Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses.

AIM: We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening. METHODS AND RESULTS: We used 7% NaCl glucose-yeast extract-peptone-fish extract broth and agar media (pH 9.5) for pour-plating and enrichment culture for 16 cheese samples produced in six European countries. HALAB were present in 9 of the 16 samples at < 20 --> 10(7) CFU g(-1). In three mould-ripened soft cheeses, HALAB counts ranged from 10(6) to 10(7) CFU g(-1) and were one order (two samples) and six orders (one sample) of magnitude greater than that of nonhaloalkaliphilic, common LAB, as enumerated on lactobacilli MRS agar. The 16S rRNA gene sequences (500 bp) of 51 of the 55 isolates examined were identical or similar to that of Marinilactibacillus psychrotolerans or Alkalibacterium olivapovliticus and related species, all of which are HALAB. CONCLUSIONS: HALAB of possible marine origin were present in various soft, semi-hard and semi-soft cheeses and were highly predominant in some mould-ripened cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: HALAB of possible marine origin are members of the microflora of various cheeses and, when dominant, may play a role in the ripening of cheeses. Microbial analysis of LAB flora in cheeses should take into consideration the presence of HALAB.

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences.

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these gamma-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Propionibacterium cyclohexanicum sp. nov., a new acid-tolerant omega-cyclohexyl fatty acid-containing propionibacterium isolated from spoiled orange juice.

A non-spore-forming, coryneform bacterium, strain TA-12T, was isolated from spoiled off-flavor orange juice. Growth of this organism occurs at pH 3.2 to 7.5, and optimum growth occurs at pH values between 5.5 and 6.5. This organism produces lactic acid, propionic acid, and acetic acid from glucose. It is catalase negative. The cells are heat resistant and can withstand a temperature of 90 degrees C for 10 min. The DNA G + C content is 66.8 mol%. This strain has as MK-9(H4) respiratory quinone system and contains meso-diaminopimelic acid in its cell wall, and omega-cyclohexyl undecanoic acid is the major cellular fatty acid. The results of a phylogenetic analysis of the 168 rRNA gene of this organism indicated that its highest level of homology is its level of homology with the representative of the classical propionibacteria, Propionibacterium freudenreichii (97.1%). Strain TA-12T is phenotypically similar to P. freudenreichii, but it produces a large amount of lactic acid and has a distinct fatty acid composition, acid tolerance, and heat resistance, which differentiate it from P. freudenreichii and other propionic acid-producing bacteria. On the basis of these findings we propose the name Propionibacterium cyclohexanicum sp. nov. for this organism. The type strain is TA-12 (= IAM 14535 = NRIC 0247).

Sulfolobus hakonensis sp. nov., a novel species of acidothermophilic archaeon.

We characterized a microbial strain that was isolated from a hot spring at a geothermal area in Hakone, Japan. This isolate, whose lobed-shaped cells were about 1.0 micron in diameter, was a facultative chemolitho-autotroph that required aerobic conditions for growth. The optimum pH was 3.0 (pH range, 1.0 to 4.0), and the optimum temperature was 70 degrees C (temperature range, 50 to 80 degrees C). Lithotrophically, this strain grew on elemental sulfur and reduced sulfur compounds. The G+C content of the genomic DNA was 38.4 mol%. This organism contained calditoglycerocaldarchaeol, which is characteristic of members of the Sulfolobaceae. The levels of 16S rRNA sequence similarity between the new isolate and Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus shibatae were less than 89.8%. Unlike S. acidocaldarius, S. solfataricus, and S. shibatae, the new isolate utilized sugars and amino acids poorly as sole carbon sources, and the levels of DNA-DNA hybridization between the new isolate and these Sulfolobus species were very low. Phenotypically, the new isolate was also distinct from the obligately lithotrophic organism Sulfolobus metallicus. We concluded that the new organism belongs to a new Sulfolobus species, for which we propose the name Sulfolobus hakonensis.

Reddish Escherichia coli cells caused by overproduction of Bacillus stearothermophilus uroporphyrinogen III methylase: cloning, sequencing, and expression of the gene.

During shotgun cloning of an amylase gene, we found a transformant of Escherichia coli with a reddish color. The transformant produced highly water-soluble red pigments the molecular masses of which were less than 3000. The plasmid harbored by the transformant contained a DNA fragment derived from a strain of Bacillus stearothermophilus. Truncation of the insert DNA showed that an 1.1-kbp Sau 3A-SalI fragment was responsible for the reddish colony. An open reading frame was found in the nucleotide sequence of the 1.1-kbp DNA fragment. The production of the red pigment was accompanied by a colorless 28-kDa protein. The sequence of the 28-kDa protein was highly homologous to bacterial uroporphyrinogen III methylases participating in corrinoid biosynthesis. The 28-kDa protein was found to be a thermostable uroporphyrinogen III methylase.

Taxonomic study of bacteria isolated from plants: proposal of Sphingomonas rosa sp. nov., Sphingomonas pruni sp. nov., Sphingomonas asaccharolytica sp. nov., and Sphingomonas mali sp. nov.

The taxonomic positions of 10 strains of 3-ketolactose-forming bacteria which were isolated from the roots of plants (Rosa sp., Psychotria nairobiensis, Ardisia crispa, Prunus persica, and apple trees) were investigated. The DNA base compositions of these strains ranged from 64.0 to 65.7 mol%, the isoprenoid quinone of each strain was ubiquinone 10, 3-hydroxy fatty acids were lacking in the cellular fatty acids of these organisms, and all of the strains contained a sphingolipid with the long-chain base dihydrosphingosin. These are characteristics of the genus Sphingomonas. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with DNA-DNA hybridization and 16S ribosomal DNA sequence comparison data, we propose the following four new species of the genus Sphingomonas: Sphingomonas rosa (type strain, IFO 15208) for the strains isolated from rose plants and formerly named [Agrobacterium rhizogenes]; Sphingomonas pruni (type strain, IFO 15498) for the strains isolated from Prunus persica; and Sphingomonas asaccharolytica (type strain, IFO 15499) and Sphingomonas mali (type strain, IFO 15500) for the strains isolated from apple trees. Two strains which were isolated from Psychotria nairobiensis and formerly named [Chromobacterium lividum] were identified as Sphingomonas yanoikuyae strains.

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences.

The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans. Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S. inulinus. The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus.

Phylogenetic diversity of the genus Cytophaga revealed by 16S rRNA sequencing and menaquinone analysis.

To clarify the intra- and intergeneric relationships of the genus Cytophaga, 16S rRNA sequences and respiratory isoprenoid quinones were determined for the type strains of the 21 validly published species and one isolate in the genus Cytophaga. The sequence analysis revealed extreme heterogeneity of this genus, which diverged into nine distinct lines of descent. Each lineage of Cytophaga was characterized by possessing either menaquinone-6 (MK-6) or MK-7. The MK-6-possessing species were located in the two lineages that were remote from MK-7 species. One of the MK-6 lineages was composed only of terrestrial species and the other only of marine species. Flavobacterium aquatile, the type species of the genus Flavobacterium, was located in the MK-6 terrestrial lineage. The terrestrial Cytophaga species with MK-6 should be transferred to the genus Flavobacterium. The marine facultative anaerobes with MK-7 were located in the bacteroides branch, and possessed signature sequences with features intermediate between the bacteroides and the flavobacteria subdivisions. Cytophaga hutchinsonii, the type species of the genus Cytophaga, had a close relationship only with Cytophaga aurantiaca. The genus Cytophaga should be restricted to these two cellulose-degrading species. The genus Cytophaga is so heterogeneous that it should be divided into several genera and higher taxa in accordance with the phylogenetic relationships.

Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer.

The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer. The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A. tumefaciens. Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M. dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92%. The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation. Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.

Zymobacter palmae gen. nov., sp. nov., a new ethanol-fermenting peritrichous bacterium isolated from palm sap.

Zymobacter palmae gen. nov., sp. nov. was proposed for a new ethanol-fermenting bacterium that was isolated from palm sap in Okinawa Prefecture, Japan. The bacterium is gram-negative, facultatively anaerobic, catalase-positive, oxidase-negative, nonsporeforming and peritrichously flagellated. It requires nicotinic acid for growth. It ferments hexoses, alpha-linked di- and tri-saccharides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannitol and sorbitol). Fifteen percent of maltose in broth medium is effectively fermented, whereas glucose with a concentration higher than 10% delayed growth initiation and decreased growth rates. Maltose is fermented to produce ethanol and CO2 with a trace amount of acids. Approximately 2 mol of ethanol are produced from 1 mol moiety of hexose of maltose. The organism possesses ubiquinone-9. The G + C content of the DNA is 55.8 +/- 0.4 mol%. Major cellular fatty acids were palmitic and oleic acids and cyclopropanic acid of C19:0. Characteristic hydroxylated acid was 3-hydroxy dodecanoic acid. The bacterium is distinct from other ethanol-fermenting bacteria belonging to the genera Zymomonas Kluyver and van Niel 1936 and Saccharobacter Yaping et al. 1990 with respect to chemotaxonomic and other phenotypic characters to warrant to compose a new genus and a new species. The type strain is strain T109 (= IAM 14233).

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria.

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character "intracytoplasmic membrane structure", that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.

Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. nov. and rejection of Methanothrix ("Methanosaeta") thermoacetophila.

Three thermophilic Methanothrix ("Methanosaeta") strains, strains PTT (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii ("Methanosaeta concilii") GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila ("Methanosaeta thermoacetophila"), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.

Purification and properties of restriction endonuclease from Deleya marina IAM 14114, a marine bacterium (DmaI).

A restriction endonuclease, designated as DmaI, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37 degrees C in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 100 mM NaCl (pH 7.5). The enzyme was stable up to 55 degrees C and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5'-CAGCTG-3', cuts between G and C and produces a flush end (isoschizomer of PvuII).

Purification, properties and determinations of recognition sequence and cleavage site of restriction endonuclease from "Agrobacterium gelatinovorum" IAM 12617, a marine bacterium (AgeI).

A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, "Agrobacterium gelatinovorum" IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved lambda and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, phi X174 RF I, M13mp 18 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37 degrees C and pH 7.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 50 mM NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5'-ACCGGT-3' and cut between A and C, producing a 5'-cohesive tetranucleotide extension.