Serological studies of a host range mutant of a lactic streptococcal bacteriophage.

A host range mutant was isolated from a bacteriophage that attacked Streptococcus cremoris 114. The mutant was able to adsorb and grow on S. cremoris 266, where the parent phage could not. The mutant phage was unable to adsorb to the original bacterial host, S. cremoris 114. The change in host range was accompanied by an alteration in the neutralization antigen as shown by a change in neutralization rate by an anti-phage serum. Serum-blocking experiments confirmed the difference in neutralization antigen between parent and mutant phages. The two phages nevertheless had similar complement fixation antigens, confirming that one was a mutant derived from the other. A distinction between complement fixation and neutralization antigens, similar to that found for the coliphages and staphylococcal phages, has therefore been demonstrated for two lactic streptococcal phages.     

Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.     

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Bacteriophage Active Against the Lactic Acid Beverage-Producing Bacterium Lactobacillus casei

A virulent bacteriophage which causes a decrease in acid production during fermentation of a lactic acid beverage named Yakult with Lactobacillus casei was isolated from the abnormal fermentation tank and named PL-1. L. casei S strain was the exclusive host cell among 18 lactic acid bacteria tested. The plaque was round with an average diameter of about 0.5 mm. It exhibited serological cross-reaction with previously isolated J1 phage. Under an electron microscope, the phage had a spermatozoon shape, with an icosahedral head (63 nm) and a long tail (12.5 by 275 nm) with about 55 striae. The free phage particles were stable at pH 5 to 8. The phage was quite sensitive to ultraviolet irradiation or to heating (60 C, 5 min), and the host was more sensitive than the phage to these treatments. Many kinds of antimicrobial chemicals were also phagocidal. Calcium ion (5 mm) was specifically essential for the phage growth cycle. A one-step growth experiment under optimum conditions (37 C and pH 6.0) showed that the eclipse period was about 75 min, that the latent period was 100 min after the phage infection, and that the average burst size was about 200. The possibility of arresting phage development in lactic acid fermentation is discussed.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk "Viili"

"Viili," a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Lysogenic Strains of Group N Lactic Streptococci

A temperate bacteriophage, designated r(1)t, was inducible from the group N lactic streptococcus, Streptococcus cremoris R(1), by ultraviolet irradiation or mitomycin C treatment. Induced lysates produced plaques on lawns of three closely related S. cremoris strains, AM(1), SK(11), and US(3). Strain SK(11) was readily lysogenized. S. cremoris AM(1) was the most reliable indicator strain, although the age of the culture used for seeding plates was critical. Zones of lysis but no plaque formation were observed on lawns of nine additional S. cremoris strains. Phage r(1)t could not be detected in filtrates of stationary-phase R(1) cultures and was near the limits of detection in logarithmically growing cultures. Phage levels were still very low (1 plaque-forming unit on AM(1) per 10 induced cells) in induced lysates of R(1) cultures. These low levels of detectable phage may be attributable to an inadequate indicator, lysogenization of the indicator, adsorption of induced phage to cellular debris, concurrent induction of other undetectable phages, or the production of high proportions of defective phages. Electron micrographs of induced R(1) lysates revealed a high incidence of incomplete phage particles, fragments, and ghosts.     

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.     

TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages.

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Identification of three different plasmid-encoded restriction/modification systems in Streptococcus lactis subsp. cremoris W56

Abstract Streptococcus lactis subsp. cremoris W56 ( S. cremoris W56) is a strain partially resistant to phage attack. Derivatives which had lost either plasmid pJW563 or pJW566 no longer expressed the restriction and modification systems encoded by these plasmids. Genetic evidence for the correlation between the plasmids and the R/M systems was obtained by transformation. In addition, a third R/M system was discovered among the transformants and was shown to be encoded by pJW565. Thus, genetic evidence for at least 3 distinct R/M systems encoded by plasmids in S. cremoris W56 is presented. One of the R/M-systems showed stronger restriction of the isometric phage p2 than of the prolate phage c2. The other two systems restricted both classes of phages with equal efficiencies.     

Partial characterization of coliphage WPK and a comparison with coliphage T3.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.     

Isolation and characterization of a new bacteriophage, KF1, immunologically cross-reactive with F-type pyocins

Pseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively. The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCl and the sedimentation coefficient of the phage were 1.54 g/ml and 392S, respectively. Some other biochemical properties were described. The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4 x 10(7). The density of the DNA in CsCl was 1.719 g/ml, the melting temperature was 95.4 degrees C. The guanine plus cytosine content was calculated to be 60 to 64%.     

一株产L-天冬氨酸酶大肠埃希氏菌的噬菌体分离和生物学特性

【目的】从大肠埃希氏菌CICC 11021S发酵液中分离一株噬菌体,对其生物学特性进行研究。【方法】采用双层平板法分离噬菌体CICC 80003;利用透射电镜观察噬菌体形态;提取噬菌体基因组,核酸内切酶处理并进行凝胶电泳;分析噬菌体最佳感染复数、一步生长曲线、p H和温度稳定性、宿主谱。考察CICC 80003对CICC 11021S生长和L-天冬氨酸酶活力的影响。【结果】CICC 80003噬菌斑圆形透明,有明显晕环;头部规则,直径约50-60 nm,尾部长约120-130 nm;基因组能被核酸内切酶Bam H I和Mlu I切开;最佳感染复数0.1,潜伏期5 min,裂解期25 min,平均裂解量约86个;最适p H值8.0;90°C温育15 min,噬菌体全部失活;能裂解大肠埃希氏菌和沙门氏菌的部分菌株。发生噬菌体污染时,CICC 11021S无法正常生长,基本检测不到L-天冬氨酸酶活力。【结论】CICC 80003属于长尾噬菌体科ds DNA噬菌体,液体环境中能够彻底裂解大肠埃希氏菌CICC 11021S。     

Some properties of 1 P+f bacteriophage for Bacillus brevis var. G.-B and its nucleic acid]

The 1 P+f phage, a virulent mutant of the moderate P+ phage for Bac. brevis var. G.-B., consists of a hexagonal head (90x90 nm) and a long non-contractile tail (340 nm). This phage is characterized by a relatively long latent period (90-110 min) and a low yield (40-50 particles per cell). The 1P+f phage is quite stable at pH values from 1 to 11, insensitive to osmotic shock, treatment with chloroform and acridine orange. The sensitivity of the phage to thermal treatment and UV-radiation has been studied. The nucleic acid of the P+f phage is double-stranded DNA of AT-type (GC equals 34.5 mole %) which contains 5-methylcytosine (0.18 mole %) and N6-methyladenine (0.32 mole%). The level of methylation of cytosine and adenine residues in DNA of the 1 P+f phage does not depend on the host studied (Bac. brevis, P- and S variants). The specificity of methylation of cytosine residues in the S and P- cells appears to be the same. DNA of the 1 P+f phage strongly differs from DNA of the host in nucleotide composition (GC equals 45.7 mole %). Nevertheless, phage DNA is very similar to DNA from Bac. subtilis in the character of pyrimidine distribution (the amount of different pyrimidine isopliths). This may testify to a somewhat common character of the nucleotide sequence organization in DNA of the phage and its host.     

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.     

Unusual structural features of the 5S ribosomal RNA from Streptococcus cremoris

The nucleotide sequence of the 5S ribosomal RNA of Streptococcus cremoris has been determined. The sequence is 5' (sequence in text) 3'. Comparison of the S. cremoris 5S RNA sequence to an updated prokaryotic generalized 5S RNA structural model shows that this 5S RNA contains some unusual structural features. These features result largely from uncommon base substitutions in helices I, II and IV. Some of these unusual structural features are shared by several of the known 5S RNA sequences from mycoplasmas. However, the characteristic bloc of deletions found in helix V of these mycoplasma 5S RNAs is not present in the 5S RNA of S. cremoris.     

Complete Genome Sequence of the Novel Lytic Avian Pathogenic Coliphage NJ01

Bacteriophages of the C3 morphotype, characterized by very long heads that exceed their width several times, are extremely rare among the Podoviridae family members and constitute only 0.5% of over 5,500 phages that have been examined by the electron microscope (H. W. Ackermann, Arch. Virol. 152:227-243, 2007; H. W. Ackermann, Arch. Virol. 146:843-857, 2001). To date, among those phages proven to be C3, only coliphage phiEco32, Lactococcus phage KSY1, Vibrio phage 71A-6, and Salmonella enterica phage 7-11, but no avian pathogenic Escherichia coli (APEC) bacteriophages, have been completely sequenced (A. Chopin, H. Deveau, S. D. Ehrlich, S. Moineau, and M. C. Chopin, Virology 365:1-9, 2007; S. A. Khan, et al., Mol. Cell Probes 15:61-69, 2001; A. M. Kropinski, E. J. Lingohr, H. W. Ackermann, Arch. Virol. 156:149-151, 2011; D. Savalia, et al., J. Mol. Biol. 377:774-789, 2008) and are available in public databases. We isolated a bacteriophage from a scale duck market in Nanjing, Jiangsu province, named NJ01, that infects APEC. Sequence and morphological analyses revealed that phage NJ01 is a C3-like bacteriophage and belongs to the Podoviridae family. Here, we announce the complete genome sequence of phage NJ01 and submit the results of our analysis.