The yeast genus Tortispora gen. nov., description of Tortispora ganteri sp. nov., Tortispora mauiana f.a., sp. nov., Tortispora agaves f.a., sp. nov., Tortispora sangerardonensis f.a., sp. nov., Tortispora cuajiniquilana f.a., sp. nov., Tortispora starmeri f.a., sp. nov. and Tortispora phaffii f.a., sp. nov., reassignment of Candida caseinolytica to Tortispora caseinolytica f.a., comb. nov., emendation of Botryozyma, and assignment of Botryozyma, Tortispora gen. nov. and Trigonopsis to the family Trigonopsidaceae fam. nov.

We describe the yeast genus Tortispora gen. nov., an early-diverging lineage in the Saccharomycetales that displays the formation of helical ascospores. The genus is based on 16 strains resembling Candida caseinolytica that were isolated from necrotic plant tissue in warm regions of the New World. Based on sequences of the D1/D2 domains of the nuclear large subunit rRNA gene, as well as other data, the strains are assigned to eight distinct species. The species are nutritionally specialized and share the unusual ability to hydrolyse casein and to grow on 1-butanol as sole carbon source. One species of the proposed new genus produces a simple ascus with a helical ascospore, whereas other species of the clade have failed to form ascospores. All species in the clade, including C. caseinolytica, are assigned to Tortispora gen. nov. The new binomials are Tortispora ganteri sp. nov., type species of the genus (SUB 86-469.5T = CBS 12581T = NRRL Y-17035T), Tortispora caseinolytica f.a., comb. nov. (UCD-FST 83-438.3T = CBS 7781T = NRRL Y-17796T), Tortispora mauiana f.a., sp. nov. (UWOPS 87-2430.3T = CBS 12803T = NRRL Y-48832T), Tortispora agaves f.a., sp. nov. (UWOPS 94-257.6T = CBS 12794T = NRRL Y-63662T), Tortispora sangerardonensis f.a., sp. nov. (UWOPS 00-157.1T = CBS 12795T = NRRL Y-63663T), Tortispora cuajiniquilana f.a., sp. nov. (UWOPS 99-344.4T = CBS 12796T = NRRL Y-63664T), Tortispora starmeri f.a., sp. nov. (G 91-702.5T = CBS 12793T = NRRL Y-63665T) and Tortispora phaffii f.a., sp. nov. (UWOPS 91-445.1T = CBS 12804T = NRRL Y-48833T). In addition, species formerly assigned to the genus Ascobotryozyma are reassigned to the genus Botryozyma. The genera Trigonopsis, Botryozyma and Tortispora are assigned to the family Trigonopsidaceae fam. nov.

Bacillus endoradicis sp. nov., an endophytic bacterium isolated from soybean root

A Gram-positive, aerobic, motile rod, designated strain CCBAU 05776T, was isolated from the inner tissues of a healthy soybean (Glycine max L.) root collected from an agricultural field in the countryside of Shijiazhuang city, Hebei Province, China. Phylogenetic analysis of the 16S rRNA gene indicated that this strain was most closely related to Bacillus muralis LMG 20238T and Bacillus simplex NBRC 15720T with similarity of 96.5 % and 96.3 %, respectively, lower than the suggested threshold (97.0 %) for separating bacterial species. In phenotypic characterization, the novel strain differed from the two most related species in that it did not hydrolyse casein or starch but could grow on MacConkey agar. It grew between 15 and 45 °C and tolerated up to 7 % NaCl (w/v). Strain CCBAU 05776T grew in media with pH 5.5 to 10 (optimal growth at pH 7.0–8.0). The predominant cellular fatty acids were iso-C15 : 0 (40.81 %) and C16 : 1ω7c alcohol (10.61 %). The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C was 40.8 mol% (T m). DNA–DNA relatedness of the novel isolate with B. muralis and B. simplex was 42.4 % and 32.7 %, respectively. Based upon the consensus of phylogenetic and phenotypic analyses, strain CCBAU 05776T represents a novel species within the genus Bacillus, for which the name Bacillus endoradicis sp. nov. is proposed. The type strain is CCBAU 05776T ( = LMG 25492T = HAMBI 3097T).

Genotypic and technological characterization of enterococci isolated from artisanal Fiore Sardo cheese

A total of 118 enterococcal strains isolated from artisanal Fiore Sardo cheese were characterized technologically and genetically. The presence of potential virulence factors was also investigated. Strains were classified as Ec. faecium (84 strains), Ec. durans (24 strains) and Ec. faecalis (10 strains). RAPD-PCR analysis with two different primers (M13 and XD9) confirmed species identification and proved useful for the detection of interstrain variations, especially among Ec. faecium isolates. Most strains could hydrolyse casein and had weak acidifying activity in milk. None of the isolates produced lipolytic reactions. Gelatinase activity was observed in two strains of Ec. faecalis and one strain of Ec. durans. β-Haemolysis on horses' blood was never detected in any of the strains, independently of species. Most strains produced tyramine from tyrosine but none decarboxylated lysine, histidine or ornithine. Overall, a wide spectrum of resistance was observed. Almost all strains were resistant to the aminoglycosides, gentamycin, kanamycin, streptomycin, neomycin, and to the semisynthetic penicillin, oxacillin, but resistance to vancomycin was not widespread among our strains: only one Ec. faecium and one Ec. durans strain were found to be vancomycin resistant. Our results show a certain diversity in technological traits of the enterococcal strains isolated from artisanal Fiore Sardo, together with a low incidence of some potentially pathogenic traits of health concern.

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris

Aminopeptidase P was purified 65·3-fold from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 with a 5·8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41600. Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position. No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue. However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

A qualitative screening procedure for the detection of casein hydrolysis by bacteria, using sodium dodecyl sulphate polyacrylamide gel electrophoresis

SummaryIncubation of bacteria with casein at 30 °C (pH 7·2) resulted in the formation of a number of peptide fragments. The peptides were separable when subjected to electrophoresis. A proteinase-positive strain ofStreptococcus lactispreferentially hydrolysed the β-casein moiety of whole casein, and after incubation for 5 min produced a characteristic pattern of four peptides on sodium dodecyl sulphate (SDS) polyacrylamide gels. After 30 min incubation only one peptide remained. This single polypeptide was then further hydrolysed to produce one (60 min) and eventually two (120 min) lower molecular weight peptides. Hydrolysis was generally complete after about 480 min. Two proteinase-negative variants of this strain did not hydrolyse casein. Crude proteinase preparations gave the same characteristic peptide patterns. SDS-polyacrylamide gel electrophoresis was used to screen more proteinase-positive strains ofStr. Lactisfor casein hydrolysing activity. Of those so far tested all produced the same peptide patterns from β-casein. Proteinase negative variants of all these strains did not hydrolyse casein.

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 102 to 105 colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 °C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 μg/mL. The medium appeared to be highly selective for P. aeruginosa with 95–100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.