Microbiota dynamics and diversity at different stages of industrial processing of cocoa beans into cocoa powder

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P < 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and Enterobacteriaceae (1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the Bacillaceae, Pseudomonadaceae, and Enterococcaceae. Eleven species of ThrS were found, but Bacillus licheniformis and the Bacillus subtilis complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing. B. subtilis complex members, particularly B. subtilis subsp. subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.     

Enhancement of the enantioselectivity of carboxylesterase A by structure-based mutagenesis

Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective CesB does not process IPG-butyrate and exhibits several-fold lower activity than CesA towards IPG-caprylate. A sequence and structure comparison allowed us to identify active site residues that may cause the difference in (enantio)selectivities of CesA and CesB towards these IPG esters. This structure-based approach led to the identification of two active site residues in CesA (F166 and F182), as promising candidates for mutagenesis in order to enhance its enantioselectivity. Mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity and without significant loss of catalytic activity. For IPG-butyrate, a CesA double mutant F166V/F182C (ER=13) was generated showing a ～13-fold increased enantioselectivity as compared to wild-type CesA (E=1). For IPG-caprylate, we designed a CesA double mutant F166V/F182Y (ER=9) displaying a ～5-fold increased enantioselectivity as compared to the wild-type enzyme (ER=2). These findings, combined with the results of molecular docking experiments, demonstrate the importance of residues at positions 166 and 182 for the enantioselectivity of CesA, and may contribute to the development of efficient biocatalysts.     

Identification and Risk Assessment for Worldwide Invasion and Spread of Tuta absoluta with a Focus on Sub-Saharan Africa: Implications for Phytosanitary Measures and Management

To support management decisions, molecular characterization of data and geo-reference of incidence records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) were combined with data on the biology and ecology of the pest to estimate its climatic suitability and potential spread at regional and global scale. A CLIMEX model was developed and used for the global prediction of current and future climate-induced changes in the distributional shifts of T. absoluta. Results revealed that temperature and moisture characterized T. absoluta population growth while the pest ability to survive the cold, hot, wet and dry stress conditions are the primary characteristics defining its range frontiers. Simulated irrigation also played an important role in the model optimization. Model predictions suggest that T. absoluta represents an important threat to Africa, Asia, Australia, Northern Europe, New Zealand, Russian Federation and the United States of America (USA). Under climate change context, future predictions on distribution of T. absoluta indicated that the invasive nature of this pest will result in significant crop losses in certain locations whereas some parts of Africa may witness diminution in ranges. The following scenarios may occur: 1) T. absoluta damage potential may upsurge moderately in areas of Africa where the pest currently exists; 2) a range diminution in temperate to Sahel region with moderate upsurge in damage potential; 3) a range expansion in tropical Africa with reasonable upsurge of damage potential. These possible outcomes could be explained by the fact that the continent is already warm, with the average temperature in majority of localities near the threshold temperatures for optimal development and survival of T. absoluta. Outputs from this study should be useful in helping decision-makers in their assessment of site-specific risks of invasion and spread of T. absoluta with a view to developing appropriate surveillance, phytosanitary measures and management strategies.     

Detecting the Influence of Initial Pioneers on Succession at Deep-Sea Vents

Deep-sea hydrothermal vents are subject to major disturbances that alter the physical and chemical environment and eradicate the resident faunal communities. Vent fields are isolated by uninhabitable deep seafloor, so recolonization via dispersal of planktonic larvae is critical for persistence of populations. We monitored colonization near 9°50′N on the East Pacific Rise following a catastrophic eruption in order to address questions of the relative contributions of pioneer colonists and environmental change to variation in species composition, and the role of pioneers at the disturbed site in altering community structure elsewhere in the region. Pioneer colonists included two gastropod species: Ctenopelta porifera, which was new to the vent field, and Lepetodrilus tevnianus, which had been rare before the eruption but persisted in high abundance afterward, delaying and possibly out-competing the ubiquitous pre-eruption congener L. elevatus. A decrease in abundance of C. porifera over time, and the arrival of later species, corresponded to a decrease in vent fluid flow and in the sulfide to temperature ratio. For some species these successional changes were likely due to habitat requirements, but other species persisted (L. tevnianus) or arrived (L. elevatus) in patterns unrelated to their habitat preferences. After two years, disturbed communities had started to resemble pre-eruption ones, but were lower in diversity. When compared to a prior (1991) eruption, the succession of foundation species (tubeworms and mussels) appeared to be delayed, even though habitat chemistry became similar to the pre-eruption state more quickly. Surprisingly, a nearby community that had not been disturbed by the eruption was invaded by the pioneers, possibly after they became established in the disturbed vents. These results indicate that the post-eruption arrival of species from remote locales had a strong and persistent effect on communities at both disturbed and undisturbed vents.     

Survival of Staphylococcus aureus ST398 in the Human Nose after Artificial Inoculation

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer.We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2–21 days) than the bovine strain 5062 (median 21 days; range 7–21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.     

The cellular computer DNA: Program or data

The classical metaphor of the genetic program written in the DNA nucleotidic sequences is reconsidered. Recent works on algorithmic complexity and logical properties of computer programs and data are used to question the explanatory value of that metaphor. Structural properties of strings are looked for which would be necessary to apply to DNA sequences if the metaphor is to be taken literally. The notion of sophistication is used to quantify meaningful complexity and to distinguish it from classical computational complexity. In this context, the distinction between program and data becomes relevant and an alternative metaphor of DNA as data to a parallel computing network embedded in the global geometrical and biochemical structure of the cell is discussed. An intermediate picture of an evolving network emerges as the most likely where the output of the cellular computing network can produce, at a different time scale, changes in the structure of the network itself by means of changes in the DNA activity patterns.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.