Patent Application: US-201414773065-A

Abstract:
a kit useful for determining the phage susceptibility of one or more strains of lactococcus lactis is described . the disclosure also describes methods for formulation of mixed defined starter cultures using strains from different phage sensitivity groups .

Description:
lactococcal strains ( table 1 ) were grown in m17 broth supplemented with 0 . 5 % glucose at 30 ° c . without agitation . phages were propagated on the relevant strains at 30 ° c . in m17 broth ( oxoid ) supplemented with 0 . 5 % glucose without agitation as previously described ( 18 ). the phages used in this study and relevant details are listed in table 1 . plaque assays were performed using the double agar method as previously described ( 15 ). this method was also applied for the host range analysis performed against a bank of lactococcal strains ( table 1 ). frequency of lysogeny assays using the erythromycin - tagged phage tp901erm ( a derivative of tp901 - 1 , also designated as tp901 - bc1034 ) was performed as previously described ( 4 ). phage purification by caesium chloride gradient was performed as previously described ( 22 ). the generated purified phage suspension ( 1 ml ) was precipitated with 10 % polyethylene glycol 8000 ( sigma - aldrich ) and 0 . 5 m sodium chloride at 4 ° c . overnight . subsequently , the suspension was centrifuged at 17 , 700 g for 15 minutes and the supernatant removed . alternatively , phage suspension was dialyzed as described for phage 2 . the peg / salt - induced precipitate was resuspended in 0 . 5 ml of te buffer ( ph 9 . 0 ) and treated with 20 μl of 20 mg · ml − 1 proteinase k for 20 minutes at 56 ° c . followed by treatment with sds at a final concentration of 2 % at 65 ° c . for 20 minutes . this mixture was then phenol / chloroform ( 25 : 24 : 1 phenol : chloroform : isoamyl alcohol , sigma aldrich ) treated at least twice and the aqueous phase precipitated with 2 . 5 volumes of ice cold 96 % ethanol and 0 . 1 volume of sodium acetate ( ph 4 . 8 ). subsequent to centrifugation , the pellet was washed in 70 % ethanol and resuspended in 100 μl of te buffer ( ph 8 . 0 ). 5 μg of dna of phages 645 , 340 , viridusjm2 ( jm2 ), pastusjm3 ( jm3 ) and p113g was extracted and verified by nanodrop quantification and confirmatory molecular id tests were conducted on the dna extract prior to shipment to the contract sequencing facility ( macrogen inc ., korea ). a 40 - to 65 - fold sequencing coverage was obtained using pyrosequencing technology on a 454 flx instrument . the files generated by the 454 flx instrument were assembled with gs assembler ( 454 , branford , conn .) to generate a consensus sequence . phages p475 , fd13 , φ7 , 936 , p272 were sequenced using the 454 roche titanium platform . these phages were sequenced as part of tagged pools of unrelated phages , built as mid - tagged rapid libraries and sequenced in one region ( half a picotitre plate ) using the gs flx titanium sequencing kit xlr70 . one phage , p680 , was sequenced as 96 base reads using the lumina highseq2000 platform , again as part of a pool of unrelated phages , tagged with an index as part of one lane of the flowcell . custom indexing primers were used to build libraries as described earlier ( 12 ). reads were assembled into contigs using clc genomics workbench 5 . 0 . 1 ( clc bio , aarhus , denmark ). quality improvement of the genome sequence involved sequencing of 15 - 25 pcr products across the entire genomes to ensure correct assembly , double stranding and the resolution of any remaining base - conflicts occurring within homopolynucleotide tracts . protein - encoding open reading frames ( orfs ) were predicted using zcurve_v and genmark . hmm followed by manual assessment and , where necessary , correction . a preliminary identification and functional annotation of orfs was performed on the basis of blastp analysis against the non - redundant protein database ( nr ) provided by the national centre for biotechnology information ( located at : http :// blast . ncbi . nlm . nih . gov / blast . cgi ). the genbank accession numbers for the phages sequenced in this study are as follows : 340 kc182542 ; 645 kc182543 ; 936 kc182544 ; fd13 kc182545 ; jm2 kc182546 ; jm3 kc182547 ; p113g kc182548 ; p272 kc182549 ; p475 kc182550 ; p680 kc182551 ; φ7 kc182552 . the relevant dna regions encompassing the cell wall polysaccharide biosynthesis operon in the sequenced lactococcal strains il1403 ( accession number : ae005176 ), kf147 ( accession number : nc — 013656 . 1 ), mg1363 ( accession number : nc — 009004 . 1 ), sk11 ( accession number : nc — 008527 . 1 ), uc509 . 9 ( accession number : cp003157 . 1 ) and cv56 ( accession number : cp002365 . 1 ) were analysed and compared using blastp analysis as described above . using this data conserved and unique regions within the operons of these strains were identified ( fig1 ). primers were designed based on llkf — 205 of il - kf ( product = 183 bp ), llmg — 0226 of mg - sk ( product = 686 bp ) and uc509 — 0206 of uc - cv cwps ( product size = 442 bp ) types as indicated in fig1 ( tables 3 and 4 ). a control was also included in which primers based on the conserved gene rmlb were used to generate a product of 891 bp to verify that the reaction was working in all samples . the multiplex pcr included these four sets of primers and was applied to the strains assessed in the host range analysis ( table 1 ) under the following conditions : 95 ° c . for 6 minutes followed by 31 cycles of 95 ° c . for 15 seconds , 57 ° c . for 30 seconds and 72 ° c . for 1 minute and a final extension step at 72 ° c . for 7 minutes . a drop of the purified phage suspension was applied to a formvar - carbon - coated copper grid for 5 min , then removed with a pipette and immediately replaced with 3 % ( vol / vol ) uranyl acetate . after 1 min , the liquid was removed with a filter paper . the grids were examined in a philips cm12 transmission electron microscope at 80 kv . all recombinant plasmids ( table 1 ) were generated in escherichia coli top10 ( invitrogen , usa ). all primers , except where stated , were ordered from eurofins mwg ( ebersberg , germany ). the variable section ( i . e . variable within c type strains ) of the cwps biosynthesis gene cluster of l . lactis 3107 , encompassing genes 3107 — 003 to 3107 — 006 , was amplified using kod dna polymerase ( invitrogen , usa ) and cloned into the low copy number , nisin - inducible vector pptpi . plasmid constructs were then transformed into the l . lactis mg1363 nisrk - containing derivative l . lactis nz9000 , in which plasmid pjp005 ( 27 ) had been introduced to allow recombineering and nisin - inducible expression . recombineering was performed in l . lactis nz9000 or derivatives thereof as previously described ( 27 ), with associated modifications as optimized for l . lactis and executing a given transformation with 500 μg of a particular oligonucleotide , which in some cases contained phosphorothioate linkages ( integrated dna technologies , leuven , belgium ). for comparative analysis of the cwps biosynthesis gene clusters that belong to the mg - sk type , as identified by multiplex pcr , relevant genomic regions encompassing the cwps biosynthesis gene cluster from lactococcal strains mg1363 ( accession number : nc — 009004 . 1 ), sk11 ( accession number : nc — 008527 . 1 ) and io - 1 ( accession number : ap012281 ) were employed . the full genome analyses of l . lactis strains 3107 , w34 , jm1 , jm2 and jm3 are currently in progress and these results will be published elsewhere . the genomic regions responsible for cwps biosynthesisin the latter five strains were identified based on blastn analysis against the reference cwps biosynthesis gene cluster of l . lactis mg1363 and submitted to genbank under the following accession numbers ; l . lactis 3107 ( kf498848 ), l . lactis w34 ( kf498852 ), l . lactis jm1 ( kf498849 ), l . lactis jm2 ( kf498850 ) and l . lactis jm3 ( kf498851 ). the presumed cwps region of each genome was analysed and compared in detail using blastp and interpro analysis . using the genomic data corresponding to the cwps biosynthesis region of the above mentioned strains , conserved and variable regions were identified . eleven phages were selected for this study in order to assess genome diversity among the 936 phages . the phages represent a broad range of 936 phages isolated across europe ( and one new zealand phage ) during a time period that spanned the 1980s until 2010 ( table 1 ). firstly , phages that have been applied in many studies of 936 phage - host interaction studies over the past decades such as p680 , p113g , p272 and 645 were selected . secondly , phage 936 was selected to serve as the prototype member of the 936 phage species for comparative purposes . furthermore , its geographical location of isolation was a consideration as the remainder of the phages are of european background ( table 1 ). finally , the geographical origin and year of isolation of the selected phages was considered , and therefore phages that had been isolated over the past thirty years in ireland ( viridusjm2 and pastusjm3 ), germany ( p680 , p113g , p272 ) and denmark ( fd13 , 645 , 340 , φ7 , p475 ) and new zealand ( 936 ) were selected . thirty four lactococcal strains ( table 1 ) were assessed for their sensitivity to the eleven phages sequenced in this study . all phages assessed in this study have a relatively narrow host range and are limited to infecting at most six different strains from this panel of thirty four possible hosts . it is also noteworthy that host range convergence was observed for certain members of the sequenced group of phages . for example , 645 and 340 have a similar host range as do p272 and p113g ( table 2 ). since these phages are derived from similar geographical locations , it is perhaps unsurprising that these apparent sub - groups of phages possess related host ranges . viridus jm2 , pastus jm3 , fd13 and 936 display very narrow host specificities infecting only a single strain among those tested . blastp analysis of the cwps clusters of the sequenced lactococcal strains identified three major cwps subgroups based on conserved sequences , allowing classification into the mg - sk , il - kf and uc - cv cwps subgroups ( fig1 ). each subgroup is defined by unique regions that were used to develop a multiplex pcr - based typing method ( see materials and methods section ). this was applied to classify the cwps type of each of the strains used in this study . of the 34 strains assessed , six strains were in this way classified as the il - kf cwps - type , fourteen belonged to the mg - sk cwps - type , while thirteen belonged to the uc - cv cwps - type . one strain ( l . lactis subsp . lactis 184 ) did not generate an amplicon for any of the three cwps types although the conserved region present in all three subtypes was amplified ( fig2 ), which may be indicative of an as yet unidentified cwps type not represented by the three sub - types presented in this study . of the eleven phages , five phages ( fd13 , 936 , φ7 , viridusjm2 and pastusjm3 ) are largely limited to infecting strains of the mg - sk cwps type ( table 2 ). conversely , the remaining six phages are almost completely limited to infecting hosts with the il - kf cwps type . there are exceptions to this generalisation , however , such as phages 645 and 340 which were shown to infect l . lactis subsp . cremoris 3107 ( mg - sk cwps ) as well as l . lactis subsp . lactis il1403 ( il - kf cwps ) and four other strains of this cwps type . these phages can infect strains of both cwps types with a relative efficiency . the same is true for phage p475 , which infects strains of the il - kf cwps type , but which also infects l . lactis w22 ( mg - sk cwps ) however at a much lower efficiency ( eop = 10 − 6 / 7 ). interestingly , none of the strains possessing the uc - cv cwps - type were infected by any of the 936 - type phages assessed in this study . the rbps of the sequenced phages were used to perform a comparative sequence analysis which also included sequences of previously sequenced 936 phage rbps . since the amino - terminal regions of these proteins are well conserved , the first 130 residues were removed from the rbp sequences and a comparison of the much more variable sequences of the rbps carboxy - terminus was performed . through this analysis , three sub - groups of rbp are identifiable ( fig3 ): group 1 corresponds to what was previously termed the sk1 - like or l . lactis subsp . cremoris - infecting phages ( 17 ); group 2 contains the bil170 - like or l . lactis subsp . lactis - infecting phages ( 17 ); while group 3 represents a distinct but small group of phages that infect primarily l . lactis subsp . lactis strains , but are also capable of infecting strains of l . lactis subsp . cremoris ( table 2 ). while there are three main rbp groups , subtle sub - groups within these groups can be distinguished ( fig2 ). the phages in this study of the rbp group 1 exclusively infect strains that possess the mg - sk cwps type . similarly , the majority of phages in this study of rbp group 2 infect strains that possess the il - kf - type cwps , while those belonging to group 3 appear to preferentially infect strains of the il - kf cwps type , though they are occasionally also capable of infecting strains with a mg - sk - type cwps as mentioned above ( the cases of p475 , 645 and 340 ). as determined , three variations of a particular genetic locus present in l . lactis strains , termed the uc - cv , il - kf , and mg - sk types , can be linked to rbp phylogeny of 936 phages . to determine if additional genetic diversity within the cwps biosynthesis gene cluster of a given cwps type exists , we analysed the genetic locus encompassing the presumed cwps biosynthetic operon of eight lactococcal genomes ( three publicly available genomes and the cwps regions of five strains from our own collection ), all belonging to the mg - sk type ( as first determined by pcr ). this comparative sequence analysis revealed the presence of a variable region within these examined cwps mg - sk type loci ( fig4 ), allowing the identification of five subtypes among members of the mg - sk type ( designated subtype c 1 to c 5 ; fig4 ) based on differences / similarities within this variable region within the various type mg - sk cwps biosynthesis loci . primers for generating the c 1 to c 5 sub - types are provided in table 5 and the generated amplicons are provided in table 6 . primers were designed based on the unique regions of each of the 5 sub - types and can be applied in a multiplex pcr approach to perform cwps mg - sk sub - typing of lactococcal strains ( table 5 ). the sequence differences between subtypes suggest that c 2 to c 5 subtype strains produce structurally different cwps compared to the previously determined c 1 type structure of l . lactis mg1363 ( 5 ). the sub - typing of lactococcal strains of the cwps mg - sk type permits a deeper insight into the specific relationships between phages and these strains and elucidates the components that phages of the 936 or p335 species recognise and specifically target . for example , the lactococcal p335 phages lc3 and tp901 - 1 cannot infect l . lactis nz9000 , which possesses the cwps c 1 sub - type . conversely , these phages can infect another lactococcal strain named 3107 , which possesses cwps c 2 sub - type . genetic swapping of the variable region of the cwps of a mg - sk subtype strain causes a change in phage sensitivities to both 936 and p335 type phages due to the high level ( 99 - 100 %) of dna sequence identity observed across conserved regions of the cwps biosynthesis gene clusters found in the c 1 subtype strain l . lactis mg1363 and the c 2 subtype strain l . lactis 3107 ( fig4 ), it was reasoned that if the variable genes found in the c 2 subtype cwps biosynthesis locus of l . lactis 3017 were to be supplied in trans to the l . lactis mg1363 nice expression system derivative l . lactis nz9000 ( c 1 subtype ), carrying a mutation in one of its variable cwps genes , the resulting recombinant strain would produce the structural equivalent of l . lactis 3107 cwps ( which is of the c 2 subtype ), thus effectively causing change of cwps subtype by this genetic swapping . to test this hypothesis , the variable region of the c 2 subtype cwps biosynthesis gene cluster was first cloned from l . lactis 3107 ( i . e . genes 3107 — 003 , 3107 — 004 and 3107 — 005 ) into the nisin - inducible plasmid pptpi , thereby generating plasmid pptpic2 . the latter plasmid was then introduced into l . lactis nz9000 - gt1 , an nz9000 derivative in which gene llnz — 01145 , which is one of the genes of the variable region within the native cwps biosynthesis mg - sk type gene cluster , had been mutated by recombineering . this mutant carries an in - frame tga stop codon in llnz — 01145 , which in turn causes the resulting mutant , designated nz9000 - gt1 , to display a phage - resistant ( to phages belonging to the 936 species ) and sedimenting phenotypes ( data not shown ), all being consistent with the expected loss of cwps biosynthesis . introduction of plasmid pptpic2 and induced expression of the variable region of the c 2 subtype cwps biosynthesis gene cluster from l . lactis 3107 on this plasmid in l . lactis nz9000 - gt1 restored wild type non - sedimenting cell growth , indicating the production of a functional cwps of subtype c 2 . to determine if the presumed c 2 subtype cwps produced in l . lactis nz9000 - gt1 pptpic2 functions as a bacteriophage - host cell surface receptor , the induced strain was challenged by plaque assay with various p335 species phages ( table 7 ), whose primary indicator strain is l . lactis 3107 . of the phages tested , only the p335 species phage φlc3 , which is unable to form plaques on wt l . lactis nz9000 or un - induced nz9000 - gt1 pptpic2 , was able to infect and form plaques on induced nz9000 - gt1 pptpic2 at an eop of 10 − 1 and can be propagated to levels of 10 7 - 10 8 pfu / ml ( data not shown ). this clearly demonstrates that the cwps of l . lactis 3107 is the host cell - surface receptor of the p335 species phage φlc3 , and that this cwps , when produced in nz9000 , is sufficient for this strain to become susceptible to φlc3 infection . interestingly , another p335 species phage , tp901erm , which also uses l . lactis 3107 as a host , was not able to form plaques on induced nz9000 - gt1 pptpic2 . however , the frequency of lysogeny of tp901erm on induced l . lactis nz9000 - gt1 pptpic2 increases 10 4 fold compared to l . lactis nz9000 - gt1 pptpi ( tp901erm can lysogenize l . lactis nz9000 at a very low frequency ( 10 − 8 )), reaching levels similar to those observed for l . lactis 3107 ( 10 − 4 ), thus showing that cwps from 3107 is also the cell surface receptor for of the p335 species phage tp901erm . the invention is not limited to the embodiment hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention . 1 . bolotin , a ., p . wincker , s . mauger , o . jaillon , k . malarme , j . weissenbach , s . d . ehrlich , and a . sorokin . 2001 . the complete genome sequence of the lactic acid bacterium lactococcus lactis s sp . lactis il 1403 . genome research 11 : 731 - 753 . 2 . boyce , j . d ., b . e . davidson , and a . j . hillier . 1995 . spontaneous deletion mutants of the lactococcus lactis temperate bacteriophage bk5 - t and localization of the bk5 - t attp site . applied and environmental microbiology 61 : 4105 - 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