Bioaccumulation and biosorption of zinc by a novel Streptomyces K11 strain isolated from highly alkaline aluminium brown mud disposal site.

Zinc biosorption and bioaccumulation by a novel extremely Zn tolerant Streptomyces K11 strain isolated from highly alkaline environment were examined. Temperature, similarly as biosorbent preparation, has negligible effect on the biosorption capacity but very strong effect on the process kinetics. Initial adsorption rate increased almost 10 times with the temperature increase from 10 to 50 °C and it was 30 times higher when non-dried biomass was used. The biosorption study revealed that the process was mainly chemically controlled, however at lower temperature intra-particle diffusion played significant role in the zinc biosorption. The experimental data fitted the Langmuir isotherm model with the maximum biosorption capacity 0.75 mmol g-1. The results of bioaccumulation onto a living biomass of Streptomyces K11 indicated very high bioaccumulation capacity of 4.4 mmol g-1. Zinc extracellular uptake (43%) slightly exceeded the intracellular accumulation (36%). High zinc bioaccumulation capacity was obviously related to extremely high zinc tolerance of Streptomyces K11.

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2).

Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.

N-terminal anchor in surface immunogenic protein of Streptococcus agalactiae and its influence on immunity elicitation.

A multiplex PCR method was developed to characterize the potential variability within the sip gene from bovine isolates of Streptococcus agalactiae. An incomplete sip gene (ncosip) was found in four isolates of S. agalactiae. According to the in silico analysis of the missing region at amino acid level, the N-terminal of surface immunogenic protein (Sip) was found to contain a LysM domain motif; whilst the incomplete Sip (NcoSip) lacked a part of this motif. Immunity elicitation against Sip was confirmed by immunization of mice. This response was partially observed also with NcoSip.

Complete genome sequence and analysis of the Streptomyces aureofaciens phage mu1/6.

The entire double-stranded DNA genome of the Streptomyces aureofaciens phage mu1/6 was sequenced and analyzed. Its size is 38.194 kbp with an overall molar G+C content of 71.2 %. Fifty-two potential open reading frames (orfs) were identified, divided into two oppositely transcribed regions. In the left arm of the mu1/6 genome, an identified putative integrase and possible regulation proteins were identified. The rightwards transcribed region contains genes organized into apparently four functional units responsible for: (i) replication, (ii) DNA packaging and head assembly, (iii) tail morphogenesis, and (iv) lysis. Putative functions were assigned to twelve orfs based on bioinformatic analysis or experimental substantiation. Comparative analysis with three complete genomes of streptomycete phages revealed resemblance with respect to the organization of their genes into functional modules. Closer relationship was observed only between mu1/6 and S. venezuelae phage VWB.

Development of specific and rapid detection of bacterial pathogens in dairy products by PCR.

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.

The use of bacteriophages in eliminating polyresistant strains of Staphylococcus aureus and Streptococcus agalactiae.

Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains.

Characterization of a complex restriction-modification system detected in Staphylococcus aureus and Streptococcus agalactiae strains isolated from infections of domestic animals.

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates of Staphylococcus aureus and Streptococcus agalactiae obtained from clinical material. Among the 100 isolates of S. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I and Sau33 I); the targeting sequence was determined as 5'-GGN CC-3' (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I, Sau93 I, Sau96* I, Sau98 I) and enzymes recognized sequence 5'-CTY RAG-3' (SmlI isoschizomer). Analysis of 40 isolates of S. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23; Sag16 I and Sag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5'-CTG CA/G-3' (PstI isoschizomer). In RMS-positive S. aureus and S. agalactiae isolates plasmid DNA capable of replication in Escherichia coli and Bacillus subtilis was also detected and isolated.

Identification of a holin encoded by the Streptomyces aureofaciens phage micro1/6; functional analysis in Escherichia coli system.

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin of Streptomyces aureofaciens phage micro1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. The holmicro1/6 gene was able to complement the defective lambda S allele in the nonsuppressing Escherichia coli HB101 strain to produce phage progeny, This fact suggests that the proteins encoded by both phage genes have analogous function, i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the lambda endolysin gains an access to its substrate, the cell wall. The concomitant expression of both S. aureofaciens holmicro 1/6 and lambda endolysin in E. coli resulted in abrupt cell lysis. This result provided further evidence that the product of holmicro 1/6 gene is a holin.

Identification and characterization of an endolysin encoded by the Streptomyces aureofaciens phage mu 1/6.

An open reading frame homologous to the genes encoding several cell-wall hydrolyzing enzymes was identified on the genome of actinophage mu 1/6. This open reading frame encoding the putative endolysin was amplified by polymerase chain reaction and cloned into the expression vector pET-21a. This gene consisted of 1182 bp encoding a 393 amino acid polypeptide with a molar mass of 42.1 kDa. The gene product was overexpressed in Escherichia coli, and then the lytic enzyme was purified by a two-step chromatographic procedure. When applied exogenously, the endolysin of phage mu 1/6 was active against all tested Streptomyces strains but did not affect other bacteria. The amino acid sequence showed a high homology with a putative amidase of the Streptomyces phase phi C31. Downstream of the endolysin gene, an open reading frame encoding an 88 amino acid protein was identified. Structural analysis of its sequence revealed features characteristics for holin.

Connection between foreign DNA replication and induced expression of the restriction-modification system in Streptomyces aureofaciens.

Tetracycline-producing strains of Streptomyces aureofaciens expressed SauLPI restriction-modification (R-M) system, which recognized specific DNA sequence 5'-GCCGGC-3' (isoschizomer Nael). The activation of the second R-M system SauLPII (5'-GAGCTC-3', isoschizomer of XhoI), which was silent during the growth cycle, after a foreign DNA transfer into this strain was observed. This phenomenon was tentatively explained as a response of the cells against the exogenous DNA entering the cells. The involvement of a SOS-like response in induction of R-M system genes in S. aureofaciens strains has been considered.

An origin of DNA replication from streptomycete phage phi U1.

A DNA fragment from phage phi U1 containing an origin of DNA replication was identified. This fragment, designated ori, was able to support the maintenance in Streptomyces lividans of a plasmid lacking a functional Gram-positive ori. The sequence of the minimal ori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in this ori fragment indicates that host factors alone were sufficient to initiate replication at ori.

Characterization of a XhoI isoschizomer in Streptomyces aureofaciens after actinophage infection.

After infection of tetracycline producing strains of S. aureofaciens with actinophages mu 1/6 and B1 some phage resistant colonies were obtained in each experiment. These colonies expressed a new restriction-modification (RM) system of type II, which was different from the common RM system (SauLPI) of these strains recognizing the sequence GCCGGC. This new RM system was not detected before in parental strains. The new endonuclease was purified from a phage resistant strain of S. aureofaciens B96, using two step column chromatography to the grade without non-specific nucleolytic activity. SauLPII endonuclease recognized and cleaved the palindromic hexanucleotide sequence 5'-C/TCGAG-3', thus it was a true isoschizomer of XhoI.

Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5'-AT/TAAT-3'.

Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5'-AT/TAAT-3' generating 5'dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of vspI, a restriction enzyme isolated from Vibrio sp.

SbvI restriction endonuclease from Streptococcus bovis.

Restriction endonuclease SbvI, an isoschizomer of HaeIII, has been isolated from rumen amylolytic bacterium Streptococcus bovis II/1. SbvI was purified from cell extract by phosphocellulose chromatography and heparin-Sepharose chromatography. The recognition sequence of SbvI was identified by digestion of pBR322, pUC9 and lambda-DNA and comparing the cleavage patterns obtained with computer-derived data. SbvI recognizes the 4-bp palindrome, 5'-GGCC-3' and cleaves DNA after the second G in the sequence, producing blunt ends.

SruI restriction endonuclease from Selenomonas ruminantium.

SruI, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT decreases AAA-3'. The recognition sequence of SruI was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and lambda DNA. The cleavage patterns obtained were compared with computer-derived data. SruI recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease SruI presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism possesses a DNA-cleaving enzyme with the same specificity as DraI or AhaIII.

Cloning and characterization of an amplified DNA sequence in chromosomal DNA of Streptomyces aureofaciens 2201.

In strain 2201 of Streptomyces aureofaciens, a high copy number amplified DNA sequence (ADS-Sa2201) was found and characterized. The amplified sequence in the chromosomal DNA of this strain forms a stretch of about 10 kb tandemly repeated 100-500 times. In this strain also, extrachromosomal copies of the repeated unit of the ADS-Sa2201 were found. In cloning experiments any autonomous replicon was found on ADS-Sa2201 and it thus can be presumed that the presence of the extrachromosomal copies of the repeated unit of ADS-Sa2201 is only a result of its excision from the chromosome. A part of the repeated unit of ADS-Sa2201 was also found in chromosomal DNA of strain 13 of S. aureofaciens. In this strain no part of ADS-Sa2201 is present extrachromosomally.

Highly transformable mutants of Streptomyces aureofaciens containing restriction-modification systems.

Streptomyces aureofaciens 13 is a mutant defective in chlortetracycline production. It was chosen as a potentially useful host for gene cloning in investigations of the organization of the biosynthetic genes for the tetracycline antibiotic pathway. From the Streptomyces aureofaciens 13 strain, three suitable clones were used for our work. The conditions for optimal formation and efficient transformation of protoplasts with plasmid DNAs have been determined. Transformation frequencies of about 10(4) to 10(5) per microgram of plasmid DNA were obtained when plasmids were isolated from Streptomyces strains. From the patterns of restriction enzyme digestion of plasmid DNA isolated from Streptomyces aureofaciens transformants, it was observed that the clones express modification systems which render plasmid DNAs resistant to cleavage by HindIII and EcoRI. Additionally, one of the clones produces the restriction endonuclease Sau13I (isoschizomer of SauI). The presence of the restriction-modification system of Sau13I does not reduce the efficiency of plasmid transformation.

The Streptomyces aureofaciens plasmid pIMB R8 and its use for shuttle vector construction.

The cryptic low copy plasmid designated as pIMB R8 was isolated from an industrial strain of the chlortetracycline producer Streptomyces aureofaciens R8/26. Using restriction endonucleases the pIMB R8 plasmid was characterized to have 15 kb. Subcloning of the Bam HI fragments of pIMB R8 into replication probe vector resulted in the identification of the replication part. The 0.7 kb Bc/I--Bg/I fragment is sufficient for normal replication, but produces about fourty times high plasmid copy numbers than the original pIMB R8. Using the replication part of pIMB R8 was constructed several shuttle cloning vectors for the E. coli-streptomycete system.