element, which detects the target compound and relays the detection to specific synthesis of so-called reporter proteins (the presence or activity of which is easy to quantify). Bioassays with bacterial reporters are a useful complement to chemical analytics because they measure biological responses rather than total chemical concentrations. Simple bacterial reporter assays may also replace more costly chemical methods as a first line sample analysis technique. Recent promising developments integrate bacterial reporter cells with microsystems to produce bacterial biosensors. This lecture presents an in-depth treatment of the synthetic biological design principles of bacterial reporters, the engineering of which started as simple recombinant DNA puzzles, but has now become a more rational approach of choosing and combining sensing, controlling and reporting DNA 'parts'. Several examples of existing bacterial reporter designs and their genetic circuitry will be illustrated. Besides the design principles, the lecture also focuses on the application principles of bacterial reporter assays. A variety of assay formats will be illustrated, and principles of quantification will be dealt with. In addition to this discussion, substantial reference material is supplied in various Annexes.

Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037.

Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore enhance PAH availability to PAH-degrading bacteria. We tested the effects of two types of biosurfactants produced by Pseudomonas sp., cyclic lipopeptides and rhamnolipids, on phenanthrene bioavailability. Bioavailability was judged from growth rates on phenanthrene and from specific induction of a phenanthrene-responsive GFP-reporter in Burkholderia sartisoli strain RP037. Co-culturing of strain RP037 with the lipopeptide-producing bacterium Pseudomonas putida strain PCL1445 enhanced GFP expression compared to a single culture, but this effect was not significantly different when strain RP037 was co-cultivated with a non-lipopeptide-producing mutant of P. putida. The addition of partially purified supernatant extracts from the P. putida lipopeptide producer equally did not unequivocally enhance phenanthrene bioavailability to strain RP037 compared to controls. In contrast, a 0.1% rhamnolipid solution strongly augmented RP037 growth rates on phenanthrene and led to a significantly larger proportion of cells in culture with high GFP expression. Our data therefore suggest that biosurfactant effects may be strongly dependent on the strain and type of biosurfactant.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A.

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral alpha-hexachlorocyclohexane (alpha-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (-) enantiomer. Concurrent formation and subsequent dissipation of beta-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral alpha-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of alpha-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.

MHC class I expression dependent on bacterial infection and parental factors in whitefish embryos (Salmonidae).

Ecological conditions can influence not only the expression of a phenotype, but also the heritability of a trait. As such, heritable variation for a trait needs to be studied across environments. We have investigated how pathogen challenge affects the expression of MHC genes in embryos of the lake whitefish Coregonus palaea. In order to experimentally separate paternal (i.e. genetic) from maternal and environmental effects, and determine whether and how stress affects the heritable variation for MHC expression, embryos were produced in full-factorial in vitro fertilizations, reared singly, and exposed at 208 degree days (late-eyed stage) to either one of two strains of Pseudomonas fluorescens that differ in their virulence characteristics (one increased mortality, while both delayed hatching time). Gene expression was assessed 48 h postinoculation, and virulence effects of the bacterial infection were monitored until hatching. We found no evidence of MHC class II expression at this stage of development. MHC class I expression was markedly down-regulated in reaction to both pseudomonads. While MHC expression could not be linked to embryo survival, the less the gene was expressed, the earlier the embryos hatched within each treatment group, possibly due to trade-offs between immune function and developmental rate or further factors that affect both hatching timing and MHC expression. We found significant additive genetic variance for MHC class I expression in some treatments. That is, changes in pathogen pressures could induce rapid evolution in MHC class I expression. However, we found no additive genetic variance in reaction norms in our study population.

The influence of bacterial activity on phosphorite formation in the Miocene Monterey Formation, California

Authigenic phosphorites from the Miocene Monterey Formation (California) including an autochthonous phosphatic laminite were analyzed for molecular biomarkers, element content, and sulfur isotopic composition of associated pyrite and sulfate to evaluate the role of bacterial activity in the precipitation of phosphate minerals. The phosphorites formed in a depositional environment typified by upwelling with dynamic bottom currents and hardground formation. Pyrite enclosed in the phosphorites shows delta S-34 values as low as -36.5 parts per thousand VCDT, which is consistent with bacterial sulfate reduction. In a three-step extraction phosphorite dissolution extraction procedure, molecular fossils of sulfate-reducing bacteria (di-O-alkyl glycerol ethers and short-chain branched fatty acids i- and ai-C-15:0, i- and ai-C-17:0, and 10MeC(16:0)) were preferentially released from the mineral lattice. This suggests that the molecular fossils were tightly bound to carbonate fluorapatite, indicating that sulfate-reducing bacteria were involved in mineral formation. A close association of sulfate-reducing bacteria with large sulfide-oxidizing bacteria, which was previously suggested to favor carbonate fluorapatite precipitation, could neither be confirmed nor excluded for the Miocene Monterey Formation phosphorites. (C) 2012 Elsevier B.V. All rights reserved.