Distribution and composition of the lysis cassette of Lactococcus lactis phages and functional analysis of bacteriophage ul36 holin

The bacteriophage lysis cassette, which comprises a lysin and a holin gene, was analyzed in 18 Lactococcus lactis phages. A muramidase motif was found in the lysins of c2-like phages, while an amidase motif was observed in the lysins of 936-like phages. Both amidase and muramidase types were detected among the P335 phages. The P335 lysins were separated into three groups based on amino acid sequence identity. A class I holin was recognized in 936-like and c2-like phages, whereas P335-like phages possess class II holins. The P335 holins were further divided into four groups based on sequence identity. Only the holins of 936-like phages contained putative dual-start motifs. The unusual lysis cassette of the highly virulent P335-like phage ul36 contains a unique holin (orf74B) upstream of a lysin which is present in several other P335-like phages. Using the λΔSthf system, we demonstrated that gpORF74B induces cell lysis at the same time as λΔSthf::S105, the effector of λ lysis. Transcriptional analysis of ul36 lysis cassette showed that first transcripts are detected 35 min after infection of L. lactis cells. The lysis clock of phage ul36 appears to be controlled by the late expression of the holin and lysin gene

Rhamnose-inducible gene expression in Listeria monocytogenes

Acid production from rhamnose is a characteristic phenotype of Listeria monocytogenes. We report the identification of the rhamnose transport and utilization operon located at lmo2846 to lmo2851, including the rhamnose-dependent promoter P(rha). Expression of reporter genes under control of P(rha) on a single copy integration vector demonstrated its suitability for inducible gene expression in L. monocytogenes. Transcription initiation from P(rha) is dose dependent, and a concentration as low as 100 µM rhamnose was found sufficient for induction. Moreover, P(rha) is subject to glucose catabolite repression, which provides additional options for strict control of expression. Infection of human THP1 macrophages revealed that P(rha) is repressed in intracellular L. monocytogenes, which is explained by the absence of rhamnose in the cytosol and possible interference by catabolite repression. The P(rha) promoter provides a novel and useful tool for triggering gene expression in extracellular L. monocytogenes, whereas intracellular conditions prevent transcription from this promoter

Complete Nucleotide Sequence and Molecular Characterization of Bacillus Phage TP21 and its Relatedness to Other Phages with the Same Name

Three different Bacillus bacteriophages designated TP21 are known from the literature. We have determined the sequence and structure of the TP21-L genome, and compared it to the other phages. The genome is 37.5 kb in size, possesses fixed invariable genome ends and features the typical modular organization of a temperate siphovirus. TP21-L is neither identical to TP21 isolated by Thorne (TP21-T), as shown by a PCR-based approach nor to TP21 isolated by He et al. (TP21-H), as estimated from phage dimensions. For reasons of clarity, we suggest renaming the different TP21 isolates

Listeria monocytogenes tyrosine phosphatases affect wall teichoic acid composition and phage resistance

Tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into two major groups: low molecular weight and conventional. The latter group also has activity as phosphoinositide phosphatases. These two kinds of PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence, and capsule/cell wall production. By annotation, Listeria monocytogenes possesses two potential low molecular weight and two conventional PTPs. Using L. monocytogenes wild-type (WT) strain 10403S, we have created an in-frame deletion mutant lacking all four PTPs, as well as four additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all four PTPs. However, the deletion mutant strain was resistant to Listeria phages A511 and P35 and sensitive to other Listeria phages. This was attributed to reduced attachment to the cell wall. The mutant lacking all PTPs was found to lack N-acetylglucosamine in its wall teichoic acid. Phage sensitivity and attachment was rescued in a complemented strain harboring a low molecular weight PTP (LMRG1707

Complete genome sequences of erwinia amylovora phages vB_EamP-S2 and vB_EamM-Bue1

Phages vB_EamP-S2 (S2) and vB_EamM-Bue1 (Bue1) infect the plant pathogen Erwinia amylovora. S2 has a genome size of 45,495 bp and belongs to the genus SP6virus. The genome size of Bue1, related to Salmonella phage Vil, is 164,037 bp. Both phages possess a depolymerase enzyme, a frequent feature of E. amylovora phages

The absence of a mature cell wall sacculus in stable Listeria monocytogenes L-form cells is independent of peptidoglycan synthesis

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan

Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP.This work was supported by grants ‘VIREVO’ CGL2016‐76273‐P (MCI/AEI/FEDER, EU) (cofounded with FEDER funds) from the Spanish Ministerio de Ciencia e Innovación and ‘HIDRAS3’ PROMETEU/2019/009 from Generalitat Valenciana. R.G.-S. was supported by a predoctoral fellowship from the Valencian Consellería de Educació, Investigació, Cultura i Esport (ACIF/2016/050) and was also a beneficiary of the BEFPI 2019 fellowship for predoctoral stays from Generalitat Valenciana and The European Social Fund. F.R.-V. was a beneficiary of the 5top100 program of the Ministry for Science and Education of Russia

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed

Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion

The increasing number of multidrug-resistant bacteria intensifies the need to develop new antimicrobial agents. Endolysins are bacteriophage-derived enzymes that degrade the bacterial cell wall and hold promise as a new class of highly specific and versatile antimicrobials. One major limitation to the therapeutic use of endolysins is their often short serum circulation half-life, mostly due to kidney excretion and lysosomal degradation. One strategy to increase the half-life of protein drugs is fusion to the albumin-binding domain (ABD). By high-affinity binding to serum albumin, ABD creates a complex with large hydrodynamic volume, reducing kidney excretion and lysosomal degradation. The aim of this study was to investigate the in vitro antibacterial activity and in vivo biodistribution and half-life of an engineered variant of the Staphylococcus aureus phage endolysin LysK. The ABD sequence was introduced at different positions within the enzyme, and lytic activity of each variant was determined in vitro and ex vivo in human serum. Half-life and biodistribution were assessed in vivo by intravenous injection of europium-labeled proteins into C57BL/6 wild-type mice. Our data demonstrates that fusion of the endolysin to ABD improves its serum circulation half-life and reduces its deposition in the kidneys in vivo. The most active construct reduced S. aureus counts in human serum ex vivo by 3 logs within 60 min. We conclude that ABD fusions provide an effective strategy to extend the half-life of antibacterial enzymes, supporting their therapeutic potential for treatment of systemic bacterial infections