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Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.

ABSTRACT: Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.

SUBMITTER: Zuniga M

PROVIDER: S-EPMC136552 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.

Zúñiga Manuel M Franke-Fayard Blandine B Venema Gerard G Kok Jan J Nauta Arjen A

Journal of virology 20021001 20

Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indic ...[more]

PMID: 12239299

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Project description:The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Project description:ABSTRACT Analysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains of Lactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1 to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall of L. lactis 3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1 L. lactis MG1363. The CWPS of L. lactis 3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-?-Glc-3-?-Galf-3-?-GlcNAc-2-?-Galf-6-?-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2 were introduced into a mutant of subtype C1 L. lactis NZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2 L. lactis 3107 and not wild-type C1 L. lactis NZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range. IMPORTANCE Despite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host.

Dataset Information

Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.

Publications

Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.

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