Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).     

Resistance Transfer Factors in sensitive strains ofS. panama

Two natural strains ofS.panama with an incomplete Resistance Factor (R factor) are described:S.panama I carries a Resistance Transfer Factor (RTF) exerting restriction on phageS.panama 47, but no resistance determinants; andS.panama 219 has a tetracycline-resistance determinant but no RTF.A number of drug-sensitive strains ofS.panama belonging to various phage types, were found to carry a factor which is able to mobilise the tetracycline-resistance determinant ofS.panama 219 and also of one strain ofE.coli and to transfer them toS.panama andE.coli K 12. This factor is not identical with the F factor, it is not a bacteriocinogenic factor and can therefore be considered as an RTF. These RTFs exert no restriction on (spp) and phage 47, and some of them are fi whilst others are fi.     

Influence of resistance-factors on the phage types ofSalmonella Panama

The resistance to antibiotics which has been increasingly observed in naturally occurringSalmonella panama, is due to an R-factor. A relationship was found between phage pattern and the presence of this R-factor. All strains belonging to phage types A, C and E are sensitive to all antibiotics and are indicated in phage-typing by wild-type phage 47 or host-range mutants of phage 47. All strains belonging to phage types B, D and F possess an R-factor and are indicated by host-modified variants of phage 47. Phage type G, indicated by a host-range mutant, and group Z contain strains with, as well as without an R-factor. Spontaneous drug-sensitive segregants of type B, D and F strains have the phage pattern A, C and E respectively. Conversely, the phage pattern of A, C and E type strains change into B, D and F respectively after infection with the R-factor ofS. panama. The theory can be advanced that type B type A+R-factor, D — C+R-factor and F = E+R-factor. This change in phage type can be considered to be due to the fact that the R-factor exerts restriction and modification of the phage which indicates theS. panama strain without the R-factor.Many of the antibiotic-resistantEscherichia coli strains found in nature possess an R-factor which can be transferred toS. panama in vitro. Relatively few of these R-factors were found to possess also the restriction marker. Thus up to the present the number ofE. coli strains possessing an R-factor which is able to create a dependable combination of phage type and drug resistance inS. panama is relatively small.     

Streptomyces aureofaciens strains as hosts for cloning of genes affecting antibiotic production

Summary Several mutants ofStreptomyces aureofaciens strain were used for protoplast regeneration and plasmid transformation. All tested mutants (excepting R 8/26) were transformable by number of plasmids and shuttle vectors. The transformation of the CTC production strains by plasmid containing cloned CTC resistance gene resulted in 1,1–4 times higher antibiotic production. From the restriction analysis of plasmid, phage and chromosomal DNAs it was estimated, that all tested mutants normally contain the modification system analogous toNae I (Roberts, 1987). Mutant R 8/26 expresses not only complete restriction-modification system mentioned above but also potential second system restricting several actinophages.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4–38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.     

Restriction/modification in Streptococcus thermophilus: isolation and characterization of a type II restriction endonuclease Sth455I

Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I. This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography. This yielded an enzyme preparation free of non-specific nucleases. The optimal reaction conditions for Sth455I are: MgCL₂, 30 mm; pH range, 8–9; incubation temperature, 37–42°C; and a high NaCl concentration, 100–200 mm. The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation. The enzyme exhibits restriction activity on the DNA of three bacteriophages of S. thermophilus and no activity on the phage lytic for strain CNRZ 455. The restriction/modification system associated with this strain is discussed.     

A novel transducing phage

Summary Two newly isolated generalized transducing phages, F126 and F130, are in many respects similar to the previously described phage F116 of P. aeruginosa strain PAO. They also share with F116 the property of not responding to host-specific restriction in strain PAO. However, while the transduction ability of phages F116 and F130 is also inert to PAO-specific restriction, transduction mediated by phage F126 is 8–120 fold reduced in restrictive conditions. In experiments designed to explore the conditions necessary to obtain restriction in F126 transduction it was found that it differed from those previously known to specify host-controlled restriction in P. aeruginosa PAO (Rolfe and Holloway, 1968). These observations suggest that more than one independent restriction system operates in strain PAO.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid. Received: 15 October 1996 / Accepted: 3 December 1996     

Conjugative transfer of tetracycline resistance in rumen streptococcal strains

In 11% of testedStreptococcus bovis strains a conjugative transfer of tetracycline resistance was observed when mating experiments were carried out on membrane filters. The recipient strain used wasS. bovis BM114 with chromosomal resistance to rifampicin. In addition, in two strains tetracycline resistance was transferred also to recipient strainEnterococcus faecium AL6. The transfer frequencies were in the range of 10⁻⁶ to 10⁻³. The donor strains were screened for the presence of plasmids and one up to four bands of plasmid DNA in all tested strains were revealed. In spite of that isolation of plasmid DNA was successful only in 53/4/114 transconjugants. Transconjugant 32/114 contained amylase activity which was higher than in the donor strain.     

A special case of lysogeny inMycobacterium avium

Phage-producing colonies of cells resistant to homologous phage F4II have been isolated after infection ofMycobacterium avium. About one per cent phage-free, sensitive clones can usually be obtained after treatment of the cell suspension with phage antiserum. Because of the relatively high free phage titer in the culture, the presence of very dense particles, probably phage equivalents, in the cytoplasm of many cells and the varied sensitivity of intracellular phage to UV irradiation at different stages of growth, one can assume that a great part of the cells in the population burst and liberate phages. The relationship between phage and the host cell is discussed as a particular case of abortive lysogeny.     

Restriction of streptomyces phage SH5 by endonuclease ShyI from Streptomyces hygroscopicus 0477

Summary DNA of the temperate Streptomyces phage SH5 (DNA molecular weight 27x10⁶) is subject to restriction-modification mediated by S. hygroscopicus 0477, S. levoris 1331 and 2340. The restriction endonuclease ShyI (isoschizomeric with SacII) isolated from S. hygroscopicus 0477 is involved in restriction of SH5·1331 and SH5·2340 DNAs in S. hygroscopicus 0477.     

Acetobacter bacteriophage A-1

A bacteriophage ofAcetobacter suboxydans was isolated and found to correspond to type A phage according to Bradley's classification. The phage contains double stranded DNA. The length of the latency period and burst size could not be precisely determined because of apparent non-synchronous release of phage from single infective cycles. The host range was determined using 24 strains ofAcetobacter andGluconobacter species. Evidence for a probable occurence of host determined restriction and modification was obtained withAcetobacter suboxydans strain ATCC 621. The phage is designated A-1 and it is the first one to be reported forAcetobacter.Abbreviations pfu plaque forming units - PTA phosphotungstic acid - GC guanarine + cytosine     

RFLP analysis of the PCR-amplified 28S rDNA inRhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.     

Characterization of phage isolates from a phage-carrying culture of Leuconostoc oenos 58N

Summary During large-scale cultivation of Leuconostoc oenos strain 58N, growth inhibition was detected and attributed to the presence of the virulent phage P581. To determine if this phage originated from a temperate phage, L. oenos 58N was exposed to mitomycin C, and this treatment led indeed to release of phages (P58II). Further examination of the lytic potential of phages P581 and P58II revealed that these two phages were able to lyse the same strains of L. oenos with the exception of the original host strain, which was only sensitive to P581. Results of DNA/DNA hybridization experiments failed to show homology between the DNA of phage P58II and the chromosomal DNA of L. oenos 58N. A phage-free culture of L. oenos 58N could be obtained after repeated subculture. These results indicate that the original L. oenos 58N was in a special type of phage-carrier state. Phages P58I and P58II were compared on the basis of morphology, lytic spectra, restriction enzyme analysis, DNA homology, genome size and protein structure and proved to be identical. It is assumed that P58I arose from the phage-carrier culture of L. oenos 58N and became virulent by some mutational event.Offprint requests to: E. K. Arendt     

Restriction and modification of phage 47 and lambda by R factors

S. panama 47 (antibiotic-sensitive, phage pattern A) was infected with R factors from a number of field strains of Enterobacteriaceae (Salmonella, Escherichia coli, Citrobacter, Enterobacter, Klebsiella andProteus) isolated from human and animal sources. These R factors could be grouped into 11 types i.e. R1 R11 on the basis of induced changes in the phage type of the recipient.R8 and R11 renderS. panama resistant to the phages A H:S. panama 47 (R3) and 47 (R6) adsorb the phages A F, but there is no phage multiplication: phages G and H are considered to be restricted and modified in these strains. The R factors R5 and R7 also exert restriction and modification on a number of the typing phages A H. The nature of the changes in phage pattern brought about by R4, R9 and R10 is not understood. R2 does not exert restriction (i.e. no change in phage pattern). The R factors were also investigated for the fi (fertility inhibition) and spp (restriction of phage ) markers.The R factors R3 R11 readily segregate, in the sense that the restriction and modification loci, and occasionally the Resistance Transfer Factor as a whole was frequently lost after R transfer. These 9 types of R factors were encountered infrequently in the present material.Resistance to tetracycline inS.panama is nearly always due to R factors of type R1. In other members of Enterobacteriaceae, notably inE.coli, R1 is less frequently found than R2.     

OCCURRENCE OF A TEMPERATE CYANOPHAGE LYSOGENIZING THE MARINE CYANOPHYTE PHORMIDIUM PERSICINUM1

A temperate cyanophage was found to lysogenize the marine cyanophyte Phormidium persicinum (Reinke) Com. (Provasoli strain). The lytic cycle was induced by the addition of mitomycin C or by brief illumination with ultraviolet light. The lytic process observed under the electron microscope showed that phage particles appeared in a nucleoplasm region 15 to 24 h after the addition of mitomycin C. The induction of the lytic process occurred simultaneously in almost all cells of every trichome. Matured phage particles were released to the medium 30 to 50 h after the addition of mitomycin C. Phage particles isolated from algal lysates had a polyhedral head (about 40 nm in diameter) with a long (about 300 nm) and noncontractile tail. The most abundant protein, presumably a structural protein, had an apparent molecular mass of about 38 kDa. The genome size estimated from restriction analysis was about 50 kbp. Phage DNA was digested with several restriction endonucleases including Sau3AI and DpnI. However, MboI failed to digest the phage DNA, suggesting that the phage DNA is highly methylated. Southern blot analysis suggested that some part of the phage was in the lytic cycle in algal cells growing under normal conditions. A possible role of temperate cyanophages in the regulation of cyanophyte populations in the marine environment is discussed.