| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Genomic Sciences Program, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624, USA
Correspondence
Todd R. Klaenhammer
klaenhammer{at}ncsu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the 3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.
Present address: Texas A&M University, Department of Nutrition and Food Science, College Station, TX 77843, USA.
A supplementary table of primers is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Moineau et al., 1996). In addition, existing phage populations can evolve resistance to phage defence systems by mutation and recombination (Bouchard & Moineau, 2000; Durmaz & Klaenhammer, 2000). Together, these selective pressures necessitate the continued development of starter cultures with enhanced phage-resistance properties.
A greater understanding of phage genomics and physiology has accelerated the development of novel and more efficacious phage-resistance systems. This information has enabled researchers to develop recombinant derivatives of starter strains through the expression of engineered phage resistance systems not previously found in nature (for a recent review, see Sturino & Klaenhammer, 2006). Interestingly, the functional components of these engineered systems have largely been phage-derived. Recently, it has been demonstrated in S. thermophilus that the utility of these techniques can be enhanced when they are directed by comparative genomic analyses (Sturino & Klaenhammer, 2002, 2004). Identification of highly conserved phage-encoded genes, pathways or processes can contribute to new defence systems effective against a wider variety of phage strains, which is of critical importance when designing defence systems for industrial applications.
S. thermophilus phages are excellent candidates for the development of such systems since commercial isolates are relatively homogeneous with regard to morphology and genomic organization (Desiere et al., 2002). All phages for this species discovered to date belong to the Siphoviridae family (morphotype B1) of viruses, having small isometric heads, long, non-contractile tails, and genomes comprised of double-stranded DNA (dsDNA). Comparative bioinformatic, hybridization and genetic analyses have found that the genome replication functions of S. thermophilus phages are catalysed by two distinct clusters of non-orthologous genes, which are exemplified by the phage Sfi21- and 7201-derived prototype modules (Brüssow et al., 1994; Lucchini et al., 1999). For several important reasons, the genes associated with the Sfi21-type genome replication module were previously found to be well suited for engineered phage-inhibitory defence strategies (Sturino & Klaenhammer, 2002). First, they have a high frequency of distribution in industrial isolates: the Sfi21-type gene cluster is found in the majority of industrial S. thermophilus phage isolates (Sturino & Klaenhammer, 2002; Brüssow & Desiere, 2001). Second, independently isolated variants exhibit striking sequence conservation (i.e. >99.9 %) at the nucleic acid level in this region. Lastly, genome replication functions have an intrinsic strategic importance since they are expressed early in the lytic cycle and may allow for the recovery of the host after the infection is aborted (Sturino & Klaenhammer, 2002).
The Sfi21-type replication module comprises a single origin of DNA replication (ori) and several ORFs that encode a putative primase, a putative helicase and a number of other proteins of undetermined function (Brüssow & Desiere, 2001). The putative primase is known to be essential for the replication of genomes composed of dsDNA (Frick & Richardson, 2001). Primases are DNA-dependent RNA polymerases, which are often hexameric, that catalyse the de novo synthesis of short oligoribonucleotide primers at sequence-specific loci located across the lagging strand. In this study, invariant and highly conserved amino acids within a consensus primase sequence were targeted by site-specific mutations in an effort to produce non-functional subunits of the oligomeric S. thermophilus phage 3-encoded putative primase. These mutant primases were then expressed in trans and their effect on phage replication was examined.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Unless otherwise indicated, bacteria were propagated as described previously (Sturino & Klaenhammer, 2002).
|
. They were all independently isolated from different dairy facilities across the continental United States and were propagated as described previously (Sturino & Klaenhammer, 2002). The EOP was calculated by dividing the p.f.u. ml–1 on the test strain by the p.f.u. ml–1 on the control strain, S. thermophilus NCK1125(pTRK687). The diameters of phage plaques were measured using a caliper and the values reported represent the mean of 60 random plaques chosen over three independent experiments. Centre of infection (COI) assays were performed as described previously and the efficiency of centre of infection (ECOI) was calculated by dividing the number of infective centres on the test strain by the number of infective centres on NCK1125(pTRK687) (Sing & Klaenhammer, 1990).
Site-directed mutagensis and plasmid construction.
The plasmids and primers used in this study are listed in Table 1, and Supplementary Table S1 (available with the online version of this paper), respectively. Conventional PCR reactions, DNA sequencing and sequence analyses were all performed as described previously (Sturino & Klaenhammer, 2002). When appropriate, PstI restriction endonuclease recognition sites were incorporated into the 5' ends of oligonucleotide primers to facilitate the cloning of PCR products. Primer-directed point mutations within specific functional protein domains or motifs were introduced by gene splicing by overlap extension (SOE) PCR as described elsewhere (Horton, 1995). All point mutations were confirmed by sequencing. Electrocompetent Escherichia coli and S. thermophilus were prepared and electroporated as described previously (Sturino & Klaenhammer, 2002).
Southern hybridizations and alkaline gel electrophoresis.
Genomic DNAs were isolated from (i) cultures 10, 20, 30, and 60 min after infection with phage 3 at an m.o.i. of 1±20 % and (ii) uninfected control cultures grown in parallel as described previously (Hill et al., 1991). Alkaline transfer of HindIII-digested genomic DNA fragments and subsequent Southern hybridizations were performed as described previously (Sturino & Klaenhammer, 2002). The digoxigenin-11-dUTP labelled probes used during hybridization studies were derived from phage 3 genomic DNA and purified using NucTrap Probe Purification columns (Stratagene).
Protein sequence manipulation and GenBank accession numbers.
The nucleic acid and deduced amino acid sequences for the 3-encoded putative primase proteins can be found in the GenBank database under the nucleotide accession number AY196178 (Sturino & Klaenhammer, 2004). Conserved domains were detected using the BLASTP (Altschul et al., 1997) and clusters of orthologous groups (COG) database tools (Tatusov et al., 2001) available at the NCBI website (http://www.ncbi.nlm.nih.gov/). Multiple alignments were performed using CLUSTAL_X (Thompson et al., 1997).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
3-derived primase is encoded by a single ORF of 1515 bp. The boundaries of this ORF are demarcated by an alternative 5'-TTG-3' translation initiation codon and a 5'-TAA-3' translation termination codon. The deduced protein sequence consists of 504 amino acids and has a predicted molecular mass of 59 kDa. The ORF is preceded by a consensus putative ribosome-binding site (RBS) (5'-AGGAGG-3') and is located upstream of an iteron-rich intergenic region, which was previously shown to contain the putative origin of DNA replication (Sturino & Klaenhammer, 2002).
A 1.5 kb fragment containing the native primase gene was amplified without its native promoter using primer set L001L (Supplementary Table S1), digested with PstI, and cloned into the PstI site of pTRK687 in the sense orientation relative to and downstream from the Lactobacillus acidophilus P6 promoter (Djordjevic et al., 1997). The resulting vector, designated pTRK809, was electroporated into S. thermophilus NCK1125 and tested for its ability to interfere with phage replication. As measured by efficiency of plaquing (EOP), the expression of the native protein did not confer resistance to phage 3 (Fig. 1) or any of the other Sfi21-type phages tested (data not shown). In addition, the expression of the parental-type primase protein did not alter the growth of NCK1125 in broth (data not shown). During standard plaque assays, replication of the pTRK687 base vector resulted in an increased plaque size relative to the NCK1125 native strain but did not affect the EOP of any of the phages tested, as was observed previously (Sturino & Klaenhammer, 2002).
|
3 primase (Fig. 2b, c). Both DNA helicase and ATPase activities are associated with the pfam01057 domain, which is required for genome replication in dsDNA parvoviruses (Nuesch & Tattersall, 1993; Wang et al., 1998). This domain was found to contain a signature glycine-rich phosphate-binding loop of sequence GNGNDGKGT near the centre of the protein (Notarnicola et al., 1995). In order to identify highly conserved residues potentially important for enzyme specificity, catalysis and/or structure, the top BLAST hits having cutoff E-values of less than 10–3 were aligned using the CLUSTAL_X program (Thompson et al., 1997). Regions of strong sequence conservation were identified (Fig. 2). The locations of these motifs relative to the conserved domains described above are shown in Fig. 2(a).
|
). Mutations were introduced by SOE PCR (Horton, 1995). Primase alleles were amplified using primer set L001L (Supplementary Table S1) and cloned into the PstI site of pTRK687 in the sense orientation relative to the P6 promoter. In an effort to increase the efficiency of translation, some of the constructs were amplified using primer set L001M (Supplementary Table S1), which mutated the (native) putative leucine (L) alternative start codon (5'-TTG-3') to the standard, methionine (M) start codon (5'-ATG-3'). These efforts, however, did not result in increased phage resistance. Phage 3 was titrated on all 13 of the point mutant constructs during standard plaque assays (Fig. 1). The various primase constructs could easily be differentiated into either phage sensitive or phage resistant based on the relative EOP and plaque diameter. Ten of 14 constructs, including pTRK809, N151am, E437A, E437D, N151am : : K238T, K238T : : E347A, N326A, and N151am : : RR340-341AA, were sensitive to phage 3, and exhibited parental-type EOP and plaque diameter. The remaining four constructs, including K238T, K238A and RR340-341AA, were completely resistant to phage 3; no plaques were formed, EOP <10–9. No phages resistant to the expression of transdominant K238T, K238A or RR340-341AA have been isolated to date, even after serial enrichment. The expression of these four constructs in trans appeared to suppress the native, phage-encoded primase protein in a dominant negative fashion.
Centre of infection assays
NCK1125(pTRK809) and the four constructs resistant to phage 3 were also tested for their ability to inhibit the formation of phage 3 infective centres. The strain harbouring pTRK809 exhibited parental-type ECOI and plaque sizes. The four strains harbouring resistance constructs exhibited a 3.5 log reduction in ECOI formation, indicating that most infected cells did not lyse or produce progeny. In addition, the sizes of plaques that did form were reduced by 80 % on all four resistant strains.
Phages isolated from plaques generated during COI experiments were titrated on the sensitive indicator host NCK1125(pTRK687) and the four hosts expressing the transdominant primase proteins. These phages plaqued normally on NCK1125(pTRK687), but no plaques were detected on K238T, K238A or RR340-341AA at any dilution, indicating that any progeny phages recovered in the COI assays are not resistant to the expression of mutant primase proteins.
Transdominant proteins interfere with intracellular phage genome synthesis in vivo
NCK1125(pTRK687) and strains expressing K238T, N151am : : K238T and RR340-341AA were infected with phage 3 at a m.o.i. of approximately 1.0. Genomic DNAs isolated from phage-infected cells over the course of the lytic cycle showed that expression of transdominant primase proteins retarded or abolished the accumulation of phage 3-specific DNA bands over time (Fig. 3). In the control NCK1125(pTRK687) parent strain (Fig. 3a, e), phage-specific dsDNA fragments began to accumulate 10 min post-infection, accrued maximally at 30 min post-infection, and decreased at 40 min due to host lysis. Phage-specific DNA bands failed to accumulate over time in hosts expressing primase proteins with the transdominant mutations K238T (Fig. 3b, f) and RR340-341AA (Fig. 3d). The transdominant phenotype was completely abolished and phage replication restored when the N151am mutation was introduced upstream of the K238T mutation (Fig. 3c). Interestingly, introduction of an E437A mutation downstream of the transdominant K238T mutation completely suppressed phage resistance. These results suggested that E437 might itself be important for protein folding, stability and/or oligomerization. Thus, the E437A mutation is believed to preclude association of the mutant subunits with the phage-encoded (wild-type) primase subunits.
|
4, 9 and 10), and a single phage encoding a 7201-type genome replication module (6) (Fig. 1). The expression of K238T, K238A and RR340-341AA all similarly inhibited the replication of Sfi21-type phages 3 (Fig. 1) and 4, 9, and 10 (data not shown), but failed to provide any protection from the 7201-type phage 6 (Fig. 1). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In the present study, multiple alignments of related primase protein sequences were used to identify critical amino acid residues potentially involved in enzyme catalysis and/or protein subunit oligomerization. Directed by this approach, invariant and highly conserved amino acids within a S. thermophilus phage primase consensus sequence were targeted by site-specific mutation(s). Characterizations of these mutant proteins led to the discovery of a novel and highly efficacious subunit poisoning system that was effective against S. thermophilus phages encoding variants of the Sfi21-type genome replication module.
The expression in trans of the K238(A/T) or RR340-341AA mutant primase proteins suppressed the function of the native, phage-encoded putative primase protein in a dominant negative fashion. The inhibition of phage genome replication and failure to complete the lytic cycle suggested that the mutant primase proteins were structurally intact and formed stable interactions with the native, phage-encoded primase protein. Alternatively, the mutant primase proteins could have formed other non-productive associations, such as substrate binding (e.g. origin of replication) in the absence of catalysis and/or inhibition of DNA replication by titrating away other phage- or host-encoded genome replication factors. Amber mutations (N151am) introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and parental plaquing sensitivity, indicating a complete suppression of primase-encoded resistance. These results indicated that translation of the transdominant mutant primase proteins was required to confer phage resistance. Given the magnitude of the resistance conferred, it was concluded that the putative primase protein is an essential enzyme required for genome replication in S. thermophilus Sfi21-type phages. Further, it was also clear that host-encoded factors were unable to complement the deficiency caused by transdominant primase expression. It is important to note that the predicted phage 3-encoded primase has not been biochemically characterized.
Several phage-encoded primase proteins, including gene product 4 (gp4) encoded by coliphage T7 (Podoviridae) (Notarnicola & Richardson, 1993) and gp
encoded by coliphage P4 (Myoviridae) (Ziegelin et al., 1995), have been characterized at the genetic and biochemical levels. Interestingly, the putative primase from phage 3 (family Siphoviridae) and its related putative orthologues lack several key functional motifs conserved among these E. coli DnaG-like primase proteins (Ilyina et al., 1992). These deficiencies notwithstanding, the phage 3-encoded enzyme described in this study has been designated a variant class of putative primase since it possesses significant regions in its carboxy terminus that exhibit weak similarities to other phage-encoded and prokaryotic polymerases, especially primases. For example, the ATPase/helicase domain (COG3387) shows similarities to the putative primase from Lactobacillus gasseri phage 
adh (Altermann et al., 1999) and the characterized primase from E. coli satellite phage P4 (Ziegelin et al., 1993, 1995).
The novel RR340-341 arginine dyad targeted in this study is located within the putative ATPase/helicase domain; however, the role of this dyad in protein function has not yet been determined. In addition, we confirm the essential role of a catalytic lysine residue (K238) in primase function. This residue is positioned within a glycine-rich phosphate-binding loop that is also located within the putative ATPase/helicase domain. This flexible structure is routinely found at the transition point between a β-strand and an -helix and forms a nucleotide-binding site (NBS) in other ATPase/helicase domains found in a variety of heterologous primase proteins (Notarnicola et al., 1995).
In heterologous phages, amino acid substitutions within the NBS motifs have previously demonstrated that these motifs are essential for phage replication both in vivo and in vitro. Transcomplementation studies in phage T7 yielded results similar to those observed here (Notarnicola et al., 1995; Notarnicola & Richardson, 1993).
When the E437A mutation was introduced downstream of the transdominant K238T mutation, phage sensitivity was completely restored. These results indicated that the E437A mutation precluded the association of the mutant primase protein with the native, phage-encoded primase. Hence, E437 is likely to be a component of the protein oligomerization domain or important for stabilization of the multimeric protein.
In a previous study, antisense RNAs directed against the phage 3-encoded putative primase gene severely retarded phage genome replication and limited the number of progeny phage released in S. thermophilus (Sturino & Klaenhammer, 2004). The expression of pri-3.10-AS, one of the largest and most effective antisense RNAs, resulted in a 2.7-log reduction in EOP and a 50 % reduction in ECOI formation, meaning that only one of every two phage-infected cells released progeny phage, even when only the first lytic cycle was impeded by antisense RNA. In contrast, the expression of transdominant primase proteins was more effective than the most effective primase-specific antisense RNA. The transdominant K238(A/T) and RR340-341AA proteins completely inhibited genome replication and reduced the EOP by more than 9 logs.
Significantly, no phages resistant to transdominant primase proteins have been isolated to date.
In conclusion, this study describes a novel, protein-based defence system effective against S. thermophilus phages encoding variants of the Sfi21-type genome replication module. Further, it appears to be the first application of transdominant mutant proteins to inhibit phage replication, via subunit poisoning, in starter culture bacteria that are widely used in industrial fermentations.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. Anné
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
adh. Gene 236, 333–346.[CrossRef][Medline]Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST, a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Bouchard, J. D. & Moineau, S. (2000). Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain. Virology 270, 65–75.[CrossRef][Medline]
Brüssow, H. & Desiere, F. (2001). Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages. Mol Microbiol 39, 213–222.[CrossRef][Medline]
Brüssow, H., Probst, A., Fremont, M. & Sidoti, J. (1994). Distinct Streptococcus thermophilus bacteriophages share an extremely conserved DNA fragment. Virology 200, 854–857.[CrossRef][Medline]
Bruttin, A., Desiere, F., d'Amico, N., Guérin, J. P., Sidoti, J., Huni, B., Lucchini, S. & Brüssow, H. (1997). Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory. Appl Environ Microbiol 63, 3144–3150.[Abstract]
Desiere, F., Lucchini, S., Canchaya, C., Ventura, M. & Brüssow, H. (2002). Comparative genomics of phages and prophages in lactic acid bacteria. Antonie Van Leeuwenhoek 82, 73–91.[CrossRef][Medline]
Djordjevic, G., Bojovic, B., Miladinov, N. & Topisirovic, L. (1997). Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA of Lactobacillus acidophilus ATCC 4356. Can J Microbiol 43, 61–69.[Medline]
Durmaz, E. & Klaenhammer, T. R. (2000). Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages. Appl Environ Microbiol 66, 895–903.
Frick, D. N. & Richardson, C. C. (2001). DNA primases. Annu Rev Biochem 70, 39–80.[CrossRef][Medline]
Hill, C., Massey, I. J. & Klaenhammer, T. R. (1991). Rapid method to characterize lactococcal bacteriophage genomes. Appl Environ Microbiol 57, 283–288.
Horton, R. M. (1995). PCR-mediated recombination and mutagenesis: SOEing together tailor-made genes. Mol Biotechnol 3, 93–99.[Medline]
Huynh, T. V., Young, R. A. & Davis, R. W. (1985). Construction and screening cDNA libraries in 
gt10 and gt11. In DNA cloning, vol. I, pp. 49–78. Edited by I. D. M. Glover. Oxford: IRL Press.
Ilyina, T. V., Gorbalenya, A. E. & Koonin, E. V. (1992). Organization and evolution of bacterial and bacteriophage primase-helicase systems. J Mol Evol 34, 351–357.[CrossRef][Medline]
Lucchini, S., Desiere, F. & Brüssow, H. (1999). Comparative genomics of Streptococcus thermophilus phage species supports a modular evolution theory. J Virol 73, 8647–8656.
Moineau, S., Borkaev, M., Holler, B. J., Walker, S. A., Kondo, J. K., Vedamuthu, E. R. & Vandenbergh, P. A. (1996). Isolation and characterization of lactococcal bacteriophages from cultured buttermilk plants in the United States. J Dairy Sci 79, 2104–2111.[Abstract]
Notarnicola, S. M. & Richardson, C. C. (1993). The nucleotide binding site of the helicase/primase of bacteriophage T7. Interaction of mutant and wild-type proteins. J Biol Chem 268, 27198–27207.
Notarnicola, S. M., Park, K., Griffith, J. D. & Richardson, C. C. (1995). A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J Biol Chem 270, 20215–20224.
Nuesch, J. P. & Tattersall, P. (1993). Nuclear targeting of the parvoviral replicator molecule NS1: evidence for self-association prior to nuclear transport. Virology 196, 637–651.[CrossRef][Medline]
Sing, W. D. & Klaenhammer, T. R. (1990). Characteristics of phage abortion conferred in lactococci by the conjugal plasmid pTR2030. J Gen Microbiol 136, 1807–1815.
Sturino, J. M. & Klaenhammer, T. R. (2002). Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages. Appl Environ Microbiol 68, 588–596.
Sturino, J. M. & Klaenhammer, T. R. (2004). Antisense RNA targeting primase interferes with bacteriophage replication in Streptococcus thermophilus. Appl Environ Microbiol 70, 1735–1743.
Sturino, J. M. & Klaenhammer, T. R. (2006). Engineered bacteriophage-defense systems in bioprocessing. Nat Rev Microbiol 4, 395–404.[CrossRef][Medline]
Tatusov, R. L., Natale, D. A., Garkavtsev, I. V., Tatusova, T. A., Shankavaram, U. T., Rao, B. S., Kiryutin, B., Galperin, M. Y., Fedorova, N. D. & Koonin, E. V. (2001). The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res 29, 22–28.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Wang, D., Yuan, W., Davis, I. & Parrish, C. R. (1998). Nonstructural protein-2 and the replication of canine parvovirus. Virology 240, 273–281.[CrossRef][Medline]
Ziegelin, G., Scherzinger, E., Lurz, R. & Lanka, E. (1993). Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities. EMBO J 12, 3703–3708.[Medline]
Ziegelin, G., Linderoth, N. A., Calendar, R. & Lanka, E. (1995). Domain structure of phage P4 alpha protein deduced by mutational analysis. J Bacteriol 177, 4333–4341.
Received 26 February 2007;
revised 30 June 2007;
accepted 6 July 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) present in various derivatives of the protein. EOP and relative plaque diameter (RPD) values are expressed as the mean±SD relative to plaques formed on NCK1125(pTRK687), the control indicator strain. Residues downstream of the N151am point mutation are not translated and are denoted by a dotted line. In an effort to increase the efficiency of translation, some of the constructs were amplified using primer set L001M (Supplementary Table S1), which mutated the (native) putative leucine (L) alternative start codon (5'-TTG-3') to the standard, methionine (M) start codon (5'-ATG-3'). These efforts, however, did not result in increased phage resistance. -, Not applicable.
