Survival of the fittest: a role for phage-encoded eukaryotic-like kinases

Phages are often thought of as mortal enemies of bacteria. This dynamic relationship has led to the evolution of a number of processes in bacteria designed to defeat these attacks. Examples of these include blocking phage attachment, CRISPR, and restriction modification systems. Temperate phages provide another source of protection by excluding infection of heterologous phage, thwarting phage production and further infection. This strategy protects the rest of the bacterial population from attack. The lambdoid phage 933W, a source of the genes encoding Shiga toxin in the highly pathogenic O157:H7 enterohemorrhagic E. coli strain, also carries a gene encoding a eukaryotic-like tyrosine kinase, Stk. In this issue of Molecular Microbiology, Friedman et al. (2011) show that Stk, through its kinase activity, excludes infection by another lambdoid phage HK97. This exclusion is very specific as it does not affect a number of other lambdoid phages. HK97 contributes to its own demise by expressing the product of an open reading frame, orf41, which is required for Stk activation. The authors further show that autophosphorylation increases the stability of Stk and suggest that autophosphorylation contributes to Stk activity. Whether or not this exclusion activity provides a selective advantage through maintenance of Stk activity is yet to be explored.     

Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W

The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.     

The Bacteriophage HK97 gp15 Moron Element Encodes a Novel Superinfection Exclusion Protein

A phage moron is a DNA element inserted between a pair of genes in one phage genome that are adjacent in other related phage genomes. Phage morons are commonly found within phage genomes, and in a number of cases, they have been shown to mediate phenotypic changes in the bacterial host. The temperate phage HK97 encodes a moron element, gp15, within its tail morphogenesis region that is absent in most closely related phages. We show that gp15 is actively expressed from the HK97 prophage and is responsible for providing the host cell with resistance to infection by phages HK97 and HK75, independent of repressor immunity. To identify the target(s) of this gp15-mediated resistance, we created a hybrid of HK97 and the related phage HK022. This hybrid phage revealed that the tail tube or tape measure proteins likely mediate the susceptibility of HK97 to inhibition by gp15. The N terminus of gp15 is predicted with high probability to contain a single membrane-spanning helix by several transmembrane prediction programs. Consistent with this putative membrane localization, gp15 acts to prevent the entry of phage DNA into the cytoplasm, acting in a manner reminiscent of those of several previously characterized superinfection exclusion proteins. The N terminus of gp15 and its phage homologues bear sequence similarity to YebO proteins, a family of proteins of unknown function found ubiquitously in enterobacteria. The divergence of their C termini suggests that phages have co-opted this bacterial protein and subverted its activity to their advantage.     

Prophage Induction Is Enhanced and Required for Renal Disease and Lethality in an EHEC Mouse Model

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production.     

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.     

Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system

Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.     

Superinfection exclusion and changes in cellular transport processes in phage infected Salmonella typhimurium.

Summary The changes induced by bacteriophage P22 in the cellular transport process(es) of the host Salmonella typhimurium (Taneja et al., 1975; Khandekar et al., 1975; Bandyopadhyay and Chakravorty, 1976) involve interactions between the superinfection exclusion system of the resident prophage and the C immunity region of the superinfecting phage. The sieA gene of the prophage interferes with the changes in the cellular transport process induced by the superinfecting phage. However, if the superinfecting phage carries active C ₁ and C ₂ genes of the superinfecting phage seem to be expressed in the sie A⁺ lysogen.     

Modelling the stability of Stx lysogens

Shiga-toxin-converting bacteriophages (Stx phages) are temperate phages of Escherichia coli, and can cause severe human disease. The spread of shiga toxins by Stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. Lysogens of Stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. We have developed a mathematical model to examine whether known differences in operator regions and binding affinities between Stx phages and lambda phage can account for the lower stability of Stx lysogens. The Stx phage 933W has only two binding sites in its left operator region (compared to three in phage lambda), but this has a minimal effect on 933W lysogen stability. However, the relatively weak binding affinity between repressor molecules and the second binding site in the right operator is found to significantly reduce the stability of its lysogens, and may account for the hair-trigger nature of the switch. Reduced lysogen stability can lead to increased frequency of genetic recombination in bacterial genomes. The development of the mathematical model has considerable utility in understanding the behaviour and evolution of the molecular switch, with implications for phage-related diseases.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic Escherichia coli, is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production, and here, we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.     

Superinfection exclusion by heteroimmune corynebacteriophages.

Superinfection of Corynebacterium diphtheriae C7(beta) by heteroimmune phage gamma is productive, whereas superinfection by gamma-bin mutants is for the most part nonproductive. Exclusion of gamma-bin phage occurred after its DNA had penetrated and was partially expressed in the heteroimmune lysogen. All of the infected cells were killed, and lysis was observed. The beta inhibitor causing exclusion was produced during the prophage state and appeared to be distinct from immune repressor. The ability of gamma-bin phage to superinfect C7(beta) productively could be restored by recombination with beta phage, indicating that both beta and gamma phages contain either indentical or similar alleles of the bin gene. The bin gene was mapped by vegetative and prophage crosses and found to be located in the region of the phage genome concerned with regulation. Both beta and gamma wild-type phages induced the resident prophage in a significant fraction of superinfeted heteroimmune lysogens. This, coupled with the fact that induction of C7(beta) abolished exclusion, suggests that the bin gene product acts as antirepressor, i.e., it reduces the level of heteroimmune repressor either directly or indirectly. The gamma-bin mutants either failed to produce antirepressor or did so with reduced efficiency. Antirepressor activity was negatively controlled by homoimmune repressor. The isolation of beta mutants that appeared bin-like suggests that beta and gamma phages contain homologous systems of exclusion and antiexclusion. Exclusion of gamm-bin by beta phage in gram-positive C. diphtheriae exhibited striking parallels to the sieB exclusion described for phages P22 and lambda in gram-negative organisms. The extended similarities of these coryngephages to lambda bacteriophage is noted.     

The phage tail tape measure protein,an inner membrane protein and a periplasmic chaperone play connected roles in the genome injection process of E. coli phage HK97

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15‐mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.     

The envelope glycoprotein of friend spleen focus-forming virus covalently interacts with and constitutively activates a truncated form of the receptor tyrosine kinase Stk

The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene Fv-2, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55(P), the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth,cell segregation,and virulence

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.