Anaerostipes butyraticus sp. nov., an anaerobic, butyrate-producing bacterium from Clostridium cluster XIVa isolated from broiler chicken caecal content, and emended description of the genus Anaerostipes.

Four butyrate-producing isolates were obtained from the caecal content of a 4-week-old broiler chicken. The 16S rRNA gene sequences were determined and confirmed the close relatedness of the four isolates, which suggested that they were derived from a single bacterial clone. Phylogenetic analysis based on 16S rRNA gene sequences showed that its closest relatives were members of cluster XIVa of the Clostridium subphylum of Gram-positive bacteria and that the closest related type strain was Anaerostipes caccae L1-92(T) (94.5 % similarity). Similarity levels of 96-98 % with sequences from uncultured bacteria from human stool samples were observed. On the basis of morphological, biochemical and phylogenetic characteristics, this strain is assigned to a novel species in the genus Anaerostipes, for which the name Anaerostipes butyraticus sp. nov. is proposed. The type strain is 35-7(T) (=LMG 24724(T) =DSM 22094(T)). An emended description of the genus Anaerostipes is also provided.

Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium.

AIMS: To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved. METHODS AND RESULTS: The adhesion of probiotic (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12), commensal (B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria (E. coli and L. monocytogenes) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2.6-27.3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower (P < 0.05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0.5-2.3%) cultures and Caco-2/HT29-MTX (0.6-3.2%) cocultures, while these values were increased in Caco-2 cultures plus mucin. CONCLUSIONS: The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.

Lysinibacillus parviboronicapiens sp. nov., a low-boron-containing bacterium isolated from soil.

A spore-forming, Gram-positive-staining, motile, rod-shaped and low-boron-containing bacterium was isolated from soil. The strain, designated BAM-582(T), can tolerate 6 % (w/v) NaCl and 50 mM boron, but optimal growth was observed without addition of boron or NaCl. The optimum temperature and pH for growth were 30 degrees C (range 10-37 degrees C) and pH 7 (range pH 6-8). A comparative analysis of the 16S rRNA gene sequence demonstrated that the isolated strain was closely related to Lysinibacillus fusiformis DSM 2898(T) (97.7 % similarity) and Lysinibacillus sphaericus IAM 13420(T) (98.2 %). Levels of DNA-DNA relatedness were 33.9 % with L. fusiformis DSM 2898(T) and 29.5 % with L. sphaericus DSM 28(T). The genomic DNA G+C content of the novel strain was 38.7 mol%. The major respiratory quinone was MK-7 and the major fatty acids were iso-C(15 : 0) (37.4 %) and anteiso-C(15 : 0) (19.0 %). Analysis of cell-wall amino acids revealed that the strain contained peptidoglycan with lysine, aspartic acid, alanine and glutamic acid, as is the case with other species of the genus Lysinibacillus. Based upon its distinctive peptidoglycan composition, phylogenetic and genotypic analyses and physiological characteristics, the strain BAM-582(T) is concluded to represent a novel species in the genus Lysinibacillus, for which the name Lysinibacillus parviboronicapiens sp. nov. is proposed (type strain BAM-582(T) =NBRC 103144(T) =KCTC 13154(T)).

Thermotalea metallivorans gen. nov., sp. nov., a thermophilic, anaerobic bacterium from the Great Artesian Basin of Australia aquifer.

A strictly anaerobic, thermophilic bacterium, designated strain B2-1(T), was isolated from microbial mats colonizing a runoff channel formed by free-flowing thermal water from a Great Artesian Basin, Australia, bore well (registered number 17263). The cells of strain B2-1(T) were slightly curved rods (3.0-3.5 x 0.6-0.7 microm) which stained Gram-negative. The strain grew optimally in tryptone-yeast extract-glucose medium at 50 degrees C (temperature growth range 30-55 degrees C) and a pH of 8 (pH growth range 6.5-9). Strain B2-1(T) grew poorly on yeast extract (0.2 %) and/or tryptone (0.2 %), which were obligately required for growth on other energy sources, including a range of other carbohydrates and organic acids, but not amino acids. The end-products of glucose fermentation were ethanol and acetate. In the presence of 0.2 % yeast extract, iron(III), manganese(IV) and elemental sulfur were reduced but sulfate, thiosulfate, sulfite, nitrate and nitrite were not reduced. Growth was inhibited by chloramphenicol, streptomycin, tetracycline, penicillin, ampicillin, sodium azide and by NaCl concentrations greater than 4 % (w/v). The DNA G+C content was 48+/-1 mol% as determined by the thermal denaturation method. 16S rRNA gene sequence analysis indicated that strain B2-1(T) was a member of the family Clostridiaceae, class Clostridia, phylum Firmicutes and was most closely related to Geosporobacter subterraneus DSM 17957(T) (89.9 % similarity). On the basis of 16S rRNA gene sequence comparisons and physiological characteristics, strain B2-1(T) is considered to represent a novel species of a new genus, for which the name Thermotalea metallivorans gen. nov., sp. nov. is proposed. The type strain is B2-1(T) (=KCTC 5625(T)=JCM 15105(T)=DSM 21119(T)).

Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., slightly halophilic and alkaliphilic marine lactic acid bacteria isolated from marine organisms and salted foods collected in Japan and Thailand.

We describe 10 new strains of marine lactic acid bacteria isolated from decaying marine algae, decaying seagrass, raw fish, salted fish and salted and fermented shrimp paste ('ka-pi') collected from a temperate area of Japan and Thailand. The isolates are Gram-positive and non-sporulating. They have motility with peritrichous flagella depending on the strains. They lack catalase and quinones. Under anaerobic conditions, lactate yields were 64-93 % of the glucose consumed; residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The pH of the fermentation medium markedly affected the product composition; at higher pH, the yield of lactate decreased (15-48 % at pH 9.0) and yields of other products increased, retaining the molar ratio. Under aerobic conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 0.5 and 4.0 % (w/v), depending on the strain, with a growth range of between 0 and 17-21 % (11 % for one isolate). The optimum pH for growth ranged between 8.0 and 9.5, with a growth range of 6.0-11.0, depending on the strains. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied three phylogenetic positions within the genus Alkalibacterium, showing 97.1-99.8 % similarity to Alkalibacterium indicireducens. DNA-DNA hybridization values (<46 %) among the 10 isolates and phylogenetically related taxa resulted in the identification of four genomic species (designated groups GS1-GS4). The G+C contents of the DNA were 41.7 mol% (group GS1), 42.2 mol% (group GS2), 41.0-43.0 mol% (group GS3) and 38.4-39.4 mol% (group GS4). The cell-wall peptidoglycan was type A4beta, Orn-d-Asp, for three genomic species (groups GS1, GS2 and GS3), and type A4beta, Orn-d-Glu, for the remaining species (group GS4). The major components of cellular fatty acids were C(16 : 0), C(16 : 1)omega9c and C(18 : 1)omega9c (oleic acid). On the bases of phenotypic characteristics, genetic distinctiveness and phylogenetic affiliations, the four genomic species, groups GS1, GS2, GS3 and GS4, were classified as four novel species within the genus Alkalibacterium, for which the names Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., respectively, are proposed. The respective type strains are T117-1-2(T) (=DSM 19181(T)=NBRC 103241(T)=NRIC 0718(T)), T143-1-1(T) (=DSM 19183(T)=NBRC 103242(T)=NRIC 0719(T)), T129-2-1(T) (=DSM 19182(T)=NBRC 103243(T)=NRIC 0720(T)) and T22-1-2(T) (=DSM 19180(T)=NBRC 103247(T)=NRIC 0724(T)).

Effect of low temperature and culture media on the growth and freeze-thawing tolerance of Exiguobacterium strains.

Bacteria of the genus Exiguobacterium have been repeatedly isolated from ancient permafrost sediments of the Kolyma lowland of Northeast Eurasia. Here we report that the Siberian permafrost isolates Exiguobacterium sibiricum 255-15, E. sibiricum 7-3, Exiguobacterium undae 190-11 and E. sp. 5138, as well as Exiguobacterium antarcticum DSM 14480, isolated from a microbial mat sample of Lake Fryxell (McMurdo Dry Valleys, Antarctica), were able to grow at temperatures ranging from -6 to 40 degrees C. In comparison to cells grown at 24 degrees C, the cold-grown cells of these strains tended to be longer and wider. We also investigated the effect of growth conditions (broth or surface growth, and temperature) on cryotolerance of the Exiguobacterium strains. Bacteria grown in broth at 4 degrees C showed markedly greater survival following freeze-thawing treatments (20 repeated cycles) than bacteria grown in broth at 24 degrees C. Surprisingly, significant protection to repeated freeze-thawing was also observed when bacteria were grown on agar at either 4 or 24 degrees C.

Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough.

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0-120 g l(-1), with the optimum at 10-20 g l(-1). The temperature range for growth at pH 7.0 was 4-50 degrees C, with the optimum at 37-40 degrees C. The optimum pH for growth was 7.0-8.0. The optimum pressure for growth was 0.1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2(T) (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA-DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

Weissella kimchii sp. nov., a novel lactic acid bacterium from kimchi.

A gram-positive, catalase-negative, non-sporulating, facultatively anaerobic, short rod-shaped bacterium, with cells measuring 0.3-0.5 x 1-2 microm and designated strain CHJ3T, was isolated from partially fermented kimchi, a traditional Korean fermented vegetable food. The strain produced CO2 gas, D-lactate from glucose and dextran from sucrose and hydrolysed aesculin and arginine. It also fermented N-acetylglucosamine, amygdalin, arbutin, cellobiose, D-fructose, galactose, beta-gentiobiose, gluconate, D-glucose, maltose, D-mannose, salicin, sucrose and D-xylose. The G+C content of the DNA was 48.2 mol%. Phylogenetic analysis of 16S rRNA showed that strain CHJ3T is a member of the genus Weissella. The nearest phylogenetic relative of strain CHJ3T was Weissella confusa, with 16S rRNA similarity of 98.3%. However, strain CHJ3T could be differentiated from W. confusa on the basis of some phenotypic characteristics, analysis of whole-cell protein patterns and DNA-DNA hybridization data. These data suggest that strain CHJ3T be classified in the genus Weissella as a novel species, Weissella kimchii sp. nov. The type strain is CHJ3T (= KCCM 41287T = DSM 14295T = KCTC 3746T).

Description of Mogibacterium pumilum gen. nov., sp. nov. and Mogibacterium vescum gen. nov., sp. nov., and reclassification of Eubacterium timidum (Holdeman et al. 1980) as Mogibacterium timidum gen. nov., comb. nov.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

Coprobacillus catenaformis gen. nov., sp. nov., a new genus and species isolated from human feces.

Three strains of Eubacterium-like isolates from human feces were characterized by biochemical tests and 16S rDNA analysis. The phenotypic characteristics of the three strains resembled those of the genus Collinsella transferred from the genus Eubacterium recently. However, Eubacterium-like strains were phylogenetically members of the Clostridium subphylum of gram-positive bacteria, and these showed a specific phylogenetic association with Clostridium ramosum and C. spiroforme. C. ramosum and C. spiroforme are gram-positive, anaerobic, spore-forming bacteria that belong to the genus Clostridium, and the G + C contents are 26.0 and 27.4 mol%, respectively. However, the three Eubacterium-like strains had G + C contents of 32.1 to 33.1 mol% and were non-spore-forming rods. Based on phenotypic characteristics, we can differentiate these species, and furthermore, a 16S rDNA sequence divergence of greater than 9% with a new related genus, Coprobacillus, is proposed for the three strains, with one species, Coprobacillus catenaformis. The type strain of C. catenaformis is JCM 10604T.

Acetobacterium tundrae sp nov, a new psychrophilic acetogenic bacterium from tundra soil.

A new psychrophilic, anaerobic, acetogenic bacterium from the tundra wetland soil of Polar Ural is described. The organism fermented H2/CO2, formate, methanol, and several sugars to acetate as the sole end-product. The temperature range for growth was 1-30 degrees C with an optimum at 20 degrees C. The bacterium showed no growth at 32 degrees C. Cells were gram-positive, oval-shaped, flagellated rods 0.7-1.l x 1.1-4.0 microm in size when grown at 1-20 degrees C. At 25-30 degrees C, the cell size increased up to 2-3 x 10-15 microm due to a defect in cell division. The DNA G+C content of the organism was 39.2 mol%. Based upon 16S rDNA analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium as a new species, for which the name Acetobacterium tundrae sp. nov. is proposed. The type strain is Z-4493 (=DSM 9173T).

Phylogenetic and phenotypic evidence for the transfer of Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov.

Three strains of Eubacterium aerofacien, JCM 10188T, JCM 7790 and JCM 7791, and 178 freshly isolated strains of the Eubacterium aerofaciens group from human faeces were characterized by biochemical tests, cell wall peptidoglycan type and 16S rRNA analysis. The Eubacterium aerofaciens group was divided into four groups by fermentation patterns of sucrose and cellobiose, and were further divided into 16 sub-groups by fermentation patterns of aesculin, salicin and amygdalin. All of the strains of the Eubacterium aerofaciens group were shown to be phylogenetically distantly related to Eubacterium limosum, which is the type species of genus Eubacterium. Eubacterium aerofaciens was shown to have a specific phylogenetic association with Coriobacterium glomerans. All the strains belonging to Eubacterium aerofaciens resembled Coriobacterium glomerans in possessing a high G + C content (60 mol%). Cell wall analysis, however, revealed the presence of different A4 beta (L-Ala)-D-Glu-L-Orn-L-Asp peptidoglycan types. Based on a 16S rRNA sequence divergence of greater than 9% with Coriobacterium glomerans and the presence of a unique peptidoglycan type, a new genus, Collinsella, is proposed for Eubacterium aerofaciens, with one species, Collinsella aerofaciens. The type strain of Collinsella aerofaciens is JCM 10188T.

Natronoincola histidinovorans gen. nov., sp. nov., A new alkaliphilic acetogenic anaerobe.

Two strains, asporogenous Z-7940 and sporogenous Z-7939, of a moderately haloalkaliphilic, obligately anaerobic, fermentative bacteria, motile, with Gram-positive cell wall structure, were isolated from soda deposits in Lake Magadi, Kenya. Both strains are mesophilic and utilize only two amino acids, histidine and glutamate, with formation of acetate and ammonium as the main end products. Strain Z-7939 in addition is able to utilize pyruvate. DNA-DNA homology between strains Z-7940 and Z-7939 was 94%, indicating that in spite of phenotypic differences they belong to the same species. They are true alkaliphiles with a pH range for growth of the type strain Z-7940 from pH 8.0 to pH 10.5, optimum at pH 9.4. Both strains obligately depend on sodium and bicarbonate ions. The optimum salt concentration for growth of the type strain is 8-10% wt/vol and the range from 4% to 16%. The G+C content of strain Z-7940 is 31.9 mol% and the strain Z-7939 is 32.3 mol%. Analysis of 16S rDNA sequence of the type strain shows it to belong to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and physiological properties, we propose a new genus and new species Natronoincola histidinovorans for these strains. The type strain is Z-7940 (=DSM 11416).

Characterization of a locus from Carnobacterium piscicola LV17B involved in bacteriocin production and immunity: evidence for global inducer-mediated transcriptional regulation.

Mutational, nucleotide sequence, and transcriptional analyses of a 10-kb fragment (carnobacteriocin locus) from the 61-kb plasmid of Carnobacterium piscicola LV17B demonstrated the presence of two gene clusters (cbnXY and cbnSKRTD) upstream of the previously sequenced carnobacteriocin B2 structural and immunity genes (cbnB2 and cbiB2). Deduced products of cbnK and cbnR have sequence similarity to proteins of Agr-type two-component signal transduction systems, and those of cbnT and cbnD have sequence similarity to proteins of signal sequence-independent secretion systems. Deduced products of cbnX, cbnY, and cbnS are class II-type bacteriocin precursors with potential leader peptides containing double-glycine cleavage sites. Genetic analysis indicated that the 10-kb locus contains information required for the production of, and immunity to, the plasmid-encoded carnobacteriocin B2 and the chromosomally encoded carnobacteriocin BM1. In addition, this locus is involved in the production of at least one additional antimicrobial compound and an inducer factor that plays a role in the regulation of carnobacteriocin B2. Transcription analysis indicated that the operons cbnXY, cbnB2-cbiB2, and cbnBM1-cbiBM1 (with the latter encoding carnobacteriocin BM1 and its immunity protein on the chromosome) and two small transcripts containing cbnS are transcribed only in induced cultures. These transcripts are coregulated and subject to inducer-mediated transcriptional control. Similar regulation of the cbn operons is mirrored by the similarity in the nucleotide sequence of their promoter regions, all of which contain two imperfect direct repeats resembling those in Agr-like regulated promoters upstream of the transcription start sites.

Characteristics of a nitropropanol-metabolizing bacterium isolated from the rumen.

We report some characteristics of a ruminal bacterium (strain NPOH1) that metabolizes 3-nitropropanol, the toxic principle of various milk vetchs that are distributed worldwide. The gram-positive bacterium was nonmotile and did not produce spores. Growth of strain NPOH1 occurred under anaerobic conditions and was supported by the electron acceptors 3-nitropropanol, 3-nitropropionate, nitrate, 2-nitropropanol, nitroethane, nitroethanol, or 3-nitro-1-propyl-beta-D-glucopyranoside (miserotoxin). Other potential electron acceptors, namely sulfate, sulfite, azide, chlorate, perchlorate, nitrite, fumarate, 2-nitrobutane, or nitrobenzene, did not support growth. Formate, lactate, and H2 stimulated growth of strain NPOH1 in the presence of the appropriate nitrocompound, whereas a variety of other potential H2 donors did not. When grown in medium containing both nitrate and either 3-nitropropanol or 3-nitropropionate, nitrate was the preferred acceptor. Strain NPOH1 reduced nitrate to nitrite and, when grown with excess reductant, nitrite was further reduced to ammonia. The products formed during the metabolism of 3-nitropropanol and 3-nitropropionate by mixed ruminal populations, 3-aminopropanol and beta-alanine, were not found in culture fluids of strain NPOH1. Analysis of total cellular fatty acid profiles and of the mole percent guanine plus cytosine suggests that strain NPOH1 is a novel bacterium. The capacity of strain NPOH1 to metabolize 3-nitropropanol suggests that this organism may play an important role in detoxification of 3-nitropropanol in the rumen.

Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway.

Carnobacteriocin B2 is a well-characterized class II bacteriocin produced by a 61-kb plasmid from Carnobacterium piscicola LV17. Export of this bacteriocin is dependent on specific ABC (ATP-binding cassette) secretion proteins. Divergicin A is a strongly hydrophobic narrow-spectrum bacteriocin produced by a 3.4-kb plasmid from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretary pathway for export (R. W. Worobo, M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 177:3143-3149, 1995). Fusion of the carnobacteriocin B2 structural gene (devoid of its natural leader peptide) behind the signal peptide of divergicin A in the expression vector pMG36e permitted production and export of active carnobacteriocin B2 in the absence of the specific secretion genes. N-terminal sequencing of purified carnobacteriocin B2 established that correct processing of the prebacteriocin occurred beyond the Ala-Ser-Ala cleavage site of the signal peptide. Carnobacteriocin B2 was produced by the wild-type strain of C. divergens, LV13, and in C. piscicola LV17C, the nonbacteriocinogenic, plasmidless variant of the original carnobacteriocin B2 producer strain. The corresponding immunity gene was included immediately downstream of the structural gene. Both of the host strains are sensitive to the bacteriocin, and both acquired immunity when they were transformed with the construct. C. divergens LV13 containing the divergicin A signal peptide-carnobacteriocin B2 fusion construct produces both divergicin A and carnobacteriocin B2 and demonstrates the first example of multiple-bacteriocin expression via the general secretory pathway. The small amount of genetic material required for independent bacteriocin expression has implications for the development of a food-grade multiple-bacteriocin expression vector for use in lactic acid bacteria.

Psychrotrophic, lactic acid-producing bacteria from anoxic waters in Ace Lake, Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only L(+)lactic acid from D(+)glucose. D(--)-ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30 degrees C and were psychrotrophic. Whole cells contained 18:1 cis 9 as a major component of their fatty acids. At 20 degrees C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (Knuc = 0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38 +/- 8%), and with Carnobacterium mobile (26 +/- 2%, 34 +/- 2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G + C content of the DNA was 32-34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol trehalose. The G + C content was 33-34%, and the Type strain is DSM 5972 (=ACAM 313).