Stephen R. Stockdale

The Lactococcal Phages Tuc2009 and TP901-1 Incorporate Two Alternate Forms of Their Tail Fiber into Their Virions for Infection Specialization

Background: Siphoviridae virions often possess lytic domains facilitating host-penetration. Results: Tuc2009 and TP901-1 virions may contain full-length or truncated tail fibers, possessing or lacking a lytic domain, respectively. Conclusion: Phages with a lytic domain infect stationary phase cells better, whereas truncated derivatives have higher adsorption efficiencies. Significance: The heterogeneous phage population serves to most effectively infect bacteria where levels of cell wall cross-linkage differ. Lactococcal phages Tuc2009 and TP901-1 possess a conserved tail fiber called a tail-associated lysin (referred to as Tal2009 for Tuc2009, and Tal901-1 for TP901-1), suspended from their tail tips that projects a peptidoglycan hydrolase domain toward a potential host bacterium. Tal2009 and Tal901-1 can undergo proteolytic processing mid-protein at the glycine-rich sequence GG(S/N)SGGG, removing their C-terminal structural lysin. In this study, we show that the peptidoglycan hydrolase of these Tal proteins is an M23 peptidase that exhibits d-Ala-d-Asp endopeptidase activity and that this activity is required for efficient infection of stationary phase cells. Interestingly, the observed proteolytic processing of Tal2009 and Tal901-1 facilitates increased host adsorption efficiencies of the resulting phages. This represents, to the best of our knowledge, the first example of tail fiber proteolytic processing that results in a heterogeneous population of two phage types. Phages that possess a full-length tail fiber, or a truncated derivative, are better adapted to efficiently infect cells with an extensively cross-linked cell wall or infect with increased host-adsorption efficiencies, respectively.

Phages of lactic acid bacteria: The role of genetics in understanding phage-host interactions and their co-evolutionary processes

Dairy fermentations are among the oldest food processing applications, aimed at preservation and shelf-life extension through the use of lactic acid bacteria (LAB) starter cultures, in particular strains of Lactococcus lactis, Streptococcus thermophilus, Lactobacillus spp. and Leuconostoc spp. Traditionally this was performed by continuous passaging of undefined cultures from a finished fermentation to initiate the next fermentation. More recently, consumer demands on consistent and desired flavours and textures of dairy products have led to a more defined approach to such processes. Dairy (starter) companies have responded to the need to define the nature and complexity of the starter culture mixes, and dairy fermentations are now frequently based on defined starter cultures of low complexity, where each starter component imparts specific technological properties that are desirable to the product. Both mixed and defined starter culture approaches create the perfect environment for the proliferation of (bacterio)phages capable of infecting these LAB. The repeated use of the same starter cultures in a single plant, coupled to the drive towards higher and consistent production levels, increases the risk and negative impact of phage infection. In this review we will discuss recent advances in tracking the adaptation of phages to the dairy industry, the advances in understanding LAB phage-host interactions, including evolutionary and genomic aspects.

Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms.

Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

Structure and assembly of TP901-1 virion unveiled by mutagenesis

Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

Lactococcus lactis phage TP901-1 as a model for Siphoviridae virion assembly.

ABSTRACT Phages infecting Lactococcus lactis pose a serious threat to the dairy fermentation sector. Consequently, they are among the most thoroughly characterized Gram positive-infecting phages. The majority of lactococcal phages belong to the tailed family of phages named the Siphoviridae. The coliphage lambda and the Bacillus subtilis phage SPP1 have been the predominant comparators for emerging siphophages both genomically and structurally and both phages recognize a membrane protein receptor. In contrast, the lactococcal P335 group phage TP901-1 attaches to cell wall surface polysaccharides. It is a typical “lambdoid” siphophage possessing a long non-contractile tail and a genomic architecture reminiscent of lambda and SPP1 despite low or undetectable sequence homology in many of its encoded products, especially those involved in host recognition. A functional analysis of the structural components of TP901-1 was undertaken based on the characterization of a series of mutants in the region encoding the capsid and tail morphogenetic elements. Through this analysis, it was possible to deduce that, despite the lack of sequence homology, the overall genomic architecture of Siphoviridae phages typified by functional synteny is conserved. Furthermore, a model of the TP901–1 assembly pathway was developed with potential implications for many tailed phages.

ΦCrAss001, a member of the most abundant bacteriophage family in the human gut, infects Bacteroides

ΦCrAss001, isolated from human faecal material, is the first member of the extensive crAssphage family to be grown in pure culture. The bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming in silico predictions of the likely host. Genome analysis demonstrated that the phage DNA is 102 kb in size, has an unusual genome organisation and does not possess any obvious genes for lysogeny. In addition, electron microscopy confirms that φcrAss001 has a podovirus-like morphology. Despite the absence of lysogeny genes, φcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium and is able to maintain itself in continuous host culture.

Viromes of one year old infants reveal the impact of birth mode on microbiome diversity

Establishing a diverse gut microbiota after birth is being increasingly recognised as important for preventing illnesses later in life. It is well established that bacterial diversity rapidly increases post-partum; however, few studies have examined the infant gut virome/phageome during this developmental period. We performed a metagenomic analysis of 20 infant faecal viromes at one year of age to determine whether spontaneous vaginal delivery (SVD) or caesarean section (CS) influenced viral composition. We find that birth mode results in distinctly different viral communities, with SVD infants having greater viral and bacteriophage diversity. We demonstrate that CrAssphage is acquired early in life, both in this cohort and two others, although no difference in birth mode is detected. A previous study has shown that bacterial OTU’s (operational taxonomic units) identified in the same infants could not discriminate between birth mode at 12 months of age. Therefore, our results indicate that vertical transmission of viral communities from mother to child may play a role in shaping the early life microbiome, and that birth mode should be considered when studying the early life gut virome.

VIGA: a sensitive, precise and automatic de novo VIral Genome Annotator.

Viral (meta)genomics is a rapidly growing field of study that is hampered by an inability to annotate the majority of viral sequences; therefore, the development of new bioinformatic approaches is very important. Here, we present a new automatic de novo genome annotation pipeline, called VIGA, to annotate prokaryotic and eukaryotic viral sequences from (meta)genomic studies. VIGA was benchmarked on a database of known viral genomes and a viral metagenomics case study. VIGA generated the most accurate outputs according to the number of coding sequences and their coordinates, outputs also had a lower number of non-informative annotations compared to other programs.

RNA Phage Biology in a Metagenomic Era

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.