Topological and phylogenetic analyses of bacterial holin families and superfamilies

Holins are small “hole-forming” transmembrane proteins that mediate bacterial cell lysis during programmed cell death or following phage infection. We have identified fifty two families of established or putative holins and have included representative members of these proteins in the Transporter Classification Database (TCDB; www.tcdb.org). We have identified the organismal sources of members of these families, calculated their average protein sizes, estimated their topologies and determined their relative family sizes. Topological analyses suggest that these proteins can have 1, 2, 3 or 4 transmembrane α-helical segments (TMSs), and members of a single family are frequently, but not always, of a single topology. In one case, proteins of a family proved to have either 2 or 4 TMSs, and the latter arose by intragenic duplication of a primordial 2 TMS protein-encoding gene resembling the former. Using established statistical approaches, some of these families have been shown to be related by common descent. Seven superfamilies, including 21 of the 52 recognized families were identified. Conserved motif and Pfam analyses confirmed most superfamily assignments. These results serve to expand upon the scope of channel-forming bacterial holins.     

Bacteriophage endolysins as a novel class of antibacterial agents

Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases produced in phage-infected bacterial cells toward the end of the lytic cycle. They reach the peptidoglycan through membrane lesions formed by holins and cleave it, thus, inducing lysis of the bacterial cell and enabling progeny virions to be released. Endolysins are also capable of degrading peptidoglycan when applied externally (as purified recombinant proteins) to the bacterial cell wall, which also results in a rapid lysis of the bacterial cell. The unique ability of endolysins to rapidly cleave peptidoglycan in a generally species-specific manner renders them promising potential antibacterial agents. Originally developed with a view to killing bacteria colonizing mucous membranes (with the first report published in 2001), endolysins also hold promise for the treatment of systemic infections. As potential antibacterials, endolysins possess several important features, for instance, a novel mode of action, a narrow antibacterial spectrum, activity against bacteria regardless of their antibiotic sensitivity, and a low probability of developing resistance. However, there is only one report directly comparing the activity of an endolysin with that of an antibiotic, and no general conclusions can be drawn regarding whether lysins are more effective than traditional antibiotics. The results of the first preclinical studies indicate that the most apparent potential problems associated with endolysin therapy (e.g., their immunogenicity, the release of proinflammatory components during bacteriolysis, or the development of resistance), in fact, may not seriously hinder their use. However, all data regarding the safety and therapeutic effectiveness of endolysins obtained from preclinical studies must be ultimately verified by clinical trials. This review discusses the prophylactic and therapeutic applications of endolysins, especially with respect to their potential use in human medicine. Additionally, we outline current knowledge regarding the structure and natural function of the enzymes in phage biology, including the most recent findings.     

Diversity,specificity and molecular evolution of the lytic arsenal of Pseudomonas phages: in silico perspective

Bacteriophages encode an arsenal of proteins to lyse bacteria by breaking their surface structures, constituting a promising alternative to antibiotics. However, the selection and bioengineering of endolysins and other phage lytic proteins need to be assisted by a previous knowledge of their molecular characteristics. In this study, all putative lytic proteins encoded in Pseudomonas phages were in silico examined to describe their diversity, host association and molecular evolution. A total of 491 proteins were identified among 223 phages, including endolysins, holins, pinholins, spanins, lipases and peptidases. Protein families and combination of functional domains were characteristic of phages belonging to the same genus, and these tended to infect a single host species. Clustering and phylogenetic analysis showed a protein grouping associated with bacterial host, and some functional domains being specific. Interestingly, most putative lytic proteins from phages infecting P. fluorescens and P. putida had negative net charges, opposed to most endolysins. Phage lifestyle also had an impact on protein variability, with transglycosylases, glucosaminidases, holins and spanins from lysogenic phages clustering into monophyletic nodes, suggesting the effect of a different selection pressure as a result of the co-option of a new function in the lysogenized bacteria.     

Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities

The mycobacterial cell wall presents significant challenges to mycobacteriophages--viruses that infect mycobacterial hosts--because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain.     

Holin of bacteriophage lambda: structural insights into a membrane lesion

The length of the infection cycle of double-stranded DNA bacteriophages is controlled by phage-encoded small integral membrane proteins, holins. Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. The paper by Savva et al . in this issue of Molecular Microbiology invites us to take a closer look at this membrane lesion. For the first time, a structural characterization of large-diameter rings formed by these peculiar membrane proteins is presented.     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.     

The Autolysin LytA Contributes to Efficient Bacteriophage Progeny Release in Streptococcus pneumoniae

Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.Streptococcus pneumoniae (pneumococcus), a common and important human pathogen, is characterized by the high incidence of lysogeny in isolates associated with infection (34, 44). Pneumococcal bacteriophages (phages) share with the majority of bacteriophages infecting other bacterial species the “holin-lysin” system to lyse the host cell and release their progeny at the end of the lytic cycle. Genes encoding both holins and lysins (historically termed “endolysins”) are indeed found in the genomes of all known pneumococcal phages (8, 28, 31, 37). Supporting this mechanism, a lytic phenotype in the heterologous Escherichia coli system was achieved only by the simultaneous expression of the Ejh holin and the Ejl endolysin of pneumococcal phage EJ-1 (8). When these proteins were independently expressed, cellular lysis was not perceived. Similar results were shown for pneumococcal phage Cp-1, not only in E. coli, but also in the pneumococcus itself (28).Phage lysins destroy the pneumococcal peptidoglycan network due to their muralytic activity, whereas holins have been shown in S. pneumoniae to form nonspecific lesions (8), most likely upon a process of oligomerization in the cytoplasmic membrane, as observed for the E. coli phage λ (13, 14, 43). It was generally proposed that holin lesions allow access of phage lysins to the cell wall (52, 54), as the majority of phage lysins, including the pneumococcal endolysins, lack a typical N-terminal secretory signal sequence and transmembrane domains (8). However, recent evidence also highlights the possibility for a holin-independent targeting of phage lysins to the cell wall, where holin lesions seem to be crucial for the activation of the already externalized phage lysins (42, 50, 51). Regardless of the mechanism operating in S. pneumoniae to activate phage lysins, holin activity compromises membrane integrity.Pneumococcal cells present their own autolytic activity, mainly due to the presence of a powerful bacterial cell wall hydrolase, LytA (an N-acetylmuramoyl-l-alanine-amidase), responsible for bacterial lysis under certain physiological conditions (47). Although other bacterial species also encode peptidoglycan hydrolases, the extensive lysis shortly after entering stationary phase caused by LytA is a unique feature of S. pneumoniae. Interestingly, LytA is translocated across the cytoplasmic membrane to the cell wall—where it remains inactive—in spite of the absence of a canonical N-terminal sequence signal (7). In the cell wall, autolysin activities are tightly regulated by mechanisms that seem to be related to the energized state of the cell membrane. In fact, depolarizing agents are able to trigger autolysis in Bacillus subtilis (16, 17), and bacteriocin-induced depletion of membrane potential triggers autolysis of some species of the genera Lactococcus and Lactobacillus, closely related to streptococci (29). It is therefore possible that the holin-inflicted perturbations of the S. pneumoniae cytoplasmic membrane upon the induction of the lytic cycle may trigger not only the lytic activity of the phage lysin, but also that of inactive LytA located in the cell wall. Accordingly, LytA could also participate in the release of phage particles at the end of the infectious cycle, especially considering its powerful autolytic activity. Previous studies have suggested a role for the host autolytic enzyme in the release of phage progeny (11, 38), but in fact, the evidence is unclear and dubious, considering that the existence of phage-encoded lysins was unknown or very poorly understood and some of the experimental conditions used to show a role of LytA could have also affected the activity of the phage lysin (38).To clarify the possible role of the bacterial autolysin in host lysis, we used the S. pneumoniae strain SVMC28, lysogenic for the SV1 prophage (34), which contains a typical “holin-lysin” cassette, and a different host strain lysogenized with the same SV1 phage. Our results show that LytA is activated by the holin-induced membrane disruption, just like the phage endolysin. In the absence of the endolysin, LytA is capable of mediating host lysis, releasing functional phage particles able to complete their life cycle. Still, sole dependence on LytA results in an altered pattern of phage release that may reduce phage fitness. Importantly, we also show that, together with the endolysin, the concurrent LytA activation is critical for optimal phage progeny release.     

Dimerization between the holin and holin inhibitor of phage lambda

Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of lambda-infected cell lysis depends solely on the 107 codon S gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.     

Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity

Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins.     

The Spanin Complex Is Essential for Lambda Lysis

Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.     

dATP/ATP, a multifunctional nucleotide, stimulates bacterial cell lysis, extracellular DNA release and biofilm development

Background

Signaling by extracellular adenosine 5′-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a “danger signal”, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this “danger signal”.

Methods and Principal Findings

Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells.

Conclusions and Significance

These data suggest that bacteria may sense extracellular dATP/ATP as a signal of “danger” and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an “inter-domain” warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells.     

Stable micron‐scale holes are a general feature of canonical holins

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.     

Characterization of the dual start motif of a class II holin gene

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S  ^λ, has a dual start motif and encodes not only the effector holin, S^λ105, but also an inhibitor, S^λ107, with a Met–Lys … extension at the terminus. The prototype class II holin gene of phage 21, S  ²¹, begins with the motif Met–Lys–Ser–Met … , and a potential RNA secondary structure overlaps the Shine–Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S  ²¹, S²¹71 and S²¹68; (ii) the shorter product is required for lysis; (iii) the longer product, S²¹71, inhibits S  ²¹ function; and (iv) the Lys-2 residue is important for the inhibitor function. Moreover, the RNA stem–loop structure is involved in the downregulation of S²¹71 synthesis. However, our results suggest that, in S  ²¹, different segments of the single consensus Shine–Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.     

Bioinformatics analysis of bacteriophage and prophage endolysin domains

Endolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

Fold designability, distribution, and disease

Fold designability has been estimated by the number of families contained in that fold. Here, we show that among orthologous proteins, sequence divergence is higher for folds with greater numbers of families. Folds with greater numbers of families also tend to have families that appear more often in the proteome and greater promiscuity (the number of unique “partner” folds that the fold is found with within the same protein). We also find that many disease-related proteins have folds with relatively few families. In particular, a number of these proteins are associated with diseases occurring at high frequency. These results suggest that family counts reflect how certain structures are distributed in nature and is an important characteristic associated with many human diseases.     

A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds

In this work we develop a microscopic physical model of early evolution where phenotype—organism life expectancy—is directly related to genotype—the stability of its proteins in their native conformations—which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the “Big Bang” scenario whereby exponential population growth ensues as soon as favorable sequence–structure combinations (precursors of stable proteins) are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species—subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution.