This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631—the first report of a full-length hemolysin gene for the species. An amplicon (∼600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.
Staphylococcus epidermidis isolated from spoiled frozen marine fish samples exhibited optimum lipase activity of 8.1 U within 72 h in batch fermentation. Inducible effect of different sugars, nitrogen sources, salts and metal ions were studied on enzyme production. Trybutyrin induced the enzyme production by twofold. Addition of lactose in the production medium further improved lipase production. Sodium chloride increased lipase production whereas the presence of metals in the media had an inhibitory effect. Cells of immobilized S. epidermidis in agar beads (3%) increased lipase production compared with free cells. The optimum temperature and pH for enzyme activity was 20°C and 7.0 respectively. Lipase retained its 85% stability at pH 6.0 and at 40°C. Immobilized cells with high lipolytic activity and stability may provide commercial advantages over conventional methods of lipase production.
The high-resolution amplified fragment length polymorphism technique (AFLP), with single PstI restriction endonuclease and two selective primers (PstI-G and PstI-GC), was used for genomotyping and study of the genomic relationships between Genista tinctoria microsymbionts sampled in England, Poland, and Ukraine. Out of 906 amplification products obtained with both selective primers, 537 markers were polymorphic and could be used to differentiate studied nodule isolates. Cluster analysis, based on AFLP patterns from PCR reaction with PstI-G and PstI-GC primers, separated Genista tinctoria rhizobia into three subgroups according to their geographic origin. The results presented in this paper emphasize the role of AFLP analysis in taxonomic and ecological studies of rhizobia.
Three hundred and eight presumed enterococcal isolates were recovered from Bryndza, a soft sheep milk cheese. The cheese samples were obtained from five different commercial distributors in Slovakia and were taken at three different seasonal intervals. All isolates were identified to the species level using genotypic tools. Species-specific PCR using ddl genes highlighted the predominance of Enterococcus faecium (176 isolates) and assigned 50 isolates to the species Enterococcus faecalis. The remaining 82 isolates were classified using repetitive element sequence-based polymerase chain reaction (PCR) with primer (GTG)5–(GTG)5-PCR, in combination with phenylalanyl-tRNA synthase gene (pheS) sequence analysis and by whole-cell protein analysis (SDS-PAGE). These strains were identified as Enterococcus durans (59 strains), Enterococcus italicus (8 strains), Enterococcus casseliflavus (3 strains), Enterococcus gallinarum (3 strains), Enterococcus hirae (1 strain), and 8 strains were members of the species Lactococcus lactis. Of the seven enterococcal species isolated, three of them, E. durans, E. faecalis and E. faecium were present in all samples studied, with E. faecium as the predominant one. The precise identification of enterococci in Bryndza cheese is an essential step in the process of evaluation of their functional properties which will be further studied and assessed.
A halotolerant and alkaliphilic Gram-negative bacterium, strain 18bAGT, that grows aerobically at the optimum temperature of 37°C, and at pH 7.5–10 (optimum 9.0), was isolated from a salt pool located in Montefredane in Campania Region (South of Italy). The isolate tolerated high concentration of NaCl up to 20%. Strain 18bAGT accumulated osmolytes and polyhydroxybutyrate, produced exopolysaccharide and possessed α-glucosidase activity. The predominant respiratory quinones were ubiquinones, Q8 and Q6(6H); phosphoethanolamine, phosphatidylglycerol and diphosphatidylglycerol were the predominant polar lipids. Major fatty acids were C16 : 1, C16 : 0, and C18 : 0. On the basis of 16S rRNA gene sequence similarity, 18bAGT was shown to belong to Halomonas genus. Analysis of 16S rRNA gene revealed a high similarity of strain 18bAGT to Halomonas venusta (DSM 4743T) and Halomonas hydrothermalis (DSM 15725T). Level of DNA-DNA relatedness between strain 18bAGT and the most related species Halomonas venusta and Halomonas hydrothermalis was 56.0% and 41.2%, respectively. The G+C content (mol%) of DNA was 53.0. The RiboPrinting patterns of Halomonas venusta and 18AGT showed a pattern similarity of 0.50. On the basis of genomic information and phenotypic characteristics strain 18bAGT represents a new species, for which the name Halomonas alkaliphila sp. nov. is proposed. The type strain is 18bAGT (=DSM 16354T =ATCC BAA-953T).
Based on MRS medium, two types of food grade (FG) culture media (FG medium I and FG medium II) for the preparation of a concentrated starter culture of Lactobacillus plantarum NRIC 0380 to manufacture a new type of instant Chinese noodle, the fermented instant Chinese noodle, were developed using FG materials. FG medium I, which is for normal static culture, contains table sugar (sucrose), Yeast peptone standard type F, Sunsoft Q-17S (emulsifier), sodium acetate, trisodium citrate and MnSO4·4–5H2O. FG medium II was designed to be used for the pH-controlled jar fermentor culture conditions. Therefore, sodium acetate and trisodium citrate as a buffer to prevent acidification of medium were omitted from FG medium I. When L. plantarum NRIC 0380 was cultured under the pH-controlled jar fermentor culture conditions, the kinetics of growth, sugar consumption and lactic acid production in FG medium II were quite similar to those observed in the Difco Lactobacilli MRS Broth. Furthermore, growths of many lactobacilli strains isolated from various fermented foods in FG medium I were also quite similar to those observed in MRS medium. Therefore, simple and practical FG media for the culture of lactobacilli were successfully established.