Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

HETEROGENEOUS GENOMIC DIFFERENTIATION IN MARINE THREESPINE STICKLEBACKS: ADAPTATION ALONG AN ENVIRONMENTAL GRADIENT

Evolutionary divergence among populations occupying ecologically distinct environments can occur even in the face of on‐going gene flow. However, the genetic underpinnings, as well as the scale and magnitude at which this differentiation occurs in marine habitats are not well understood. We investigated the patterns and degree of genomic heterogeneity in threespine sticklebacks (Gasterosteus aculeatus) by assessing genetic variability in 20 nongenic and 20 genic (associated with genes important for freshwater adaptation) microsatellite loci in samples collected from 38 locations spanning the entire Baltic Sea coast to the North Sea boundary. Population divergence (F_ST ≈ 0.026) and structuring (five genetic clusters) was significantly more pronounced in the genic as compared to nongenic markers (F_ST ≈ 0.008; no genetic clusters). Patterns of divergence in the genic markers—45% of which were identified as outliers—correlated with local differences in salinity. Yet, a strong positive correlation between divergence in genic and nongenic markers, and their association with environmental factors suggests that adaptive divergence is reducing gene flow across the genome. Apart from providing a clear demonstration of heterogeneous genomic patterns of differentiation in a marine species, the results are indicative of adaptive population structuring across the relatively young Baltic Sea in spite of ample opportunities for gene flow.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.     

Oxygen consumption of the aquatic leaf beetles Macroplea mutica and Macroplea appendiculata is low and not influenced by salinity

Leaf beetles of the genus Macroplea live permanently under water. Species‐specific preferences for either freshwater or brackish water are available in the literature. To detect potential physiological differences, the oxygen consumption of Macroplea mutica and Macroplea appendiculata from habitats with differing salinities is measured at two different salinities (0 and 10). The specific oxygen consumption does not depend on oxygen saturation of the water (values in the approximate range of 25–100% occur during the experiments). There is no difference between species or sampling locations. Similarly, the salinity during the measurements does not affect the oxygen consumption of the beetles, either when compared as salinity per se (0 versus 10), or when classified as home salinity or atypical salinity. Comparisons with other chrysomelid beetles and aquatic insects (using available published data) reveal that the two Macroplea species have relatively low metabolic rates. This finding is discussed in the light of activity patterns and morphology, especially the reduction of flight muscles, which comprise a highly metabolically active tissue.     

Production of piscicolin 126 by Carnobacterium maltaromaticum UAL26 is controlled by temperature and induction peptide concentration

Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25°C, but at temperatures less than 19°C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25°C. Bacteriocin production in UAL26 grown at 15°C can be induced by addition of 10⁻¹⁰ M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25°C requires 10⁻⁷ M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Larval development and emergence sites of farm‐associated Culicoides in the United Kingdom

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of internationally important arboviruses of livestock including bluetongue virus (BTV). Information on the habitats used by Culicoides for larval development is valuable for establishing targeted vector control strategies and for improving local scale models of vector abundance. This study combines emergence trap collections of adult Culicoides identified using molecular markers and physiochemical measurements of habitats to investigate larval development sites of Palaearctic Culicoides in South East England. The known range of larval habitats for several Culicoides species is extended and the potential BTV vector species C. obsoletus and C. scoticus are confirmed to co‐occur in many larval habitats. The presence of emerging C. obsoletus was favoured by increasing substrate moisture level [odds ratio (OR) 6.94 (2.30; 20.90)] and substrate pH [OR 4.80 (1.66; 13.90)] [bias‐corrected D_xy: 0.68; area under the curve (AUC): 0.86] rather than any particular larval habitat type, as expected for a species with relatively wide larval habitat preference. Of the newly emerged sub‐genus Avaritia individuals collected, 23% were observed to have a degree of abdominal pigmentation commonly inferred to indicate parity. If consistent across species and locations, this observation represents a potential source of error for age structure analyses of Culicoides populations.     

Thermoregulation and activity patterns of two syntopic cicadas,Tibicen chiricahua and T. duryi (Homoptera: Cicadidae), in central New Mexico

Summary Tibicen chiricahua and T. duryi are cicada species that are active as adults early each summer in central New Mexico, and are often syntopic in pinyon-juniper woodlands. Both species regulate thoracic temperature (T_th) within fairly narrow limits by utilizing behavioral mechanisms and evaporative cooling. However, syntopic populations of these two species were found to regulate at different T_th despite having synchronous annual and daily activity periods; overall mean T_th of T. chiricahua was 3.1°C higher than it was for T. duryi. Interspecific differences in evaporative cooling abilities and rates of passive heat exchange could not account for this difference in T_th. Part of the difference in T_th resulted from the fact that individuals of the two species were active in thermally distinct microhabitats. Within each species, mean T_th varied among behavior categories, and differences in how the two species allocated their time between activities also contributed to the interspecific difference in T_th. Though T. duryi is restricted to pinyon-juniper habitats such as the one in this study, T. chiricahua is also found in warmer habitats. The difference in T_th in the syntopic populations probably reflects interspecific differences in thermal preferences and thermal optima that are adaptive over their respective habitat ranges. The degree of dependence of T_th on ambient temperature (T_am) varied between activities within both species, with the least dependence exhibited during singing. Singing involves intense activity of tymbal muscles, which apparently can only function effectively over a relatively narrow range of temperature.     

Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml⁻¹), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0). Received 02 December 1996/ Accepted in revised form 25 August 1997     

Purification and characterization of a bacteriocin from Lactobacillus rhamnosus L34

Bacteriocins are ribosomally synthesized peptides having considerable potential as a food preservative because of their strong antagonistic activity against many food spoilage and pathogenic organisms. A bacteriocin from Lactobacillus rhamnosus isolates was purified using ammonium sulphate precipitation and molecular exclusion chromatography techniques. Ammonium sulphate precipitation resulted in higher yield of bacteriocin, but the specific activity and fold purification were higher for molecular exclusion chromatography. The bacteriocin exhibited inhibition against food-borne pathogens and spoilage microorganisms, including both Gram-positive and -negative bacteria. Amylase, lipase and catalase did not alter the antimicrobial activity but proteolytic enzymes inactivated the bacteriocin. It was heat stable and exhibited activity in a pH range of 2–8 with maximum activity at pH 5.0. Molecular weight of bacteriocin was found to be ~5.6 kDa using SDS-PAGE. HPLC profile showed a single peak further attesting the purity of the bacteriocin.     

Resistance to freshwater exposure in White Sea Littorina spp. II: Acid-base regulation

Parameters of acid-base and energy status were studied by in vivo ³¹P-nuclear magnetic resonance spectroscopy in three White Sea Littorina spp. (L.littorea, L. saxatilis and L. obtusata) during prolonged anaerobiosis in freshwater. Intracellular pH decreased significantly, especially during the early period of anaerobiosis, but later the decrease in intracellular pH slowed down considerably, suggesting a capacity for intracellular pH regulation in all three species. There was a trend for intracellular pH to fall most rapidly in the least freshwater-resistant species, L. obtusata, as compared to the most resistant, L. littorea. Non-bicarbonate, non-phosphate buffer values estimated by the homogenate technique were similar in the three studied species (28–37 mmol pH⁻¹ kg⁻¹ wet weight) and did not change during freshwater exposure. The CaCO₃ buffer value of the foot tissues was considerably higher (171–218 mmol pH⁻¹ kg⁻¹ wet weight) and decreased significantly during freshwater exposure. The contribution of the multiple tissue buffering systems to intracellular pH regulation in Littorina spp. shifts between different stages of freshwater exposure. Initially, the non-bicarbonate, non-phosphate tissue buffering system seems to be of major importance for metabolic proton buffering at intracellular pH between 7.5 and 7.0. During later stages of anaerobiosis and at lower intracellular pH, the CaCO₃ buffer is involved in proton buffering. Decrease in the CaCO₃ buffer value during freshwater exposure was in quantitative agreement with the amount of metabolic protons buffered, thus suggesting that CaCO₃ tissue stores may serve as a major buffering system during prolonged anaerobiosis in Littorina spp. Accepted: 23 December 1999     

DNA SEQUENCE VARIATION IN THE RIBOSOMAL INTERNAL TRANSCRIBED SPACER REGION OF FRESHWATER CLADOPHORA SPECIES (CHLOROPHYTA)1

Freshwater species of Cladophora (Chlorophyta) are globally distributed and occupy an unusually wide range of ecological habitats. Delineating species is difficult because most easily observed morphological traits are highly variable and because sexual reproduction has not been clearly documented. Synthesizing ecological data on freshwater Cladophora species is problematic because it is unclear whether freshwater Cladophora species comprise many genetically distinct species or a few ecologically and morphologically variable and/or plastic species. We determined nucleotide sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron of freshwater Cladophora species from a wide range of habitats and geographic locations. We compared these sequences to those derived from culture collections of C. fracta and C. glomerata, the two most commonly reported freshwater Cladophora species. Cladophora fracta and C. glomerata had very similar ITS sequences (95.3%). All other sequences were identical to those from the C. fracta or C. glomerata culture collections with the exception of one California sample that was similar to both C. fracta (95.6%) and C. glomerata (92.4%). ITS genotypes did not correlate with morphology or geography. This analysis shows that common freshwater Cladophora species comprise very few (possibly one) ecologically and morphologically variable species.     

High abundance of Trochospongilla horrida (Porifera, Spongillidae) in the Rhine (Germany) 1992–1995

Trochospongilla horrida (Porifera, Spongillidae) was the dominant freshwater sponge in the Rhine between Karlsruhe and Bonn between 1992 and 1995 are reported. It showed an annual life cycle, with an inactive phase during winter. The life cycle was controlled by temperature. Gemmules were produced in high numbers, and played a significant role as resting bodies. Their role as distribution units was negligible because they were tightly fixed to the substratum. Trochospongilla horrida preferred relatively stable habitats, not affected by fluctuations in water level; it was restricted to habitats with a slightly alkaline pH. Laboratory experiments showed a delayed germination of the gemmules, which explains why the highest abundance of the species occured considerably after the first hatching colonies.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

Winter tourism increases stress hormone levels in the Capercaillie Tetrao urogallus

Montane and alpine habitats in Europe remained relatively undisturbed until the beginning of the last century. Today, outdoor recreation activities are a major economic factor in alpine regions. Many tourism areas coincide with winter habitats of shy and endangered species. The Western Capercaillie Tetrao urogallus has suffered from rapid population declines during recent decades over much of its range. In central Europe, many Capercaillie are exposed to intensive human recreation activities in their habitats, which may contribute to this decline. However, little is known about their susceptibility to human recreation activities. This study assessed whether human recreation in winter evokes physiological stress responses in several populations of Capercaillie. During two winters, we sampled 1130 Capercaillie droppings in Germany and Switzerland of populations at various distances from winter recreation activities and measured concentrations of faecal corticosterone metabolites. Capercaillie in relatively dense and homogeneous mountain forests dominated by Norway Spruce Picea abies showed markedly increased stress hormone levels closer to locations with winter recreation activity. However, this physiological response to human recreation was not detectable in forests dominated by various pine species and a heterogeneous structure. Capercaillie may be particularly sensitive to recreation because any factor affecting their fine‐tuned physiological and behavioural adaptations to survive under harsh winter conditions may lead to harmful fitness costs.     

Nitrification in Dutch heathland soils

A survey was conducted over a range of 17 Dutch heathland locations, subdivided into 41 sites dominated by either dwarf-shrubs (Calluna vulgaris or Erica tetralix) or grass species (Deschampsia flexuosa or Molinia caerulea). Among the habitats of the dominant plant species relatively little differences in general soil properties were observed. The P status of Deschampsia sites was relatively high as well as the NO₃ ^–-N concentrations in the 0–10 cm layer (FH included) at the grass-dominated sites. At sites with a dead or degenerating dwarf-shrub vegetation, NH₄ ⁺-N concentrations reached very high levels.Net production of nitrate was observed during incubation of intact 0–10 cm soil cores (FH-layer included) in the laboratory for all sites, even though in some instances, particularly at Calluna and Erica sites, no nitrate was initially measured. Generally, a higher nitrification rate was found for the grass-dominated sites, and for Deschampsia in particular. The net production of nitrate was highly significantly correlated with net N mineralization, being a reasonable predictor of nitrification in a simple regression model (R²=0.47; P<0.001). Net nitrification was also significantly correlated with the NO₃ ^–-N initially present at the start of the growing season (R=0.65; P<0.001) and with the labile organic P content of the soil (R=0.65; P<0.001). By including initial NO₃ ^–-N and labile organic P, together with net N mineralization and pH, in a multiple regression model, net nitrate production could be predicted with a much higher precision (R²=0.75; P<0.001). Although apparent nitrification was not significantly correlated with pH, the latter contributed significantly to the multiple regression equation for the prediction of the former.The influence of the labile organic P pool may act via its positive correlation with microbial biomass, thus more or less reflecting the potential mineralization/nitrifying capacity of a particular site.     

Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.     

Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by Carnobacterium maltaromaticum UAL307

Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Chronic effects of low insecticide concentrations on freshwater caddisfly larvae

The insecticide load in surface waters does not ordinarily reach concentrations acutely toxic to aquatic fauna. The effects of the low insecticide concentrations typical of natural habitats are still not clear, for they often appear only after relatively long exposure times. To test such a situation, the insecticides lindane and parathion were introduced into a static-with-renewal outdoor aquaria system at concentrations about four and five orders of magnitude lower than their respective 96-h LC₅₀s, and their chronic (about 90 days) effects on the survival rate of freshwater caddisfly larvae were observed. The emergence and hence survival rate of Limnephilus lunatus Curtis was significantly reduced by lindane at 0.1 ng l^–1, a value nearly five orders of magnitude lower than the 96-h LC₅₀. Parathion, with acute and subacute toxicity similar to that of lindane, did not significantly alter the emergence rate of this species. In contrast, this substance did produce a significant reduction in emergence rate of the closely related species Limnephilus bipunctatus Curtis at 1 ng l^–1, even though this species was significantly less susceptible than L. lunatus to parathion at high concentrations. We conclude that chronic insecticide exposure can be hazardous to freshwater macroinvertebrates even at unexpectedly low concentrations. The low-concentration effects may depend on both species and substance and therefore cannot be predicted from toxicity data at higher concentrations.