PT Unknown AU Ali, YHM TI Molecular studies on bacteriophages of the foodstarter culture Streptococcus thermophilus PY 2009 PU Christian-Albrechts-Universität zu Kiel WP https://macau.uni-kiel.de/receive/diss_mods_00003451 LA en DE Streptococcus thermophilus; Starter Culture; Bacteriophages; Lysogeny; Multiplex-PCR; Phage lipoprotein; Starterkultur; Bakteriophagen; Lysogenie; Phagenlipoprotein AB Lactic acid bacteria play divergent and important roles for human beings: On the one hand they include major bacterial pathogens like Streptococcus pyogenes and S. pneumoniae and on the other hand many species are used as starter cultures for the manufacture of fermented food (i.e., dairy products, meat, vegetables, wine, sour dough, cocoa) and silage. S. thermophilus is one of the most important lactic acid bacteria required for the manufacture of yogurt, mozzarella and other cheese varieties (e.g. Cheddar and Swiss-type cheeses). Bacteriophage attacks are always a serious problem in industrial fermentations leading to significant financial losses. Characterization of bacteriophage populations is therefore the first step of efficient phage control in dairies and other food fermentation industries. The aim of the work described in Chapter 1 was to identify and characterize a new temperate S. thermophilus phage (i.e., phage TP-778) that was induced from its lysogenic host strain S. thermophilus SK778. Phage TP-778 revealed the typical morphology of all known S. thermophilus phages with isometric heads and long non-contractile tails. It was classified as a pac-type phage with a genome size of ca. 44 kb. No prophage-cured strain could be obtained, indicating that correct excision of prophage DNA from the host chromosomal DNA was prevented. Phage TP-778 could be propagated lytically on the non-lysogenic S. thermophilus strain B106, and a lytically propagated derivative (i.e., TP-778L) was isolated and also characterised as described in this chapter. When phage TP-778 DNA was used as a probe for Southern blot analysis of the chromosomal DNAs of a set of non-lysogenic S. thermophilus strains, an unexpected high background of unspecific hybridization signals appeared in the DNAs of all strains. This indicated that the phage TP-778 genome could (1) either carry DNA homologous to the chromosomal DNA of S. thermophilus starter strains or (2) that the phage would be capable to package (and consequently transduce) chromosomal DNA during phage proliferation. In order to address these questions, a detailed DNA sequence analysis was performed for the lysogeny module of the phage genome and its flanking regions from the phage lysin gene to the phage cro regulatory protein gene. This genomic region was selected since it should reveal all genetic determinants for prophage integration/excision (i.e. integrase gene), for establishing the lysogenic/lytic phage life cycle (i.e., regulatory genes of the genetic switch) and furthermore the junction sites between prophage and bacterial DNA (i.e., attL and attR). The 2 neighbouring prophage regions from the left prohage/chromosome junction site (attL-site) to cro gene (fragment attL-cro) and from the lysin gene to the expected attR-site (fragment lysin-attR) located on the opposite flanking region of the prophage DNA were amplified by PCR and sequenced. Sequence analysis of the lysin-attR prophage DNA revealed the presence of about 1.9-kb prophage remnant DNA. A truncated 398-bp integrase gene was present on the phage remnant. A functional attR-site was also deleted from this prophage fragment, indicating that integration or excision of the prophage could not occur by the site-specific recombination via homologous attP and attB sites. Hence, this fragment was renamed as lysin-attR fragment. While only a non-functional (truncated) integrase was identified on the lysin-attR prophage fragment, a complete 1080-bp integrase gene was identified on the attL-cro DNA fragment. This core region of the TP-778 lysogeny module was highly homologous to the corresponding region of the well-characterized S. thermophilus temperate phage TP-J34 (Neve et al., 2003) and did also code for a unique lipoprotein (Orf142TP-778). Hence it was concluded that prophage TP-778 is directly associated with a prophage remnant in the lysogenic host strain. In order to confirm that TP-778 can package extra DNA, a 16S rDNA region was amplified from purified phage particles treated with DNase I to destroy any non-packaged chromosomal DNA contaminations. The positive PCR reaction was confirmed by Southern blot analysis using the 16S rDNA probe. It was concluded that phage TP-778 and its lytic derivative (TP-778L) were both capable of packaging chromosomal host DNA. The core region of the lysogeny module of lytically propagated phage derivative TP-778L was also analysed by DNA sequencing. Phage TP-778L contained a 398-bp truncated recombinant integrase gene remnant. By alignment with the intact 1080-bp integrase gene of phage TP-778 and with the 398-bp integrase gene fragment present on the prophage remnant, it was shown that the TP-778L integrase fragment exhibited a recombinant structure originating from a homologous recombination event between the two integrase determinants present in the lysogenic SK778 cells. In Chapter 2 comparisons of genes ltpTP-778 (orf142TP-778) of phage TP-778 and ltpTP-J34 (orf142TP-778) of phage TP-J34 and their corresponding gene products are presented. The lipoprotein gene ltpTP-778 of phage TP-778 is closely related to the corresponding gene ltpTP-J34 of phage TP-J34. Previously, ltpTP-J34 has been shown to code for a unique superinfection exclusion system. This phage resistance mechanism was also active in a lactococcal host background against the isometric-headed phage P008 but not aggainst the prolate-headed phage P001 (Sun et al., 2006). The amino acid sequences of both deduced lipoproteins differed only by 10 amino acids. The ltpTP-778 gene was cloned into the expression vector pMG36e. The resulting recombinant plasmid pYAL1 was also transformed into the Lactococcus lactis strain Bu2-60 in order to assess its effect of ltpTP-778 on the phage resistance phenotype of the cells. Gene expression of LtpTP-778 in strain Bu2-60 was confirmed by SDS-PAGE analysis, but the protein did not react with a polyclonal antiserum raised against LtpTP-J34 coded by phage TP-J34. When 3 Bu2-60(pYAL1) transformants were challenged with phages P008 and P001, 2 of them were partially resistant to phage P001 but not to phage P008. The 3rd transformant was still sensitive to both phages. The deviation of phage resistance phenotypes observed in Bu2-60 transformants harbouring the ltpTP-778 or the ltpTP-J34 gene was suggested to be due to the variability of one or more of the 10 amino acids that differ in the lipoproteins coded by ltpTP-778 and ltpTP-J34. Due to the high economical losses that may result from a phage infection of starter cultures, a rapid and sensitive method is required for phage detection and identification in the dairy. In Chapter 3, a rapid and reliable multiplex-PCR method is described allowing the simultaneous detection of S. thermophilus phages and their differentiation into the 2 well-known pac- and cos-type subgroups. Since pac- and cos-type phages are composed of different structural proteins, 2 sets of primers were designed from the internal highly conserved region of the major head protein gene of 8 completely sequenced S. thermophilus pac-type phages (TP-J34, O1205, Sfi11, 2972) and cos-type phages (Sfi21, 2701, Sfi19, DT1). The 2 primer sets could be used simultaneously in a multiplex PCR assay to detect and distinguish S. thermophilus phages, because the PCR-products differed in fragment size (432-bp product for pac-type phages versus 514-bp product of cos-type phages). The reliability of the multiplex PCR protocol was validated and confirmed by the following 3 controls: (I) restriction enzyme analysis of the DNA of cos- and pac-type DNA phages with and without a heating step at 74°C required for the melting of fragments harbouring cos-sites, (II) SDS-PAGE analysis of the structural proteins of pac-type phages (3 major bands) and cos-type phages (2 major bands), and furthermore by (III) immuno electron microscopy of the phages treated with a polyclonal antiserum raised against the pac-type phage TP-778. The multiplex PCR yielded results in a short time of approximately 2.5 h. The method is highly sensitive with a limit of detection as low as ca. 103 phages per ml of acidic whey. The protocol was also used in a colony PCR approach to detect and identify 3 new lysogenic S. thermophilus strains harbouring inducible pac-type prophages within a set of 60 S. thermophilus strains isolated from traditional Egyptian yoghurt (Zabady) samples. When a number of newly isolated S. thermophilus phages was screened by the multiplex-PCR developed in Chapter 3, phage P738 failed to deliver a PCR product. In order to confirm that this phage represents a new phage species for S. thermophilus phages, a detailed characterization was performed for this phage as described in Chapter 4. Phage P738 was originally propagated on the well-described S. thermophilus strain S4 but could also infect a set of 15 other S. thermophilus strains illustrating its broad host range. Notably, it was not possible to propagate phage P738 lytically in liquid cultures at the standard incubation temperature of 40°C used for these thermophilic cultures. Efficient cell lysis and phage proliferation was only possible at a lower temperature of 30°C. Phage P738 had a unique morphotype with an isometric head (57 nm ), a remarkably short, non-contractile tail (124 nm length x 10 nm ) and a distinct tail fiber (54 nm length). By SDS-PAGE analysis it was documented that phage P738 revealed a distinct structural protein profile with one major protein band (33 kDa) and 5 minor protein bands (sizes: 50 kDa, 60 kDa, 72 kDa, 91 kDa, 150 kDa). By pulsed-field gel electrophoresis and restriction enzyme analysis, phage P738 was identified as a pac-type phage with a genome size of ca. 35 kb. The P738 DNA did neither hybridize with the DNA from other S. thermophilus reference phages (phages TP-J34, TP-778L, P53) nor with the DNA of different L. lactis reference phages (phages BK5-T, P335, r1t, TP901-1, P001, P008, sk1, P446). A 2.8-kb EcoRV fragment of the P738 genome was cloned into the plasmid vector pJET1.2/blunt. Using a primer walking strategy, the DNA sequence was determined from a 6.2 kb genomic region. Seven open reading frames (orfs) were identified which were transcribed in the same direction. By in silico DNA sequence analysis it was shown that the genes putatively code for the large subunit of terminase (TerL) and furthermore for the structural proteins required for head morphogenesis. These putative proteins revealed high similarity with proteins from S. pyogenes prophages. Only very little similarity was found to the corresponding gene products of S. thermophilus phages. By MALDI-TOF mass spectrometry of the phage structural proteins, 2 protein bands were identified that could be correlated with orf430 (50-kDa product; portal protein) and with orf302 (33-kDa product; major head protein). A P738 specific set of PCR primers was selected for targeting the structural gene of the major head protein of this new S. thermophilus phage species and was included in the multiplex PCR tool developed in Chapter 3. PI Kiel ER