Innovations in food preservation in pastoral zones.

Food preservation makes a significant contribution to food security and food safety in pastoral communities with limited access to external food sources. Raw materials are preserved by heating, drying, smoking, pickling, salting, curing or fermentation with microorganisms. This article describes preservation techniques in the pastoral context, targeting the major dietary components of milk, meat and cereals; related health risks; and potential innovations for food preservation. Sustainable elimination of pathogenic microorganisms, preventing re-contamination, sporulation and the growth of zoonotic and foodborne microorganisms, is necessary to enhance food safety and ensure food security by reducing post-harvest losses and food waste. However, modern preservation procedures are difficult to adapt to the lifestyles of pastoralists and so are rarely implemented or accepted. Innovations should therefore focus on improving existing accepted procedures by promoting synergistic combinations to compensate for the disadvantages of these traditional techniques and ensure the quality of the raw material right up until consumption. Drying and spontaneous fermentation are key preservation techniques among pastoralists that serve as opportunities for innovation and can be shared across pastoral communities. Further potential for innovation lies in the unique, largely uncharacterised, microflora biodiversity of fermented products. The characterisation, safety assessment and conservation of these microorganisms are needed to develop locally adapted starter cultures that retain or improve on the desired characteristics of the finished product. Careful sensitisation of stakeholders, the study of social acceptance and capacitybuilding at all levels are required to achieve the sustainable implementation of such innovations, which will contribute to enhanced food security and safety.

Dans les communautés pastorales, dont l'accès aux sources d'approvisionnement alimentaire extérieures est limité, la conservation des denrées alimentaires joue un grand rôle pour assurer la sécurité alimentaire et l'innocuité des aliments. Les méthodes permettant de conserver les aliments crus sont le traitement thermique, la déshydratation et le séchage, le fumage ou fumaison, le saumurage, le salage et la fermentation. Les auteurs décrivent les techniques de conservation pratiquées par les sociétés pastorales, axées sur les composantes les plus importantes du régime alimentaire des populations concernées, à savoir le lait, la viande et les céréales, ainsi que les risques sanitaires qui leur sont associés et les innovations potentielles dans ce domaine. Afin de minimiser les pertes post-récolte et le gaspillage alimentaire et d'améliorer ainsi l'innocuité des aliments et la sécurité alimentaire, il est indispensable d'éliminer durablement les agents pathogènes, d'empêcher la survenue de nouvelles contaminations et d'éviter les risques de sporulation et de croissance de micro-organismes zoonotiques ou d'origine alimentaire. Cependant, les procédures modernes de conservation sont difficiles à concilier avec le mode de vie des pasteurs, ce qui explique qu'elles soient mal acceptées et rarement mises en pratique. Les efforts d'innovation devront donc porter en priorité sur l'amélioration des procédures actuellement acceptées et encourager le recours simultané à plusieurs méthodes afin de créer une synergie qui permette d'atténuer les inconvénients des techniques traditionnelles tout en garantissant la qualité des aliments crus jusqu'au moment de leur consommation. La déshydratation et la fermentation spontanée, deux méthodes majeures de conservation dans les communautés pastorales, offrent un potentiel d'innovation qui peut être largement diffusé parmi ces communautés. La biodiversité exceptionnelle de la microflore présente dans les produits fermentés doit encore faire l'objet d'une caractérisation exhaustive afin d'exploiter le potentiel d'innovation qui lui est associé. Pour qu'un levain naturel constitué de flore bactérienne puisse être élaboré localement en perpétuant, voire en améliorant les caractéristiques souhaitées du produit final, il faut préalablement caractériser ces micro-organismes, évaluer leur innocuité et les conserver. La mise en oeuvre durable de ces innovations requiert une sensibilisation minutieuse des parties prenantes, l'analyse de leur acceptabilité par la société et le renforcement des capacités à tous les niveaux, ce qui contribuera à améliorer la sécurité alimentaire et l'innocuité des aliments.

En las comunidades pastorales, que tienen escaso acceso a fuentes alimentarias externas, la conservación de los alimentos contribuye en gran medida a la seguridad y la higiene alimentarias. Para conservar un alimento crudo es posible cocinarlo, secarlo, ahumarlo, encurtirlo, salarlo, curarlo o hacerlo fermentar con microorganismos. Tras describir las técnicas de conservación utilizadas en medios pastorales, prestando especial atención a los componentes básicos del régimen alimentario (esto es, leche, carne y cereales), los autores exponen los riesgos sanitarios conexos y las posibles innovaciones en la materia. Para mejorar la inocuidad de los alimentos y garantizar la seguridad alimentaria es indispensable librarlos duraderamente de microorganismos patógenos, lo que evita la contaminación reiterativa e impide la esporulación y el crecimiento de microorganismos zoonóticos y de transmisión alimentaria, cosa que a su vez reduce el volumen de pérdidas tras la cosecha y el de alimentos echados a perder. Sin embargo, resulta difícil adaptar los modernos procedimientos de conservación a los modos de vida de las sociedades pastorales, lo que hace que rara vez sean implantados o aceptados. Por ello las innovaciones deben ir dirigidas ante todo a mejorar los procedimientos ya existentes y aceptados promoviendo combinaciones sinérgicas para compensar las desventajas de esas técnicas tradicionales y garantizar la calidad de los alimentos crudos hasta el momento en que sean consumidos. El secado y la fermentación espontánea, que son técnicas fundamentales de conservación entre los pastores, ofrecen oportunidades de innovación que las diversas comunidades pastorales pueden compartir. Otro posible yacimiento de innovación reside en la singular biodiversidad de la microflora de los productos fermentados, que en gran parte aún no está caracterizada. La caracterización de estos microorganismos, la evaluación de su inocuidad y su conservación son otros tantos procesos necesarios para obtener cultivos iniciadores adaptados al contexto local que retengan o mejoren las características deseadas del producto final. Para posibilitar una aplicación duradera de tales innovaciones, que contribuirán a lograr mayores cotas de seguridad e higiene alimentarias, es preciso sensibilizar cuidadosamente a los interesados, estudiar la aceptación social de los procedimientos en cuestión e impartir formación a todos los niveles.

Influence of pH and the degree of protonation on the inhibitory effect of fatty acids in the ruminal methanogen Methanobrevibacter ruminantium strain M1.

AIMS: To investigate the relationship between the protonation of medium-chain fatty acids (MCFA) and their inhibitory effect on a ruminal methanogen species. METHODS AND RESULTS: Cell suspensions of Methanobrevibacter ruminantium M1 in 1 mg dry matter (DM) ml(-1) were supplemented with lauric acid (C12 ) and myristic acid (C14 ) at a concentration of 8 µg ml(-1) with different pH levels of the potassium-free buffer, where the calculated degrees of protonation of C12 and C14 varied from 0·3 to 50% and from 1 to 76% respectively. Methane formation, ATP efflux, potassium leakage and cell viability were monitored 15, 30 and 45 min after the reaction started. Declining methane formation rate, increasing ATP efflux and potassium leakage, and decreasing survival of M. ruminantium were observed with increasing degrees of protonation, i.e. with decreasing pH. CONCLUSIONS: The inhibition of methanogenesis by C12 and C14 is more efficient at a pH of 5-6 as compared to pH 7. SIGNIFICANCE AND IMPACT OF THE STUDY: Methane mitigation strategies in ruminants which use supplementation of feed with MCFA such as C12 and C14 may be more effective in a low rumen pH environment. This finding is helpful in designing diets to effectively decrease methane emissions by ruminants.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway.

OBJECTIVE: This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). MATERIAL AND METHOds: The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. RESULTS: After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. CONCLUSIONS: Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Risk factors for symptoms of gastrointestinal illness in rural town Isiolo, Kenya.

This study assesses risk factors for food-borne gastrointestinal illness indicated by diarrhoea and/or vomiting using 14-day recalls among children and young adults. The study was set in Isiolo, a rural town of Kenya, inhabited mainly by pastoralists of different ethnic groups. The preparation methods of milk at the household level were also investigated. The study was cross-sectional and involved 900 participants from randomly selected households. They were interviewed using a structured questionnaire. An unmatched nested case-control study was constructed by randomly selecting three controls for each case. Potential risk factors for gastrointestinal illness were analysed using both univariate and multivariate logistic regression models with random effect on ethnic groups. The study results showed that consumption of mutton, carrots, Irish potatoes, raw camel milk, boiled camel milk and fermented camel milk were important risk factors for diarrhoea and/or vomiting, whereas the consumption of boiled goat milk, boiled cow milk, spinach, washing of hands with soap and the presence of proper drainage system had protective effects (odds ratio < 1). We conclude that in this setting, primarily vegetables and the camel milk market chain pose the greatest risks for symptoms of food-borne gastrointestinal illness.

Co-cultivation of a bacteriocin-producing mixed culture of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 isolated from baby faeces.

AIMS: We investigated the kinetics of growth, metabolism and antimicrobial activity of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 previously isolated as a consortium from human baby faeces and producing antimicrobial proteinaceous compounds. METHODS AND RESULTS: Cell growth, antimicrobial activity, glucose consumption, organic acid production and pediocin-gene expression were monitored during pure and mixed strain batch cultures with controlled pH (6.0) at 37 degrees C. The balance of the two strains in mixed cultures was stable, yielding high cell count of 10(9) CFU ml(-1) after a very short incubation time of 4 h for UVA1 and 7 h for RBL67, and RBL67 was not affected by the high production of pediocin by UVA1 during co-culture (up to 12.8 microg ml(-1)). Furthermore, a real-time PCR assay was developed and allowed gene-expression analysis of the pediocin-gene pedA. CONCLUSIONS: The co-culture of RBL67 and UVA1 showed high stability, cell yields and bacteriocin production during batch cultures and has potential for use as a probiotic mixture with antibacterial properties. In addition, a gene-expression real-time PCR assay was successfully developed, used for the relative quantification of the pediocin transcript pedA and demonstrated to be a valuable complementary tool to activity assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study reporting of a stable mixed culture of two bacteriocin-producing strains of human origin.

Genetic and phenotypic diversity of Bifidobacterium thermacidophilum fecal isolates from newborns.

This study was undertaken to genetically identify and phenotypically characterize 14 bifidobacteria isolated from 20 breast-fed newborns. These isolates showed 98%-99% similarity to Bifidobacterium thermacidophilum subsp. suis based on 16S rDNA. Further analysis by pulsed-field gel electrophoresis of chromosomal DNA digested with XbaI revealed 4 distinct restriction patterns. The predominant pattern, shared by 8 (57%) isolates, produced a macro-restriction profile with about 13 large fragments ranging in size from >242.5 to 23.1 kb, whereas the other 6 displayed 3 distinct restriction profiles all characterized by more micro- than macro-restriction, with fragments ranging in size from 97 to 9.4 kb. Phenotypic characteristics, including carbohydrate fermentation profile, maximal growth temperature, and antibiotic susceptibility, varied widely even among strains showing the same restriction profile. The presence of B. thermacidophilum in stools of newborn infants may indicate the potential of these bacteria for aiding the development of the intestinal ecosystem.

Bt176 corn in poultry nutrition: physiological characteristics and fate of recombinant plant DNA in chickens.

A genetically modified Bt176 corn hybrid, which contains an insecticidal protein against the European corn borer, and its conventional, nonmodified counterpart were evaluated in 4 separate trials to verify substantial equivalence in feeding value and animal performance. Thirty-six individually kept laying hens and 3 replicates of 94 broiler chickens each, assigned to 12 cages, were fed 2 different hen and broiler diets containing either 60% conventional or 60% Bt176 corn. The nutrient compositions of the 2 corn hybrids and the 2 corn diets revealed no major differences. Furthermore, metabolism and performance data revealed no significant differences between the birds that received the conventional, nonmodified corn, and those that received the modified corn diets. The detection of the genetic modification, by PCR, in feed obtained from insect-resistant Bt corn, in tissues and products from animals fed Bt corn is described. In all evaluated chicken tissues of muscle, liver, and spleen, the corn-chloroplast ivr gene fragment was amplified. It can be deduced from these findings and from other studies that the transfer of DNA fragments into the body is a normal process that takes place constantly. Nevertheless, no recombinant plant DNA fragments such as recombinant bla or cry1A(b) fragments could be found. Bt-gene specific constructs from the Bt corn were not detected in any of the poultry samples, neither in organs, meat, nor eggs.

Effects of mixtures of lauric and myristic acid on rumen methanogens and methanogenesis in vitro.

AIMS: To identify the most effective mixture of non-esterified lauric (C12) and myristic (C14) acid in suppressing ruminal methanogenesis, and to investigate their effects on the methanogenic population. METHODS AND RESULTS: C12/C14 mixtures were incubated with rumen fluid using the Hohenheim gas test apparatus. Methane production and the numbers of Archaea declined with an increasing proportion of C12. With a 2 : 1 proportion of C12/C14, the maximum methane-suppressing effect (96%) was achieved similar to that with C12 alone. The proportions of the individual methanogenic orders of total methanogens were altered by varying the C12/C14 ratio. CONCLUSIONS: Although C14 alone had no effect on methanogenesis, C14 enhanced the methane-suppressing effect of C12 in certain mixtures. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support strategies for an environment-friendly ruminant nutrition as it was demonstrated that part of the less palatable C12 could be replaced by C14 without losing its methane-suppressing potential.

Characterization of the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis.

A D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with D-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. K(m) values for D-xylulose 5-phosphate and D-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

Propionicin SM1, a bacteriocin from Propionibacterium jensenii DF1: isolation and characterization of the protein and its gene.

We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1. The heat-stable protein was strongly bactericidal against P. jensenii DSM20274. On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P. jensenii DF1. It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA. Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da). The corresponding gene was named ppnA. It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1. No sequence homology is detectable with known proteins. However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993). Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria. The highest identity values were found for ORF1 with the theta replicase (acc. no. U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc. no. AF060871) found in Rhodococcus rhodochrous (46.4%).

Description of Saccharomyces turicensis sp. nov., a new species from kefyr.

The new species Saccharomyces turicensis sp. nov. isolated from different kefyr grains is described. Although its morphological properties differ, its physiological characteristics come close to those of Saccharomyces bayanus Saccardo and Saccharomyces pastorianus Reess ex E. C. Hansen. However, electrophoretic karyotyping and restriction fragment length polymorphism of the internal transcribed spacer region yield clear differences. Sequences (270 nucleotides) of the D2 domain at the 5'-terminal end of the large subunit ribosomal RNA gene reveal 98.0% identity with Saccharomyces exiguus. Since strains of a particular yeast species usually show less than 1% substitution in the D2 domain, the yeast in question is considered to be a new species. The name Saccharomyces turicensis is proposed indicating the place Zürich (Turicum in Latin) where the yeast had been isolated.

Transcriptional analysis of the rubrerythrin and superoxide dismutase genes of Clostridium perfringens.

We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from Clostridium perfringens containing the superoxide dismutase-encoding gene, sod. Previously, rubrerythrin from C. perfringens had been isolated and its gene (rbr) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152-7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of rbr and sod of C. perfringens. Thus, rbr and sod each represent a monocistronic operon. Their transcription start points were located by primer extension analyses. sod transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither sod nor rbr transcription was influenced by oxidative stress.

Acquired antibiotic resistance in lactic acid bacteria from food.

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. theta-type replicating plasmids of the pAMbeta1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29,871 bp resistance plasmid detected in Lactococcus lactis subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabolic traits to generate food-grade vectors.

Classification and identification of propionibacteria based on ribosomal RNA genes and PCR.

A rapid method was developed to differentiate the genus Propionibacterium from other genera by using a modified multiplex-PCR (MPCR) approach. Three 16S rRNA-targeted oligonucleotide primers were designed to amplify simultaneously two DNA-fragments in the MPCR assay. The universal primer pair bak11w and bak4 (corresponding to the E. coli 16S rRNA positions 8-25 and 1522-1540, respectively) was used in combination with the primer pair bak4 and gd1 (5'-TGCTTTCGATACGGGTTGAC-3'). The later sequence corresponding to a 16S rRNA motif that is unique for the genus Propionibacterium. Propionibacteria were identified by the amplification of a Propionibacterium-genus specific 900-bp fragment whereas MPCR with DNA from other bacteria generated only a DNA fragment of 1500 bp in amplifications with the two universal primers. The whole procedure including cell lysis, MPCR amplification and analysis can be performed within 1 day, detection limits are at approximately 10(3) cfu propionibacteria (or 35 pg DNA). In addition, the taxonomic situation of the genus Propionibacterium was reexamined using a cycle sequencing strategy. Based on the 16S rDNA, a phylogenetic tree of all the Propionibacterium type strains was reconstructed.

Biochemical and genetic characterization of the acetaldehyde dehydrogenase complex from Acetobacter europaeus.

The aldehyde dehydrogenase complex, which catalyzes the oxidation of acetaldehyde to acetic acid, was purified to apparent homogeneity from the membrane fraction of the industrial vinegar-producing strain Acetobacter europaeus. The determined Km for acetaldehyde was 2.1 mM. SDS-PAGE of the enzyme complex showed the presence of three different subunits with molecular masses of 79, 46, and 17 kDa, respectively. The two larger subunits contained heme. The difference spectrum indicated a cytochrome c, a heme B, and a [2Fe-2S] cluster. The nucleotide sequence of several cloned fragments of a 6-kb chromosomal DNA segment from A. europaeus was determined. It contains three consecutive open reading frames that correspond to proteins with calculated molecular masses of 84.1, 49.0, and 16.7 kDa; these were assigned to the purified proteins and named aldH, aldF, and aldG, respectively. The N-terminal sequence of the 79-kDa subunit was detected within the predicted amino acid sequence of AldH, which indicated the presence of a leader peptide. Cotranscription of the three genes was shown by Northern hybridization. Sequence analysis and experimental evidence allowed the assignment of the following cofactors to the respective subunits of the aldehyde dehydrogenase complex: heme C to AldF, [2Fe-2S] cluster to AldG, and heme B and a molybdopterin cofactor to AldH. Part of an open reading frame, gdhA, was detected upstream of the operon that showed high similarities to the C-terminal part of several pyrroloquinoline-chinone-dependent glucose dehydrogenases.

Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.

Rubrerythrin from Clostridium perfringens: cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function.

The food-borne pathogen Clostridium perfringens, which is an obligate anaerobe, showed growth under conditions of oxidative stress. In protein extracts we looked for superoxide dismutase (SOD) activities which might scavenge highly toxic superoxide radicals evolving under such stress conditions. Using the classical assay to detect SOD activity on gels after electrophoresis of C. perfringens proteins, we obtained a pattern of three major bands indicating SOD activity. The protein representing the brightest band was purified by three chromatographic steps. On the basis of 20 amino acids determined from the N terminus of the protein, we designed a degenerate oligonucleotide probe to isolate the corresponding gene. We finally sequenced an open reading frame of 195 amino acids (molecular mass, 21,159 Da) with a strong homology to the Desulfovibrio vulgaris rubrerythrin; therefore, we assumed to have cloned a rubrerythrin gene from C. perfringens, and we named it rbr. The C-terminal region of the newly detected rubrerythrin from C. perfringens contains a characteristic non-heme, non-sulfur iron-binding site -Cys-X-X-Cys-(X)12-Cys-X-X-Cys- similar to that found in rubrerythrin from D. vulgaris. In addition, three -Glu-X-X-His- sequences could represent diiron binding domains. We observed SOD activity in extracts of Escherichia coli strains containing the recombinant rbr gene from C. perfringens. A biological function of rubrerythrin as SOD was confirmed with the functional complementation by the rbr gene of an E. coli mutant strain lacking SOD activity. We therefore suppose that rubrerythrin plays a role as a scavenger of oxygen radicals.

Evidence for a defective prophage on the chromosome of Methanobacterium wolfei.

Evidence shows the presence on the chromosome of Methanobacterium wolfei of a defective prophage which, by DNA-DNA hybridization, is closely related to the virulent archaeophage psi M1 of Methanobacterium thermoautotrophicum Marburg. Partial sequencing of a M. wolfei 16S rRNA gene and phylogenetic analysis indicated that this organism is more closely related to other representatives of the genus Methanobacterium than to M. thermoautotrophicum Marburg. The chromosomal region of M. wolfei encoding the putative prophage was found to be deleted for two non-contiguous segments of the phage psi M1 genome and thus encompassed only 80 to 90% of the psi M1 DNA. The prophage region was mapped to a 30 kb restriction fragment on the physical map of the M. wolfei chromosome. A randomly chosen DNA fragment was cloned from phage psi M1 DNA, as was its homologous counterpart from the chromosome of M. wolfei. The 126-bp region present in both clones exhibited 100% sequence identity.

Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg.

A gene (sod) encoding superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identify was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod, separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium.

Tryptophan gene cluster of Methanobacterium thermoautotrophicum Marburg: molecular cloning and nucleotide sequence of a putative trpEGCFBAD operon.

A recombinant cosmid carrying the Methanobacterium thermoautotrophicum Marburg trp genes was selected by complementation of Escherichia coli trp mutations. A 7.3-kb fragment of the cloned archaeal DNA was sequenced. It contained the seven trp genes, arranged adjacent to each other in the order trpEGCFBAD. No gene fusions were observed. The trp genes were organized in an operonlike structure, with four short (5- to 56-bp) intergenic regions and two overlapping genes. There was no indication for an open reading frame encoding a leader peptide in the upstream region of trpE. The gene order observed in the M. thermoautotrophicum trp operon was different from all known arrangements of the trp genes in archaea, bacteria, and eucarya. The encoded sequences of the Methanobacterium Trp proteins were similar in size to their bacterial and eucaryal counterparts, and all of them contained the segments of highly similar or invariant amino acid residues recognized in the Trp enzymes from bacteria and eucarya. The TrpE, TrpG, TrpC, TrpA, and TrpD proteins were 30 to 50% identical to those from representatives of other species. Significantly less sequence conservation (18 to 30%) was observed for TrpF, and TrpB exhibited a high degree of identity (50 to 62%) to the sequences of representatives of the three domains. With the exception of TrpB, the beta subunit of tryptophan synthase, tryptophan was absent from all Trp polypeptides.