Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml⁻¹), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0). Received 02 December 1996/ Accepted in revised form 25 August 1997     

Characteristics and Genetic Determinants of Bacteriocin Activities Produced by Carnobacterium piscicola CP5 Isolated from Cheese

Carnobacterium piscicola CP5, isolated from a French mold-ripened soft cheese, produced a bacteriocin activity named carnocin CP5, which inhibited Carnobacterium, Enterococcus and Listeria spp. strains, and among the Lactobacillus spp. only Lactobacillus delbrueckii spp. [24]. The activity was purified by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography followed by reverse-phase high-performance liquid chromatography (RP-HPLC). This latter step separated two peaks with anti-listerial activity (CP51 and CP52). Carnocin CP51 was partially sequenced, and the N-terminal part revealed the presence of the “pediocin-like consensus” sequence-Tyr-Gly-Asn-Gly-Val-. Then, a degenerated 24-mer oligonucleotide probe was constructed from the N-terminal sequence and used to detect the structural gene. It was localized on a plasmid of about 40 kb. Cloning of restriction fragments of this one, followed by DNA sequencing, revealed the presence of the second anti-Listeria bacteriocin gene (CP52). By comparing sequences in data banks and confirming results with PCR reactions, carnocin CP51 shared homologies with carnobacteriocin BM1, and carnocin CP52 was similar to carnobacteriocin B2, both produced by C. piscicola LV17 [2]. However, carnobacteriocin A from C. piscicola LV17 gene was lacking in C. piscicola CP5, and the two microorganisms have been isolated from different ecological environments: C. piscicola CP5 and C. piscicola LV17 were isolated from soft cheese and vacuum-packed meat respectively. This fact could allow different application perspectives for C. piscicola CP5. Received: 16 April 1997 / Accepted: 9 May 1997     

Properties of a bacteriocin produced by Carnobacterium piscicola CP5

Summary Carnobacterium piscicola CP5 produced a bacteriocin named carnocin CP5 that inhibited Carnobacterium, Enterococcus and Listeria spp. and among the Lactobacillus spp. only Lactobacillus delbrueckii ssp. Carnocin CP5 was stable 1h at 100°C at pH 7.0. It was inactivated by numerous proteolytic enzymes. Production of carnocin, CP5 occured in MRS broth regulated at pH 7.0. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa, but around 5 kDa after action of SDS or urea. Novobiocin treatment led to non-producer variants.     

Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.     

Large scale purification protocol for carnocin KZ 213 from Carnobacterium piscicola

Large scale purification of a class IIa bacteriocin has been developed to recover pure carnocin KZ 213 produced by Carnobacterium piscicola 213. Most previous protocols reported in the literature for the purification of small peptides have used reversed phase chromatography but scale-up is difficult. The first step of this new protocol is hydrophobic interaction chromatography, the second and third steps are cation exchange chromatography. The protocol leads to a complete recovery of carnocin KZ 213 with 95% purity and to a concentration factor of 83. From 10 l culture supernatant, 5.8 mg carnocin KZ 213 have been produced with a specific activity of 8,500 UA g⁻¹. The protocol is easy to implement for larger volumes.     

Purification and cloning of piscicolin 61, a bacteriocin fromCarnobacterium piscicola LV61

Piscicolin 61, a bacteriocin produced byCarnobacterium piscicola LV61, inhibits the growth of strains ofCarnobacterium, Lactobacillus, andEnterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculatedM _r from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

Purification and characterization of lanatoside 15′-O-acetylesterase from Digitalis lanata Ehrh.

Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s⁻¹ · (g protein)⁻¹. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s⁻¹ · (g protein)⁻¹. Received: 25 March 1997 / Accepted: 3 July 1997     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

In vitro Inhibition of Clinical Isolates of Otitis Media Pathogens by the Probiotic Streptococcus salivarius BLIS K12

Otitis media is a common childhood infection, frequently requiring antibiotics. With high rates of antibiotic prescribing and increasing antibiotic resistance, new strategies in otitis media prevention and treatment are needed. The aim of this study was to assess the in vitro inhibitory activity Streptococcus salivarius BLIS K12 against otitis media pathogens. Efficacy of the bacteriocin activity of S. salivarius BLIS K12 against the otitis media isolates was assessed using the deferred antagonism test. Overall, 48% of pathogenic isolates exhibited some growth inhibition by S. salivarius BLIS K12. S. salivarius BLIS K12 can inhibit the in vitro growth of the most common pathogens.

     

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid leader peptide with six Arg⁺ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0 to pH 10.0. Received: 9 January 1997 / Received revision: 27 March 1997 / Accepted: 13 April 1997     

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h. Received: 27 January 1997 / Received revision: 18 March 1997 / Accepted: 13 April 1997     

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L⁻¹) was obtained in 9 h with 20 g L⁻¹ glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L⁻¹. Maximum bacteriocin activity (2.9–3.2 MBU L⁻¹) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h⁻¹ and specific bacteriocin production rate increased linearly with dilution rate. Received 31 July 1996/ Accepted in revised form 01 November 1996     

Enantioselective hydration of 2-arylpropionitriles by a nitrile hydratase from Agrobacterium tumefaciens strain d3

The enantioselective nitrile hydratase from the bacterium Agrobacterium tumefaciens d3 was purified and completely separated from the amidase activity that is also present in cell extracts prepared from this strain. The nitrile hydratase had an activity optimum at pH 7.0 and a temperature optimum of 40 °C. The holoenzyme had a molecular mass of 69 kDa, the subunits a molecular mass of 27 kDa. The enzyme hydrated various 2-arylpropionitriles and other aromatic and heterocyclic nitriles. With racemic 2-phenylpropionitrile, 2-phenylbutyronitrile, 2-(4-chlorophenyl)propionitrile, 2-(4-methoxy)propionitrile or ketoprofen nitrile the corresponding (S)-amides were formed enantioselectively. The highest enantiomeric excesses (ee >90% until about 30% of the respective substrates were converted) were found for the amides formed from 2-phenylpropionitrile, 2-phenylbutyronitrile and ketoprofen nitrile. For the reaction of the purified nitrile hydratase, higher ee values were found than when whole cells were used in the presence of an inhibitor of the amidase activity. The enantioselectivity of the whole-cell reaction was enhanced by increasing the reaction temperature. Received: 20 June 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C. Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998     

Purification and N-Terminal Amino Acid Sequence of Dextranicin 24, a Bacteriocin of Leuconostoc sp.

Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH₂₋ K G V L G W L S M A S S A L T G P Q Q . . . Received: 26 January 1996 / Accepted: 28 March 1996     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin‐like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin‐positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin⁺ trait is maintained in animals at the same location. The HC5‐like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5‐like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.