Mobile genetic elements in the genome of the beneficial rhizobacterium Pseudomonas fluorescens Pf-5
© Mavrodi et al; licensee BioMed Central Ltd. 2009
Received: 30 September 2008
Accepted: 13 January 2009
Published: 13 January 2009
Pseudomonas fluorescens Pf-5 is a plant-associated bacterium that inhabits the rhizosphere of a wide variety of plant species and and produces secondary metabolites suppressive of fungal and oomycete plant pathogens. The Pf-5 genome is rich in features consistent with its commensal lifestyle, and its sequence has revealed attributes associated with the strain's ability to compete and survive in the dynamic and microbiologically complex rhizosphere habitat. In this study, we analyzed mobile genetic elements of the Pf-5 genome in an effort to identify determinants that might contribute to Pf-5's ability to adapt to changing environmental conditions and/or colonize new ecological niches.
Sequence analyses revealed that the genome of Pf-5 is devoid of transposons and IS elements and that mobile genetic elements (MGEs) are represented by prophages and genomic islands that collectively span over 260 kb. The prophages include an F-pyocin-like prophage 01, a chimeric prophage 03, a lambdoid prophage 06, and decaying prophages 02, 04 and 05 with reduced size and/or complexity. The genomic islands are represented by a 115-kb integrative conjugative element (ICE) PFGI-1, which shares plasmid replication, recombination, and conjugative transfer genes with those from ICEs found in other Pseudomonas spp., and PFGI-2, which resembles a portion of pathogenicity islands in the genomes of the plant pathogens Pseudomonas syringae and P. viridiflava. Almost all of the MGEs in the Pf-5 genome are associated with phage-like integrase genes and are integrated into tRNA genes.
Comparative analyses reveal that MGEs found in Pf-5 are subject to extensive recombination and have evolved in part via exchange of genetic material with other Pseudomonas spp. having commensal or pathogenic relationships with plants and animals. Although prophages and genomic islands from Pf-5 exhibit similarity to MGEs found in other Pseudomonas spp., they also carry a number of putative niche-specific genes that could affect the survival of P. fluorescens Pf-5 in natural habitats. Most notable are a ~35-kb segment of "cargo" genes in genomic island PFGI-1 and bacteriocin genes associated with prophages 1 and 4.
Recent analyses of bacterial genomes have revealed that these structures are comprised of a mixture of relatively stable core regions and lineage-specific variable regions (also called genomic islands (GIs)), which commonly contain genes acquired via horizontal gene transfer. In bacteria, horizontal gene transfer occurs via conjugation, DNA uptake, transduction and lysogenic conversion, and is mediated largely by mobile genetic elements (MGEs). MGEs are present in most sequenced genomes and can account for the bulk of strain-to-strain genetic variability in certain species . MGEs are part of a so-called "flexible gene pool" and shape bacterial genomes by disrupting host genes, introducing novel genes and triggering various rearrangements. One class of MGEs is derived from bacteriophages and a second is derived from plasmids. Both classes may be associated with integrase genes, insertion sequence (IS) elements and transposons, thus forming elements that are mosaic in nature . Our current knowledge of the impact of MGEs on their hosts comes primarily from pathogenicity islands in which bacteriophages, plasmids and transposons act as carriers of genes encoding toxins, effector proteins, cell wall modification enzymes, fitness factors, and antibiotic and heavy metal resistance determinants in pathogenic bacteria. Much less is known about the diversity and role of MGEs in nonpathogens, in which these elements may enable their hosts to adapt to changing environmental conditions or colonize new ecological niches.
The present study presents the results of an in-depth structural analysis of the MGEs in the genome of Pseudomonas fluorescens Pf-5, which is the largest Pseudomonas genome sequenced to date http://www.pseudomonas.com. Strain Pf-5 is a model biological control agent that inhabits the rhizosphere of plants and suppresses diseases caused by a wide variety of soilborne pathogens [3–15]. The original analysis of the Pf-5 genome  focused primarily on the strain's metabolic capacity and on the pathways involved in the production of secondary metabolites. The latter encompass nearly six percent of the genome and include antibiotics that are toxic to plant pathogenic fungi and Oomycetes and contribute to Pf-5's broad-spectrum biocontrol activity. The aim of the present study was to more thoroughly analyze and annotate sections of the Pf-5 genome that contain MGEs. Here, we describe one transposase, six regions containing prophages (termed Prophage 01 to 06) and two genomic islands that are present in the Pf-5 genome.
Results and discussion
Phage-related elements and genomic islands of P. fluorescens Pf-5 genome
Presence of integrase
Type of feature
PFL_1210 to PFL_1229
PFL_1842 to PFL_1846
Defective prophage in tRNASer
PFL_1976 to PFL_2019
PFL_2119 to PFL_2127
Defective prophage in tRNAPro
PFL_3464 to PFL_3456
Defective prophage in tRNACys
PFL_3739 to PFL_3780
Lambdoid prophage in tRNASer
Genomic island 1
PFL_4658 to PFL_4753
Putative mobile island PFGI-1 in tRNALys
Genomic island 2
PFL_4977 to PFL_4984
Genomic island in tRNALeu
Prophage 01 of Pf-5 and homologous prophages in closely related strains
Interestingly, a prophage element found in the identical spot (between mutS and cinA) in the genome of P. fluorescens SBW25 http://www.sanger.ac.uk/Projects/P_fluorescens has a similar overall organization but contains a P2-like bacteriophage tail cluster (orf5 through orf18) similar to that in phage CTX (Fig. 1), thus resembling another class of phage tail-like bacteriocins, the R-type pyocins of P. aeruginosa . Furthermore, a homologous region from P. fluorescens Pf0-1 (CP000094) contains both the lambda-like and P2-like tail clusters (Fig. 1), making it similar to the hybrid R2/F2 pyocin locus from P. aeruginosa PAO1 . The differences in organization of the putative phage tail-like pyocins among these prophages clearly indicate that the corresponding loci are subject to extensive recombination, with a possible recombination hotspot between two highly conserved DNA segments comprised of the phage repressor (prtR) and holin (hol) genes, and the endolysin (lys) gene (Fig. 1).
In strains Pf-5 and Q8r1-96, the putative prophage 01-like pyocins are integrated between mutS and the cinA-recA-recX genes (Fig. 1), which suggests that these elements might be activated during the SOS response, as is the putative prophage gene cluster integrated into the mutS/cinA region of P. fluorescens DC206 . The mutS/cinA region is syntenic in several Gram-negative bacteria , and comparisons reveal that prophage 01-like elements occupy the same site in the genomes of P. fluorescens Pf0-1, P. fluorescens SBW25, and P. entomophila L48 , whereas unrelated prophages reside upstream of cinA in P. putida F1 (GenBank CP000712) and P. syringae pv. tomato DC3000 . The latter strain, as well as P. putida KT2440 , carry SfV-like bacteriophage tail assembly clusters elsewhere in the genome.
Prophage 03 of P. fluorescens Pf-5
Prophage 06 and other prophage regions of P. fluorescens Pf-5
Prophage 06 is the largest prophage region of P. fluorescens Pf-5 and encodes a 56-kb temperate lambdoid phage integrated into tRNASer(see Additional file 4). It is mosaic in nature with no homologues present in strains Pf0-1 or SBW25. P. fluorescens Pf-5 carries four genomic copies of tRNASer, of which tRNASer(2) and tRNASer(3) are associated with prophages carrying integrases of different specificity (see Additional file 5). The anticodon, V- and T-loops of tRNASer(2) are parts of the 104-bp putative attB site of prophage 06, whereas the T-loop of tRNASer(3) forms part of the 60-bp putative attachment site of prophage 02. The latter is a prophage remnant that spans 8.4 kb and consists of a gene encoding an ATP-dependent nuclease (PFL_1842) and a phage integrase gene with two internal frameshift mutations (see Additional file 6). The mobility of prophage 06 probably is mediated by a lambda-type integrase encoded by PFL_3794, which resides adjacent to the putative attR site. Prophage 06 contains gene modules that are involved in head morphogenesis (capsid proteins PFL_3764 and PFL_3765), DNA packaging (terminase PFL_3766), DNA recombination (a NinG-like protein, PFL_3773 and a putative NHN-endonuclease, Orf1) and tail morphogenesis (tail tip fiber proteins PFL_3744 and PFL_3751, tail length tape measure protein PFL_3753, and minor tail proteins PFL_3749, PFL_3750, and PFL_3752). The tail assembly module resembles the corresponding region from Burkholderia thailandensis bacteriophage φE125 , although in prophage 06 the module is split by the integration of four extra genes (Fig. 5B). Prophage 06 also contains a regulatory circuit with genes for a Cro/C1 repressor protein (PFL_3780) and two putative antirepressor proteins (PFL_3747 and PFL_3746); a gene for a putative cytosine C5-specific methylase (PFL_3792); and lysis genes encoding holin (PFL_3770) and endolysin (PFL_3798). However, since the endolysin gene is localized beyond the putative attR site it is not clear whether it represents part of the prophage 06 genome or a remnant from integration of a different phage (see Additional file 4). Finally, prophage 06 contains two genes, PFL_3740 and PFL_3796, which probably arose through gene duplication and encode putative conserved phage-related proteins that are 88% identical to one another.
Prophages 04 and 05 are prophage remnants with reduced size and/or complexity that carry several mutated phage-related genes (Tables 1, see Additional files 7 and 8). Prophage 04 (13.5-kb) has an average G+C content of 56.3% and contains a putative phage integrase gene adjacent to tRNAPro (1). It also carries two genes (PFL_2122 and PFL_2123) that encode minor tail assembly proteins, a gene encoding Cro/C1 repressor, and the bacteriocin gene llpA1 (PFL_2127). Interestingly, the repressor gene and llpA1 are highly similar to their counterparts from prophage 01, suggesting that they arose via gene duplication. Prophage 05, a 2.6-kb prophage remnant, has a G+C content of 55.3% and carries genes encoding a truncated phage integrase and a putative phage tail protein (PFL_3464) (see Additional file 8). The region is flanked by 84-bp direct repeats, one of which probably represents the attB site and partially overlaps with the anticodon and T loops of tRNACys.
Genomic island PFGI-1
Location and integrase
Integrative conjugative elements (ICEs) are a rapidly growing class of strain-specific mosaic MGEs that can profoundly impact the adaptation and evolution of bacterial species . ICEs vary in size from 10 to 500 kb, encode for mobility loci, and commonly exhibit anomalous G+C content and codon usage. Typical ICEs carry phage-like integrase genes that allow for site-specific integration, most often into tRNA genes, as well as plasmid-like replication and recombination functions and conjugative machinery that contributes to horizontal transfer. Finally, they often carry gene clusters encoding functions that are not essential for the host but that provide an advantage under particular environmental conditions. There is increasing evidence that ICEs derived from plasmids and encoding host-specific pathogenicity traits as well as traits essential for survival in natural habitats are widely distributed among members of the genus Pseudomonas [29–34].
Genes involved in plasmid replication, recombination, conjugative transfer, and possible origin of PFGI-1
Whether PFGI-1 exists in strain Pf-5 or in any other Pseudomonas host as an episome is not known. However, the first two-thirds of PFGI-1 contain putative plasmid replication, partitioning and conjugation genes that are readily aligned at the DNA level with those from plasmid pKLC102 of P. aeruginosa C . The putative origin of replication, oriV, is situated immediately upstream of PFL_4669 and spans about 1,100 bp. Plasmid origins of replication often contain arrays of specific ~20 bp repeats, called iterons, that serve as binding sites for the cognate replication initiator Rep protein and are involved in replication and partitioning [39, 40]. In addition to plasmid-specific iterons, some plasmid origins contain A+T-rich repeats where host replication initiation factors bind and open DNA, as well as repeats serving as binding sites for the host DnaA initiator protein. The putative oriV from PFGI-1 exhibits typical features of a plasmid replication origin. The first half is A+T-rich and has four conserved direct repeats of a perfect 23-bp palindrome (5'-CTGAGTTCGGA ATCCGAACTCAGT-3'). The second half is represented by a G+C-rich stretch that overlaps with the region between PFL_4668 and PFL_4669 and contains four conserved 46-bp direct repeats, each of which includes an imperfect 21-bp inverted repeat (5'-AGTG TTGTGG GCCACA CCACT-3'). The putative oriV is flanked by genes encoding proteins involved in plasmid replication, recombination and segregation, including putative homologues of DnaB and ParB (PFL_4663 and PFL_4666), a nucleoid associated protein (PFL_4665), topoisomerase TopA (PFL_4672), a putative helicase (PFL_4674), a single-strand binding protein (PFL_4671), and a partitioning protein, ParA (Fig. 6). PFGI-1 does not encode a Rep protein, and it is not clear whether it replicates by a theta-type or strand displacement mechanism, although the latter has been suggested for pKLC102 . Like some conjugative plasmids, PFGI-1 carries homologues of the stress-inducible genes umuC (PFL_4692) and umuD (PFL_4691), which encode a putative lesion bypass DNA polymerase and a related accessory protein, respectively. Such genes may be involved in plasmid DNA repair and umuDC-mediated mutagenesis, which could allow plasmids to adapt more quickly to new bacterial hosts .
PFGI-1 also contains a cluster of 10 genes, pilLNOPQRSTUVM (PFL_4675 through PFL_4683) (Fig. 6), that spans over 10 kb and closely resembles part of the pil region from the self-transmissible E. coli plasmid R64 . In E. coli, these genes are involved in production of thin flexible sex pili required for mating and transfer of R64 in liquid media. The similarity between the pil clusters of R64 and PFGI-1 suggests that the latter encode mating pili rather than type IV pili involved in bacterial twitching motility, adherence to host cells, biofilm formation and phage sensitivity . P. fluorescens Pf-5 has the capacity to produce type IV pili, and the corresponding biosynthetic genes are located in at least three clusters found outside of PFGI-1. The PFGI-1 pil cluster contains genes for pilin protein PilS (PFL_4680), prepilin peptidase PilU (PFL_4681), outer membrane protein PilN (PFL_4676), nucleotide binding protein PilQ (PFL_4678), integral membrane protein PilR (PFL_4679), and pilus adhesin PilV (PFL_4682). Unlike R64, PFGI-1 does not include a shufflon region that determines recipient specificity in liquid matings via generation of different adhesin types [42, 44]. Finally, PFGI-1 carries genes encoding proteins that may be involved in conjugal DNA transfer. PFL_4696 and PFL_4706 encode for TraG-like coupling proteins that may function as membrane-associated NTPases, which during conjugation would mediate transport of DNA covalently linked to a putative relaxase protein (the product of PFL_4751).
Recent studies have demonstrated that ICEs are a major component of a flexible gene pool of different lineages of Gram-negative Proteobacteria [45–47]. Metabolically versatile members of the Pseudomonadaceae are no exception, with ICEs having been identified among strains of P. aeruginosa [29–32], P. syringae [36, 48], and P. fluorescens . Comparison of PFGI-1 with islands from other Pseudomonas spp. reveals at least six highly conserved gene clusters (Fig. 7). These regions include: genes encoding partitioning (parA and parB) and single-strand binding (ssb) proteins, pilot proteins (traG), relaxase (PFL_4751), integrase (int), the putative origin of replication, and at least 26 conserved hypothetical ORFs. The pil operon is another syntenic cluster shared by PFGI-1 and the pathogenicity islands pKCL102 and PAPI-1, and PPHGI-1 of P. aeruginosa and and GI-6 of P. syringae. These findings further confirm the results of a recent study by Mohd-Zain et al. , who compared the evolutionary history of 33 core genes in 16 GIs from different β- and γ-Proteobacteria and found that despite their overall mosaic organization, many genomic islands including those from Pseudomonas spp. share syntenic core elements and evolutionary origin.
Putative phenotypic traits encoded by PFGI-1
As a rule, ICEs carry unique genes that reflect the lifestyles of their hosts. In P. aeruginosa and P. syringae, ICEs encode pathogenicity factors that allow these bacteria to successfully colonize a variety of hosts, as well as metabolic, regulatory, and transport genes that most probably enable them to thrive in diverse habitats [29, 30, 32, 33, 36, 50]. An unusual self-transmissible ICE, the clc element from the soil bacterium Pseudomonas sp. B13, enables its host to metabolize chlorinated aromatic compounds [34, 46, 51].
In PFGI-1, a unique ~35 kb DNA segment that is absent from pKLC102 and other closely related ICEs (Figs. 6 and 7) encodes "cargo" genes that are not immediately related to integration, plasmid maintenance or conjugative transfer. Some of these genes are present in a single copy and do not have homologues elsewhere in the Pf-5 genome. About half of PFGI-1 "cargo" genes also are strain-specific and have no homologues in genome of P. fluorescens Pf0-1.
How could genes encoded by PFGI-1 contribute to the survival of P. fluorescens Pf-5 in the rhizosphere? Some of them might facilitate protection from environmental stresses. For example, nonheme catalases similar to the one encoded by PFL_4719 (Fig. 6) are bacterial antioxidant enzymes containing a dimanganese cluster that catalyzes the disproportionation of toxic hydrogen peroxide into water and oxygen . PFGI-1 also carries a putative cardiolipin synthase gene (PFL_4745) and a cluster of four genes, cyoABCD (PFL_4732 through PFL_4735), that encode components of a cytochrome o ubiquinol oxidase complex. In P. putida, cardiolipin synthase was implicated in adaptation to membrane-disturbing conditions such as exposure to organic solvents , whereas the cytochrome o oxidase complex was shown to be highly expressed under low-nutrient conditions such as those found in the rhizosphere, and to play a crucial role in a proton-dependent efflux system involved in toluene tolerance [54, 55]. Finally, PFGI-1 cargo genes with predicted regulatory functions include a GGDEF-motif protein (PFL_4715), a two-component response regulator with a CheY domain (PFL_4716) and a sensor histidine kinase (PFL_4750). Although the exact role of these genes remains unknown, the GGDEF domain proteins represent an emerging class of bacterial regulators involved in the synthesis of bis-(3'-5')-cyclic dimeric GMP, a global signaling messenger , and two-component signal-transduction systems are widely employed by Gram-negative bacteria to sense changes in the environment and appropriately modulate the expression of certain genes .
Genomic island PFGI-2
Genomic island 02, or PFGI-2, spans 16.8 kb and has an average G+C content of 51.5%. It is flanked by imperfect 51-bp direct repeats, one of which partially overlaps with tRNALeu(6) and probably represents the attB site (see Additional file 10). Although P. fluorescens Pf-5 does not have a type III protein secretion pathway, approximately half of PFGI-2 (i.e. an 8.1-kb DNA segment spanning genes PFL_4977 to PFL_4980) closely resembles a gene cluster found in the exchangeable effector locus (EEL) of a t ripartite type III secretion pa thogenicity i sland (T-PAI) from the plant pathogen P. viridiflava strain ME3.1b  (see Additional file 10). Even the presence of a putative phage integrase gene (PFL_4977) (see Additional files 5 and 10) and integration into tRNALeu immediately downstream of the tgt and queA genes is typical of T-PAI islands from P. viridiflava  and P. syringae . In addition to T-PAI-like genes, PFGI-2 contains a putative phage-related MvaT-like (PFL_4981) transcriptional regulator, a superfamily II helicase (PFL_4979), a putative nucleoid-associated protein (PFL_4983), and a putative nuclease (PFL_4984). None of the aforementioned homologues of PFGI-2 genes in P. viridiflava have been characterized experimentally to date, making in difficult to deduce the function, if any, of this genome region. It also is possible that PFGI-2 is inactive and simply represents a T-PAI-like remnant anchored in the Pf-5 chromosome.
Transposons of P. fluorescens Pf-5
Unlike the genomes of other Pseudomonas spp., that of P. fluorescens Pf-5 is devoid of IS elements and contains only one CDS (PFL_2698) that appears to encode a full-length transposase. Three other transposase-like CDSs (PFL_1553, PFL_3795, and PFL_2699) found in the Pf-5 genome contain frameshifts or encode truncated proteins. PFL_2698 and PFL_2699 encode IS66-like transposases and are found within a large cluster (PFL_2662 through PFL_2716) of conserved hypothetical genes. Corrupted transposases encoded by PFL_1553 and PFL_3795 belong to the IS5 family and are associated with gene clusters encoding a putative filamentous hemagglutinin and prophage 06, respectively.
Recent analyses have revealed that most sequenced bacterial genomes contain prophages formed when temperate bacteriophages integrate into the host genome . In addition to genes encoding phage-related functions, many prophages carry non-essential genes that can dramatically modify the phenotype of the host, allowing it to colonize or survive in new ecological niches [60, 61]. Our knowledge of the ecological role of Pseudomonas prophages is limited, but data from other bacterial species suggest ways in which prophage regions could affect the survival of P. fluorescens Pf-5 in natural habitats.
Temperate bacteriophages similar to those encoded by prophages 03 and 06 are capable of development through both lysogenic and lytic pathways, and the presence of prophages can protect the host from superinfection by closely related bacteriophages . On the other hand, the lytic pathway ultimately results in phage-induced host cell lysis, and it has been reported that the presence of virulent bacteriophages can adversely affect rhizosphere-inhabiting strains of P. fluorescens [62–64]. Similarly bacteriophage tail-like bacteriocins such as the one encoded by prophage 01 are capable of killing both closely and more distantly related strains of bacteria, presumably through destabilization of the cell membrane [65–69].
Temperate bacteriophages and bacteriophage-like elements also are an important part of the bacterial flexible gene pool and actively participate in horizontal gene transfer [60, 70]. Among the putative lysogenic conversion genes in P. fluorescens Pf-5 are two copies of llpA, located adjacent to prophages 01 and 04. These genes encode low-molecular weight bacteriocins resembling plant mannose-binding lectins that kill sensitive strains of Pseudomonas spp. via a yet-unidentified mechanism . The fact that both llpA copies reside near prophage repressor genes, as well as the involvement of a recA-dependent SOS response in LlpA production by a different strain of Pseudomonas , suggests that the association of llpA genes with prophages is not accidental and that the prophages may be involved in the regulation of bacteriocin production in P. fluorescens Pf-5.
The analysis of MGEs revealed at least 66 CDSs not present in the original Pf-5 genome annotation (data are summarized in supplemental Tables). The bulk of these newly predicted CDSs fall in the category of conserved hypothetical genes of bacterial or phage origin. Predicted products of the remaining novel CDSs exhibit similarity to proteins of diverse enzymatic, regulatory, and structural functions and include a phage integrase, an ATP-dependent DNA ligase, an endonuclease, plasmid partitioning and stabilization proteins, a NADH-dependent FMN reductase, an acytransferase, a PrtN-like transcriptional regulator, a Com-like regulatory protein, a P-pilus assembly and an integral membrane protein.
Taken together, the analyses of six prophage regions and two GIs in the Pf-5 genome indicate that these structures have evolved via exchange of genetic material with other Pseudomonas spp. and extensive recombination. Transposition is unlikely to have played a major role in this evolution, as the genome of Pf-5 is nearly devoid of transposons and IS elements that are common in certain other Pseudomonas genomes. Most of the 260 kb comprising MGEs in the Pf-5 genome is similar to corresponding regions in the genomes of other Pseudomonas spp. Nevertheless, the MGEs also include regions unique to the Pf-5 genome that could contribute to the bacterium's fitness in the soil or rhizosphere.
Strains and plasmids
Wild type variants of P. fluorescens Pf-5 , P. fluorescens SBW25 , and P. fluorescens Q8r1-96  were used in the study. Pseudomonas strains were grown at 28°C in King's B medium , while E. coli strains were grown in LB  or 2xYT  at 25°C or 37°C. When appropriate, antibiotic supplements were used at the following concentrations: tetracycline, 12.5 μg/ml; chloramphenicol, 35 μg/ml; and ampicillin, 100 μg/ml.
DNA manipulations and sequence analyses
Plasmid DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation were carried out using standard protocols . All primers were developed with Oligo 6.65 Software (Molecular Biology Insights, West Cascade, Colo.), and routine PCR amplifications were performed with Taq DNA polymerase (Promega, Madison, Wisc.) according to the manufacturer's recommendations.
Sequencing of prophage 01 from P. fluorescens Q8r1-96 was carried out essentially as described by Mavrodi et al. . Briefly, the Q8r1-96 gene library was screened by PCR with oligonucleotide primers col1 (5' GCT GCT GGG CAA TGG TAA CAC 3') and col2 (5' CTG CCG ACT GCT CAC CTA TC 3') and a positive cosmid clone was shotgun sequenced by using the EZ::TN™ <Kan-2> transposition system (Epicentre Technologies, Madison, Wisc.). DNA sequencing was carried out by using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif.), and sequence data were compiled and analyzed with Vector NTI 9.1.0 (Invitrogen Corp., Carlsbad, Calif.) and OMIGA 2.0 (Accelrys, San Diego, Calif.) software packages. Database searches for similar protein sequences were performed using the NCBI's BLAST network service, and searches against PROSITE, Profile, HAMAP, and Pfam collections of protein motifs and domains were carried out by using the MyHits Internet engine . Signal peptides were predicted with SignalP v. 3.0. . The nucleotide sequence of prophage 01 from P. fluorescens Q8r1-96 has been deposited in GenBank under accession number EU982300.
The 3.12-kb prophage 01 probe was amplified by PCR from P. fluorescens Q8r1-96 genomic DNA with the oligonucleotide primers orf11-1 (5' CAT TCG TGT GCC GCT GTT CTA 3') and orf14-2 (5' TGA CCA GGC GAA CAG CGT CTG 3'). The 1.79-kb P. fluorescens SBW25-specific prophage 01 probe was amplified from genomic DNA of SBW25 with oligonucleotides SBW3 (5' GAA CTC ACC AGC GTC CTT AAC 3') and SBW4 (5' GGG CAG CTC CTT GGT GAA GTA 3'). Amplification was carried out with Expand Long DNA polymerase (Roche Applied Science, Indianapolis, IN) according to manufacturer's recommendations. The cycling program included a 2-min initial denaturation at 94°C followed by 25 cycles of 94°C for 10 sec, 60°C for 30 sec, and 68°C for 3 min. Amplified DNA was gel-purified with a QIAEX II gel extraction kit (Qiagen, Santa Clarita, Calif.) and labeled with the Biotin High Prime System (Roche Applied Science). Genomic DNA was purified using a cetyltrimethylammonium bromide miniprep protocol , digested with Eco RI and Pst I, separated by electrophoresis in a 0.8% agarose gel, and transferred onto a BrightStar-Plus nylon membrane (Ambion, Inc., Austin, TX) in 0.4 M NaOH. The membranes were pre-hybridized and hybridized at 58°C in a solution containing 5 × SSC , 4 × Denhardt's solution , 0.1% SDS, and 300 μg per ml of denatured salmon sperm DNA (Sigma). After hybridization, the membranes were washed twice in 2 × SSC, 0.1% SDS at room temperature, twice in 0.2 × SSC, 0.1% SDS at room temperature, and once in 0.2 × SSC, 0.1% SDS at 60°C.
Full-length hol genes from strains Pf-5 and Q8r1-96 were amplified by using KOD Hot Start DNA polymerase (Novagen, Inc.) and oligonucleotide primer pairs holupPf5 (5' AGG GAC CTC TAG AAA CAT CGT TA 3') – holowPf5 (5' TTT TGG ATC CGG TGA GTC AAG GCT G 3') and hol-xba (5' GAC CAG TCT AGA CAT GCT CAT CA 3') – hol-low (5' TTT TGG ATC CGC GGT ATC GCT T 3'), respectively. Full-length lys genes from Pf-5 and Q8r1-96 were amplified by using primer sets lysupPf5 (5' CGC CAT TCT AGA TTA CTG AAC AA 3') – lyslowPf5 (5' TTT TGG ATC CGC AGG ACC TTC AGA C 3') and lysQ8-up (5' CGG ACA TCT AGA ATC ATG CAC TTG 3') – tail13 (5' GCC GCT TGG GTG ATT TGA TT 3'), respectively. The cycling program included a 2-min initial denaturation at 94°C followed by 35 cycles of 94°C for 15 sec, 59°C for 30 sec, and 68°C for 1 min, and a final extension at 68°C for 3 min. PCR products were gel-purified and cloned into the Sma I site of the plasmid vector pCR-Blunt (Invitrogen) under the control of the T7 promoter. The resultant plasmids were single-pass sequenced to confirm the integrity of cloned genes and electroporated into E. coli Rosetta/pLysS (Novagen) with a Gene Pulser II system (Bio-Rad Laboratories, Hercules, Calif.). Plasmid-bearing E. coli clones were selected overnight on LB agar supplemented with ampicillin and chloramphenicol, and suspended in 2xYT broth supplemented with antibiotics to give an OD600 of 0.1. After incubation with shaking for one hour at room temperature, gene expression was induced in the broth cultures with 3 mM IPTG. The induced cultures were incubated with shaking for another 5 hours and the cell density was monitored by measuring OD600 every 30 min. To disrupt cell membranes in endolysin-expressing cultures, a drop of chloroform was added after four hours of induction. Two independent repetitions were performed with each strain.
The authors are grateful to Dr. Olga Mavrodi for help with the screening of Q8r1-96 gene library for ssh6-positive cosmid clones. This work was supported by the US Department of Agriculture, National Research Initiative, Competitive Grants Program (grants number 2001-52100-11329, 2003-35319-13800 and 2003-35107-13777).
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