The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.

BackgroundSpanins are phage lysis proteins required to disrupt the outer membrane. Phages employ either two-component spanins or unimolecular spanins in this final step of Gram-negative host lysis. Two-component spanins like Rz-Rz1 from phage lambda consist of an integral inner membrane protein: i-spanin, and an outer membrane lipoprotein: o-spanin, that form a complex spanning the periplasm. Two-component spanins exist in three different genetic architectures; embedded, overlapped and separated. In contrast, the unimolecular spanins, like gp11 from phage T1, have an N-terminal lipoylation signal sequence and a C-terminal transmembrane domain to account for the topology requirements. Our proposed model for spanin function, for both spanin types, follows a common theme of the outer membrane getting fused with the inner membrane, effecting the release of progeny virions.ResultsHere we present a SpaninDataBase which consists of 528 two-component spanins and 58 unimolecular spanins identified in this analysis. Primary analysis revealed significant differences in the secondary structure predictions for the periplasmic domains of the two-component and unimolecular spanin types, as well as within the three different genetic architectures of the two-component spanins. Using a threshold of 40% sequence identity over 40% sequence length, we were able to group the spanins into 143 i-spanin, 125 o-spanin and 13 u-spanin families. More than 40% of these families from each type were singletons, underlining the extreme diversity of this class of lysis proteins. Multiple sequence alignments of periplasmic domains demonstrated conserved secondary structure patterns and domain organization within family members. Furthermore, analysis of families with members from different architecture allowed us to interpret the evolutionary dynamics of spanin gene arrangement. Also, the potential universal role of intermolecular disulfide bonds in two-component spanin function was substantiated through bioinformatic and genetic approaches. Additionally, a novel lipobox motif, AWAC, was identified and experimentally verified.ConclusionsThe findings from this bioinformatic approach gave us instructive insights into spanin function, evolution, domain organization and provide a platform for future spanin annotation, as well as biochemical and genetic experiments. They also establish that spanins, like viral membrane fusion proteins, adopt different strategies to achieve fusion of the inner and outer membranes.Electronic supplementary materialThe online version of this article (10.1186/s12859-018-2342-8) contains supplementary material, which is available to authorized users.

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.spanin | membrane fusion | spheroplast | holin | endolysin P hage lysis, the most common cytolytic event in the biosphere, has been extensively studied in phage λ, where four genes encoding five proteins (Fig. 1A) effect a three-step lytic process that releases the progeny virions (1, 2). The infection cycle suddenly terminates when the S105 holins, small membrane proteins encoded by gene S, are redistributed into large 2D foci, resulting in the formation of μm-scale holes in the cytoplasmic membrane (3). This event, called holin "triggering," occurs at a time specific to the allelic state of S and is temporally regulated by the proportion of a second S product, the antiholin S107 (4, 5). The R endolysin is then able to escape through the holes to attack the peptidoglycan (PG). Because the PG layer confers shape and mechanical integrity to the cell, holin and endolysin function was long thought to be necessary and sufficient for lysis (6, 7). However, recent genetic and physiological studies revealed that two other λ proteins, Rz and Rz1, are also required (Fig. 1B) (8). Rz and Rz1 are a type II integral membrane protein (N-in, C-out) and an outer membrane (OM) lipoprotein, respectively (9-11). Interacting by the C-termini of their periplasmic domains, Rz and Rz1 form a complex spanning the entire periplasm, designated as the spanin complex to denote its topology in the envelope. Accordingly, Rz and Rz1 are designated as the inner membrane (IM) (i-spanin) and OM (o-spanin) subunits of the spanin complex ( Fig. 1 A and B) (12). Experiments with GFP-Rz chimeras and biochemical analysis of envelope proteins indicate that the spanin complexes accumulate in the envelope throughout the morphogenesis period of the infection cycle (8, 13). The available data indicate that, after destruction of the PG by the endolysin, the spanin complex functions to disrupt the OM. In the absence of spanin function, the infection cycle termin...

The increased prevalence of drug-resistant, nosocomial infections, particularly from pathogenic members of the complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterize phage Petty, a novel podophage that infects multidrug-resistant and Genome analysis reveals that phage Petty is a 40,431bp ϕKMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single subunit endolysin, but lacks canonical spanins to disrupt the outer membrane. Analysis of other ϕKMV-like genomes revealed that spanin-less lysis cassettes are a feature of phages infecting within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). Gene, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4 kDa protein gp was cloned and expressed, and its activity against EPS in solution was determined. The enzyme degraded purified EPS from its host strain AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant strains. Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data needs to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this study, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported able to infect both and The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome, and confirm that, unlike the rounding of λ lysogens lacking functional spanin genes, cells infected with phage Petty lyse by bursting. This suggests phages like Petty employ a different mechanism to disrupt the outer membrane of hosts during lysis.

Coliphage lambda proteins Rz and Rz1 are the inner membrane and outer membrane subunits of the spanin complex—a heterotetramer that bridges the periplasm and is essential for the disruption of the outer membrane during phage lysis. Recent evidence suggests the spanin complex functions by fusing the inner and outer membrane. Here, we use a genetics approach to investigate and characterize determinants of spanin function. Because Rz1 is entirely embedded in the +1 reading frame of Rz, the genes were disembedded before using random mutagenesis to construct a library of lysis-defective alleles for both genes. Surprisingly, most of the lysis-defective missense mutants exhibited normal accumulation or localization in vivo, and also were found to be normal for complex formation in vitro. Analysis of the distribution and nature of single missense mutations revealed subdomains that resemble key motifs in established membrane-fusion systems, i.e., two coiled-coil domains in Rz, a proline-rich region of Rz1, and flexible linkers in both proteins. When coding sequences are aligned respective to the embedded genetic architecture of Rz1 within Rz, genetically silent domains of Rz1 correspond to mutationally sensitive domains in Rz, and vice versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp 28 in OM disruption. Additionally, gp 28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp 28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp 28 protein prior to lysis. Gp 28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp 28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp 28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering. Escherichia coli is a Gram-negative facultative anaerobic bacterium which is commonly found as a member of the commensal gut flora in mammals 1. While commensal E. coli strains generally do not cause disease in humans, multiple strains of E. coli have acquired virulence factors and may cause disease with symptoms ranging from mild discomfort to life-threatening bacteremia. These strains have been categorized into several pathotypes, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) 2. Enterotoxigenic E. coli strains can be distinguished from other E. coli pathotypes by the presence of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (HT) 2. These enterotoxins induce traveler's diarrhea (TD), characterized by mild to severe watery diarrhea 2 , which may be accompanied by nausea, vomiting, abdominal pain, fever or blood in stool 3. TD is one of the most common illnesses contracted by people from developed countries during international travel, with ETEC being the major causative agent of TD in Latin America, Africa, South Asia and the Middle East 4. TD typically self-resolves or may be successfully treated with antibiotics, but the global increase in the emergence of antibiotic resistance warrants evaluation of alternative treatment approaches 4. Bacteriophages (phages) are the natural viral predators of bacteria. Due to the ongoing emergence of multidrug-resistant bacteria and limited development of new antibiotics, there has been a renewed interest in the use of phages as antimicrobials 5-9. However, knowledge of the basic biology of phages outside of a number of well-studied model organisms is limited. Phages are highly diverse, and they are constantly co-evolving with their bacterial hosts 10,11. In the ...

We have identified common and inexpensive lab reagents that confer increased aerosol survivability on phi6 and other phages. Our results suggest that soluble protein is a key protective component in nebulizing medium.