Wilfried J. J. Meijer

The ø29-family of phages

SUMMARY Continuous research spanning more than three decades has made the Bacillus bacteriophage φ29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage φ29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5′ ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to φ29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5′ ends. These φ29-like phages can be divided into three groups. The first group includes, in addition to φ29, phages PZA, φ15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of φ29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of φ29-like phages, which is included in this review.

Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage ϕ29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the ϕ29 genome and the B. subtilis chromosome. Binding of Spo0A near the ϕ29 DNA ends, constituting the two origins of replication of the linear ϕ29 genome, prevents formation of ϕ29 protein p6‐nucleoprotein initiation complex resulting in inhibition of ϕ29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA‐binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage ϕ29 development and the bacterial chromosome replication during the onset of sporulation are discussed.

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication.

An early expressed operon, located at the right end of the linear bacteriophage φ29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in φ29‐related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N‐terminally located transmembrane‐spanning domain is required for its membrane localization. The variant p16.7A, in which the N‐terminal membrane anchor was replaced by a histidine‐tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a φ29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivoφ29 DNA replication is discussed.

Molecular basis for the exploitation of spore formation as survival mechanism by virulent phage ϕ29

Phage ϕ29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that ϕ29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host‐encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected ϕ29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses ϕ29 development by repressing the main early ϕ29 promoters via different and novel mechanisms and also by preventing activation of the single late ϕ29 promoter.

Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Inhibition of Bacillus subtilis natural competence by a native, conjugative plasmid-encoded comK repressor protein

Under certain growth conditions, Bacillus subtilis can develop natural competence, the state in which it is able to bind, adsorb and incorporate exogenous DNA. Development of competence is a bistable process and is subject to complex regulation. Rok is a repressor of the key transcriptional activator of competence genes, comK, and limits the size of the subpopulation that develops competence. Here we report the finding that the large conjugative B. subtilis plasmid pLS20 harbours a rok homologue rok(LS20). Although the deduced product of rok(LS20) is considerably shorter than the chromosomally encoded Rok protein, we show that ectopic expression of the plasmid-encoded Rok(LS20) leads to inhibition of competence by repressing comK, and that the effects of the plasmid and chromosomally encoded Rok proteins are additive. We also show that pLS20 inhibits competence in a rok(LS20) -dependent manner and that purified Rok(LS20) preferentially binds to the comK promoter. By analysing the available databases we identified several additional rok-like genes. These putative rok genes can be divided into two groups and we propose that rok(LS20) is the prototype of a newly identified subgroup of nine rok genes. Finally, we discuss the possible role of the plasmid-located rok and its relatedness with other rok genes.

Phage φ29 Terminal Protein Residues Asn80 and Tyr82 Are Recognition Elements of the Replication Origins

Initiation of phage φ29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the φ29 terminal protein (TP) and the φ29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various φ29 TP mutants were generated at conserved amino acid residues from positions 61 to 87.In vitro φ29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with φ29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.

The integral membrane protein p16.7 organizes in vivo φ29 DNA replication through interaction with both the terminal protein and ssDNA

Remarkably little is known about the in vivo organization of membrane‐associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage φ29 as a model system. Previously, we demonstrated that the φ29‐encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane‐associated φ29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the φ29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C‐terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA‐binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo φ29 DNA replication is presented.

DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.

The accumulation of subgenomic phage φ29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage φ29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single‐stranded self‐complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double‐stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5′‐TGTTtCAC‐GTGgAACA‐3′ being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the φ29 genome. In addition, the almost identical sequence 5′‐TgGTTTCAC‐GTGGAAtCA‐3′ was found once. These five DSEs are all located in the right half of the φ29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic φ29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.

kinC/D-mediated heterogeneous expression of spo0A during logarithmical growth in Bacillus subtilis is responsible for partial suppression of φ29 development

The host of the lytic bacteriophage φ29 is the spore‐forming bacterium Bacillus subtilis. When infection occurs during early stages of sporulation, however, φ29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. Recently, we have shown that Spo0A, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic φ29 cycle in cells having initiated sporulation. Surprisingly, we found that φ29 development is suppressed in a subpopulation of logarithmically growing culture and that spo0A is heterogeneously expressed during this growth stage. Furthermore, we showed that kinC and, to a minor extent, kinD, are responsible for heterogeneous expression levels of spo0A during logarithmical growth that are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of φ29. Whereas spo0A was known to be heterogeneously expressed during the early stages of sporulation, our findings show that this also occurs during logarithmical growth. These insights are likely to have important consequences, not only for the life cycle of φ29, but also for B. subtilis developmental processes.