Summary At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S2168. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character. Holins are small phage-encoded membrane proteins that control the length of the bacteriophage infection cycle by determining the time of host lysis (1, 2). Almost all of the experimental work done on holin function at the physiological or molecular level has been focused on a few holin genes: lambda S, phage 21 S 21 , and T4 t. These genes encode the holins S105, S 21 68, and T, which are, respectively, the prototypes of three distinct holin classes, distinguished by their experimentally determined membrane topologies (Fig. 1A). S105 has class I topology, with three transmembrane domains (TMDs) arranged N-out C-in, whereas S 21 68 has class II topology, with two TMDs arranged N-in C-in. As of the last major review, ϳ90% of the Ͼ100 genes from phages and prophages of eubacteria that have been assigned as putative holins fit into either class I or class II, based on predicted topology using widely accepted algorithms (1). These predicted holins are very diverse, with 12 and 14 unrelated sequence families grouped into classes I and II, respectively. Class III topology, with a single TMD and a large periplasmic domain, is restricted to the sequence homologs of protein T, the holin of T4. These proteins are found only in phages of the large myophage class of T4-like phages and in the large siphophage class of T5-like phages.Despite this diversity, holins share some universal functional features, based mainly on extensive work that has been done with these three holin paradigms. Holin genes are turned on at the beginning of viral morphogenesis, so that the holins accumulat...

For most phages, holins control the timing of host lysis. During the morphogenesis period of the infection cycle, canonical holins accumulate harmlessly in the cytoplasmic membrane until they suddenly trigger to form lethal lesions called holes. The holes can be visualized by cryo-electron microscopy and tomography as micrometer-scale interruptions in the membrane. To explore the fine structure of the holes formed by the lambda holin, S105, a cysteine-scanning accessibility study was performed. A collection of S105 alleles encoding holins with a single Cys residue in different positions was developed and characterized for lytic function. Based on the ability of 4-acetamido-4=-((iodoacetyl) amino) stilbene-2,2=-disulfonic acid, disodium salt (IASD), to modify these Cys residues, one face of transmembrane domain 1 (TMD1) and TMD3 was judged to face the lumen of the S105 hole. In both cases, the lumen-accessible face was found to correspond to the more hydrophilic face of the two TMDs. Judging by the efficiency of IASD modification, it was concluded that the bulk of the S105 protein molecules were involved in facing the lumen. These results are consistent with a model in which the perimeters of the S105 holes are lined by the holin molecules present at the time of lysis. Moreover, the findings that TMD1 and TMD3 face the lumen, coupled with previous results showing TMD2-TMD2 contacts in the S105 dimer, support a model in which membrane depolarization drives the transition of S105 from homotypic to heterotypic oligomeric interactions.H ost lysis at the end of the bacteriophage infection cycle is one of the most common cellular fates in the biosphere. For most phages, the holin controls the timing of lysis and thus the length and fecundity of the infection cycle. The best-studied holin is the S105 protein of phage lambda, one of two products of the lambda S gene ( Fig. 1 and 2), the other being the S107 antiholin (1-4). Throughout the morphogenesis period of the infection cycle, S105 accumulates in the membrane without detectable effect on cell physiology or membrane integrity. Suddenly, at an allele-specific time, the S105 population is said to "trigger" to form lethal membrane lesions. Triggering is detectable by a sudden halt in culture growth and respiration, collapse of the membrane potential, massive ion leakage into the medium, and loss of viability. Moreover, triggering can be imposed prematurely by causing a sudden reduction in the membrane potential, using energy poisons like 2,4-dinitrophenol (DNP) and cyanide (5). The physiological effects observed for the cell are due to the formation of micrometer-scale membrane lesions, or holes, approximately 1 to 3 per cell and averaging Ͼ340 nm in diameter (6), the largest membrane lesions described in biology. The formation of these massive holes allows folded, fully active cytoplasmic endolysins to be released nonspecifically into the periplasm, resulting in rapid degradation of the peptidoglycan (2, 7). The term "hole" was chosen to distinguish these lesions from...

Host cell lysis is the terminal event of the bacteriophage infection cycle. In Gram-negative hosts, lysis requires proteins that disrupt each of the three cell envelope components, only one of which has been identified in Mu: the endolysin gp22.

SummaryA multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.