Repeated sequences and the sites of genome rearrangements in bacteriophages ofLactobacillus delbrueckii subsp.lactis

Summary We have sequenced the KIS-element, a 1.5 kb insertion segment present in the genome ofLactobacillus delbrueckii subsp.lactis phage LL-K, but absent from its close relative, phage LL-H. The KIS-element showed some sequence features of a transposable element: it was flanked by direct repeats of a 20 nt long sequence which was in the genome of LL-H as a target sequence. The KIS-element contained two putative ORFs. The C-terminal part of ORF333 consisted of clusters of direct repeats, capable of coding Lys/Arg-Gly-Asp motifs, which are known to be able to bind to glycoproteins. A homologous counterpart of the KIS-element was also found in the genome of prolate-headedL. delbrueckii subsp.lactis phage JCL1032, even though the phage JCL1032 is not a close relative of phage LL-K. The nucleotide sequence comparison between KIS-element and its homologous counterpart in JCL1032 showed that there have occurred several genome rearrangements at the repeat clusters.     

Safety evaluation of Lactobacillus delbrueckii subsp. lactis UO 004, a probiotic bacterium

Lactobacillus delbrueckii subsp. lactis UO 004 was evaluated for its use as a potential probiotic from a safety point of view. The strain did not exhibit mucinolytic or other enzymatic activities that might be detrimental, such as those involving glycosidases (beta-D-glucosaminidase or alpha-D-galactosidase) or arylamidases (factor Xa and quimotrypsin-like activities), frequently present in Lactobacillus strains isolated from patients with endocarditis, although it was able to express protein Ca and kallikrein-like activities. On the other hand, the presence of the strain did not interfere with the growth of certain species of normal intestinal microbiota, such as Enterococcus fecalis, Escherichia coli, Bifidobacterium bifidum or Bacteroides fragilis. Moreover, the potential probiotic strain UO 004 is sensitive to antibiotics with transmissible resistance mechanisms in Lactobacillus such as chloramphenicol, erythromycin, tetracycline and vancomycin. In addition, strain L. delbrueckii UO 004 was not able to translocate towards the intestinal barrier of mice or produce changes in their activity or general health status.     

Impact of bile salt adaptation of Lactobacillus delbrueckii subsp. lactis 200 on its interaction capacity with the gut

In a previous work, bile-salt-resistant derivatives were obtained from non-intestinal lactobacilli. The aim of this work was to investigate the impact of bile adaptation of Lactobacillus delbrueckii subsp. lactis 200 on morphology, surface properties, in vivo interaction capacity with the gut and ability to activate the gut immune response. Electron microscopy studies, growth kinetics in the presence of bovine and porcine bile, the capacity to deconjugate bile acids, hydrophobicity, autoaggregation and co-aggregation capacities were studied for the parental strain and its bile-resistant derivative in vitro. Additionally, survival in intestinal fluid, the interaction with the gut and the immunomodulating capacities were studied in mice. Bile salt adaptation conferred upon the adapted strain a higher capacity to withstand physiological concentrations of bile salts and greater survival capacity in intestinal fluid. However, bile salt exposure reduced cell hydrophobicity, autoaggregation and adhesion capacities, resulting in reduced persistence in the intestinal lumen and delayed capacity to activate the gut immune response. Insight into the effects of bile salts upon the interaction and immunomodulating capacity of lactobacilli with the gut is provided, relating in vitro and in vivo results.     

Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539

Summary.  A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages. Received October 16, 1998 Accepted April 20, 1999     

Characterization of bacteriocin HV219, produced by Lactococcus lactis subsp. lactis HV219 isolated from human vaginal secretions

Bacteriocin HV219, produced by Lactococcus lactis subsp. lactis HV219, is active against Gram-positive and Gram-negative bacteria. Activity was lost when treated with proteolytic enzymes, SDS, Triton X-114 and Triton X-100, but not at pH 2.0 to 10.0 or after 20 min at 121 degrees C. Growth in the presence of yeast extract as sole nitrogen source yielded 3200 AU/ml. No bacHV219 activity was recorded in MRS broth with maltose, mannose, lactose or sucrose as sole carbohydrate, but fructose yielded 1600 AU/ml. K(2)HPO(4) at 10.0 g/l yielded 3200 AU/ml. Addition of 1.0 mg/l cyanocobalamin, l-ascorbic acid and thiamine to MRS broth yielded 3200 AU/ml, 1600 AU/ml and 1600 AU/ml, respectively. The mode of activity is bacteriolytic, as confirmed by atomic force microscopy.     

Two DNA antirestriction systems of bacteriophage P1, darA, and darB: characterization of darA- phages

Bacteriophage P1 is only weakly restricted when it infects cells carrying type I restriction and modification systems even though DNA purified from P1 phage particles is a good substrate for type I restriction enzymes in vitro. Here we show that this protection against restriction is due to the products of two phage genes which we call darA and darB (dar for defense against restriction). Each of the dar gene products provides protection against a different subset of type I restriction systems. The darA and darB gene products are found in the phage head and protect any DNA packaged into a phage head, including transduced chromosomal markers, from restriction. The proteins must, therefore, be injected into recipient cells along with the DNA. The proteins act strictly in cis. For example, upon double infection of restricting cells with dar+ and dar- P1 phages, the dar+ genomes are protected from restriction while the dar- genomes are efficiently restricted.     

Characterization of reiterated human DNA with respect to mammalian X chromosome homology

Recombinants containing human repetitive DNA sequences were identified by dot hybridization and classified with respect to presence on the X chromosome and homology to mouse DNA. Using genomic probes that differ in number of X chromosomes, we observed extensive homology between human autosomal and X sequences. Hybridization to genomic probes that differ in species of origin indicate that these reiterated sequences have diverged between mouse and man. Eleven recombinants, each containing a different reiterated sequence(s), were hybridized in situ to metaphase chromosomes of mouse and man. These studies indicate that reiterated DNA which is homologous to the human X chromosome is more similar to DNA of human autosomes than to any murine chromosome. Therefore, it seems that reiterated DNA sequences on the human X chromosome have diverged as much during mammalian evolution as sequences on human autosomes. Moreover, the extensive modification of the original mammalian X has not interferred with the X inactivation process.     

Influence of growth conditions on the production of a bacteriocin by Lactococcus lactis subsp. lactis ST34BR, a strain isolated from barley beer

Bacteriocin ST34BR, a small polypeptide of 2.9 kDa produced by Lactococcus lactis subsp. lactis ST34BR, inhibits the growth of Enterococcus faecalis, Escherichia coli, Lactobacillus plantarum, Lactobacillus casei, Pseudomonas aeruginosa and Staphylococcus aureus. MRS broth, adjusted to pH 6.0 yielded 6,400 AU/ml, compared to 400 AU/ml recorded in BHI broth, M17 broth, 10% (w/v) soy milk, and 8% and 10% (w/v) molasses. At pH of 4.5 only 800 AU/ml was produced. Based on comparative studies in MRS broth, without organic nitrogen, supplemented with different combinations of tryptone, meat extract and yeast extract, tryptone was identified as the stimulating nitrogen compound. Growth in the presence of 20 g/l glucose, maltose, mannose or sucrose yielded bacteriocin levels of 6,400 AU/ml, whereas the same concentration of lactose and fructose yielded 3,200 AU/ml and 1,600 AU/ml, respectively. No difference in bacteriocin ST34BR activity was recorded in MRS broth supplemented with 2 g/l K2HPO4 and 2 g/l, 5 g/l, 10 g/l or 50 g/l KH2PO4. However, 20 g/l KH2PO4 increased bacteriocin ST34BR production to 12,800 AU/ml. Glycerol at 1g/l to 10 g/l in MRS broth reduced bacteriocin activity to 3,200 AU/ml, whilst 20 g/l and 50 g/l yielded only 1,600 AU/ml. The presence of cyanocobalamin, L-ascorbic acid, thiamine and DL-6,8-thioctic acid in MRS broth at 1.0 ppm, respectively, did not result in increased activity levels.     

Citrate metabolism in starved cells of Streptococcus lactis subsp. diacetylactis

The effect of starvation on citrate metabolism by fully grown cells of Streptococcus lactis subsp. diacetylactis was studied by three different procedures. An 8-hour starvation period in fermented medium decreased citrate uptake rate but diacetyl and acetoin levels remained constant over a fermentation without starvation period: about 19 mg l^-1 diacetyl and 130 mg l^-1 acetoin were produced after utilization of 8.8 mM citrate. When fermentation products were discarded from the medium before starvation, diacetyl and acetoin levels reached 30 mg l^-1 and 240 mg l^-1, respectively. When the cells were allowed to rest on an isotonic diluent, citrate uptake rate slowed down, but the diacetyl plus acetoin yield remained higher since the cells accumulated as much acetoin (120 mg l^-1) and more diacetyl (50 mg l^-1). Peak production of diacetyl still coincided with citrate exhaustion whereas a delay in acetoin production was observed.     

Ultrastructure of Bacillus licheniformis bacteriophage BLE and its DNA

A new bacteriophage has been isolated and its form has been described. As far as we know, it is active on Bacillus licheniformis only and is called BLE. The phage particles were negatively stained with phosphotungstic acid. The head of phage BLE is bacilliform and is about 50 nm wide and 130 nm in length. The noncontractile tail is 240 nm in length and 6.3 nm thick and has a cross-striated sheath. The base plate of the tail exhibits the shape of a six-pointed star. From the phage BLE, phenol-extracted DNA molecules were prepared for electron microscopy by the cytochrome c film technique of A. Kleinschmidt and R. K. Zahn (Z. Naturforsch. B, 14, 770–779). Some DNA molecules probably of circular form were observed. Measurement of contour lengths of DNA molecules gave a value 21 ± 0.8 gm, which corresponds to a molecular weight of (41.1 ± 1.5) × 103.     

Complete genomic sequence of a T4-like bacteriophage, phiAS4, infecting Aeromonas salmonicida subsp. salmonicida

A newly identified virulent phage (named phiAS4) infecting Aeromonas salmonicida subsp. salmonicida was isolated from river water in Korea. Morphological analysis of phiAS4 by transmission electron microscopy revealed that it belonged to the family Myoviridae. The genome of phiAS4 comprised a linear double-stranded DNA of 163,875 bp with a G + C content of 41.3%, and genomic analysis revealed 271 putative ORFs, 67 putative promoters, 25 putative terminator regions, and 16 tRNA-encoding genes. Most of the ORFs of phiAS4 showed a high degree of similarity to those of Aeromonas phage 25, which belongs to the T4-like group. Moreover, the comparison of the genome of phiAS4 with those of its relatives demonstrated that phage phiAS4 is closely related to members of the T4-like group and can be classified as a new member of the T4-like phages infecting bacteria of the family Aeromonadaceae.     

Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: prevalence, morphology, host range, and DNA homology

Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillus phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (n = 107) and Turkey (n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus, L. gasseri, and L. jensenii. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple Lactobacillus species. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradley's phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic Lactobacillus strains released phages spontaneously at a high frequency of 10(-3) to 10(-4) PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range, which can be lytic against other vaginal lactobacilli regardless of their geographic origin.     

Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni(2+) affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.     

Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.     

Legionella pneumophila serogroup Lansing 3 isolated from a patient with fatal pneumonia, and descriptions of L. pneumophila subsp. pneumophila subsp. nov., L. pneumophila subsp. fraseri subsp. nov., and L. pneumophila subsp. pascullei subsp. nov

Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3.     

The DNA of bacteriophage 643: isolation and properties of the DNA of a bacteriophage infecting lactic Streptococci

The DNA extracted from bacteriophage 643, which infects group N strains of Streptococci, could be separated into four components by electrophoresis on agarose gels. Electron microscopy established the predominant form of the DNA to be a relaxed, circular molecule of molecular weight 14.9 X 10(6). The four species observed on electrophoresis are believed to be a circular monomeric form, a linear form derived from it, a circular dimeric form, and the corresponding linear dimer. Two DNA components were demonstrated by sedimentation velocity in the analytical ultracentrifuge and by zone sedimentation in sucrose gradients. These corresponded to a circular monomeric form and a circular dimeric form; no evidence for supercoiled forms was found by sedimentation in either neutral or denaturing solvents. Bacteriophage 643 DNA was sensitive to cleavage by single-strand-specific nuclease S1 and could be labeled in vitro by the PolI-catalyzed incorporation of [alpha-32P]dATP into the molecule under conditions that did not permit nick translation, suggesting that the circular duplex molecule is interrupted by a single-stranded gap.     

Characterization of 1706, a virulent phage from Lactococcus lactis with similarities to prophages from other Firmicutes

The virulent lactococcal phage 1706, isolated in 1995 from a failed cheese production in France, represents a new lactococcal phage species of the Siphoviridae family. This phage has a burst size of 160 and a latent period of 85 min. Its linear double-stranded DNA genome was composed of 55,597 bp with a 33.7% G+C content. Its deduced proteome (76 ORFs) shared limited similarities to other known phage proteins. SDS-PAGE coupled with LC-MS/MS analyses led to the identification of 15 structural proteins. The most striking feature of the 1706 proteome was that 22 ORFs shared similarities with proteins deduced from the genome of either Ruminococcus torques and/or Clostridium leptum. Both are Firmicutes bacteria found in the gut flora of humans. We also identified a four-gene module in phage 1706, most likely involved in host recognition that shared similarities with lactococcal prophages. We propose that the virulent phage 1706 infected another bacterial genus before picking up a lactococcal host recognition module.     

Structure and synthesis of a lipid-containing bacteriophage. IX. Serological disparity between bacteriophage PM2 and its host cell components

Several immunological properties of bacteriophage PM2 were determined. The structural components of this virus are antigenically unrelated to those of its normal host cell, Pseudomonas BAL-31. Furthermore, the virion contains at least two antigenic components. Both intact bacteriophage PM2 and its constituents appear to be only weakly immunogenic. A virus neutralizing antiserum of low potency (K < 200) could be obtained with the former, but no neutralizing antibodies were detected in the sera of rabbits injected with detergent-disrupted virus.