Conventional taxonomy of lactobacilli surviving radurization of meat

All of the 113 catalase-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium. The majority of the strains (100) were identified as Lactobacillus sake. These were divided into four sub-groups based on their sugar fermentation pattern: group IA1 (melibiose (+), maltose (-), amygdalin (-), 76 strains); group IA2 (melibiose (+), maltose (-), amygdalin (+), 14 strains); group IB1 (melibiose (+), maltose (+), amygdalin (+), four strains); group IB2 (melibiose (+), maltose (+), amygdalin (-), six strains). Of the remaining strains, two produced L(+)-lactic acid and were identified as L. farciminis, three were identified as L. curvatus and eight showed characteristics of both L. sake and L. curvatus and were designated 'L. sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested. Most L. sake strains produced significant amounts of H2O2. Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance. The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L. sake species in radurized meat are discussed.     

Circadian rhythmicity and photoperiodism in the pitcher-plant mosquito: can the seasonal timer evolve independently of the circadian clock?

The two major rhythms of the biosphere are daily and seasonal; the two major adaptations to these rhythms are the circadian clock, mediating daily activities, and the photoperiodic timer, mediating seasonal activities. The mechanistic connection between the circadian clock and the photoperiodic timer remains unresolved. Herein, we show that the rhythmic developmental response to exotic light:dark cycles, usually used to infer a causal connection between the circadian clock and the photoperiodic timer, has evolved independently of the photoperiodic timer in the pitcher-plant mosquito Wyeomyia smithii across the climatic gradient of eastern North America from Florida to Canada and from the coastal plain to the mountains. We conclude that the photoperiodic timing of seasonal events can evolve independently of the daily circadian clock.     

Glutathione Participates in the Regulation of Mitophagy in Yeast

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4) that target namely peroxisomes (5, 6), chromatin (7, 8), endoplasmic reticulum (9), ribosomes (10), and mitochondria (3, 11–13). Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14–16). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS⁴ production (17). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18) caused by the loss of efficiency of the maintenance process (degradation/biogenesis de novo) is often considered as a major cause of cellular aging (19–23). In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following induction/blockade of apoptosis (23), suggesting that autophagy of mitochondria was required for cell survival following mitochondria injury (14). Consistent with this idea, a direct alteration of mitochondrial permeability properties has been shown to induce mitochondrial autophagy (13, 24, 25). Furthermore, inactivation of catalase induced the autophagic elimination of altered mitochondria (26). In the yeast Saccharomyces cerevisiae, the alteration of F₀F₁-ATPase biogenesis in a conditional mutant has been shown to trigger autophagy (27). Alterations of mitochondrial ion homeostasis caused by the inactivation of the K⁺/H⁺ exchanger was shown to cause both autophagy and mitophagy (28). We have reported that treatment of cells with rapamycin induced early ROS production and mitochondrial lipid oxidation that could be inhibited by the hydrophobic antioxidant resveratrol (29). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30): these authors showed that starvation stimulated ROS production, namely H₂O₂, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H₂O₂. This provided a possible connection between the mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3, 12). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3, 12, 32, 33). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33).The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria.     

A major QTL for resistance against Fusarium head blight in European winter wheat

We report on the verification of a resistance quantitative trait locus (QTL) on chromosome 1BL (now designated Qfhs.lfl-1BL) which had been previously identified in the winter wheat cultivar Cansas. For a more precise estimation of the QTL effect and its influence on plant height and heading date lines with a more homogeneous genetic background were created and evaluated in four environments after spray inoculation with Fusarium culmorum. Qfhs.lfl-1BL reduced FHB severity by 42% relative to lines without the resistance allele. This QTL did not influence plant height, but significantly delayed heading date by one day. All of the most resistant genotypes of the verification population carried this major QTL displaying its importance for disease resistance. This resistance QTL has not only been found in the cultivar Cansas, but also in the three European winter wheat cultivars Biscay, History and Pirat. A subsequent meta-analysis confirmed the presence of a single QTL on the long arm of chromosome 1B originating from the four mentioned cultivars. Altogether, the results of the present study indicate that Qfhs.lfl-1BL is an important component of FHB resistance in European winter wheat and support the view that this QTL would be effective and valuable in backcross breeding programmes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A human genome structural variation sequencing resource reveals insights into mutational mechanisms

Understanding the prevailing mutational mechanisms responsible for human genome structural variation requires uniformity in the discovery of allelic variants and precision in terms of breakpoint delineation. We develop a resource based on capillary end sequencing of 13.8 million fosmid clones from 17 human genomes and characterize the complete sequence of 1054 large structural variants corresponding to 589 deletions, 384 insertions, and 81 inversions. We analyze the 2081 breakpoint junctions and infer potential mechanism of origin. Three mechanisms account for the bulk of germline structural variation: microhomology-mediated processes involving short (2-20 bp) stretches of sequence (28%), nonallelic homologous recombination (22%), and L1 retrotransposition (19%). The high quality and long-range continuity of the sequence reveals more complex mutational mechanisms, including repeat-mediated inversions and gene conversion, that are most often missed by other methods, such as comparative genomic hybridization, single nucleotide polymorphism microarrays, and next-generation sequencing.     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

The contribution of an hourglass timer to the evolution of photoperiodic response in the pitcher-plant mosquito, Wyeomyia smithii

Abstract Photoperiodism, the ability to assess the length of day or night, enables a diverse array of plants, birds, mammals, and arthropods to organize their development and reproduction in concert with the changing seasons in temperate climatic zones. For more than 60 years, the mechanism controlling photoperiodic response has been debated. Photoperiodism may be a simple interval timer, that is, an hourglasslike mechanism that literally measures the length of day or night or, alternatively, may be an overt expression of an underlying circadian oscillator. Herein, we test experimentally whether the rhythmic response in Wyeomyia smithii indicates a causal, necessary relationship between circadian rhythmicity and the evolutionary modification of photoperiodic response over the climatic gradient of North America, or may be explained by a simple interval timer. We show that a day-interval timer is sufficient to predict the photoperiodic response of W. smithii over this broad geographic range and conclude that rhythmic responses observed in classical circadian-based experiments alone cannot be used to infer a causal role for circadian rhythmicity in the evolution of photoperiodic time measurement. More importantly, we argue that the pursuit of circadian rhyth-micity as the central mechanism that measures the duration of night or day has distracted researchers from consideration of the interval-timing processes that may actually be the target of natural selection linking internal photoperiodic time measurement to the external seasonal environment.     

Cloning and heterologous expression of hematin-dependent catalase produced by Lactobacillus plantarum CNRZ 1228

Lactobacillus plantarum CNRZ 1228 exhibited heme-dependent catalase activity under environmental conditions similar to those encountered during sausage fermentation. The 1,455-bp catalase gene (katL) was cloned and encoded a protein of 484 amino acids. Expression of katL in a heterologous host showed that katL encodes a functional catalase. PCR screening of selected strains of lactic acid bacteria for katL indicated the presence of similar genes in other strains of lactobacilli.