RESUMO
Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.
Assuntos
Bacteriófagos/fisiologia , Escherichia coli/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/efeitos dos fármacos , Vias Biossintéticas/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , DNA/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Essenciais , Genoma Bacteriano , Mutação/genética , Fenótipo , Reprodutibilidade dos Testes , Supressão GenéticaRESUMO
d-Aminoacyl-tRNA deacylase (DTD) hydrolyzes d-amino acids mistakenly attached to tRNAs and, thus, has been implicated in perpetuating protein homochirality. Fifty years after the discovery of DTD, it has now been shown that its function extends beyond 'chiral proofreading' because it also eliminates glycine that has been erroneously coupled to tRNAAla.
Assuntos
Aminoaciltransferases/metabolismo , Escherichia coli/enzimologia , Glicina/metabolismo , RNA de Transferência/metabolismoRESUMO
In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.
Assuntos
Fagos Bacilares/fisiologia , Bacillus anthracis/virologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Parasita , Ligação Viral , Fagos Bacilares/genética , Bacillus cereus/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Deleção de Genes , Teste de Complementação Genética , Mutagênese Insercional , Filogenia , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ÏVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ÏJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. METHODS: Host-range analysis and plaque morphology screening were done for both ÏJA1 and ÏVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. RESULTS: The host specificity of ÏJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ÏVchO139-I produced a larger halo around the plaques than ÏJA1. Restriction profiles of ÏJA1 and ÏVchO139-I genomes were also different. The genomes of ÏJA1 and ÏVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ÏJA1 and ÏVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. CONCLUSIONS: The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ÏJA1 could be demonstrated experimentally. Our data pave the way for potential use of ÏJA1 and ÏVchO139-I in Vibrio cholerae typing and control.
Assuntos
DNA Viral/química , DNA Viral/genética , Variação Genética , Genoma Viral , Podoviridae/genética , Vibrio cholerae O139/virologia , Análise por Conglomerados , Especificidade de Hospedeiro , Humanos , Dados de Sequência Molecular , Filogenia , Podoviridae/isolamento & purificação , Podoviridae/fisiologia , Análise de Sequência de DNA , Ensaio de Placa ViralRESUMO
BACKGROUND: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. METHODS: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. RESULTS: Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. CONCLUSIONS: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).
Assuntos
Fagos Bacilares/fisiologia , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Mutação , Sequência de Aminoácidos , Bacillus anthracis/ultraestrutura , Bacillus anthracis/virologia , Bacteriólise , Sequência de Bases , Mapeamento Cromossômico , Ordem dos Genes , Dados de Sequência Molecular , Óperon , Fenótipo , Plasmídeos/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3' overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3' end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and -1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3' ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.
Assuntos
Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Viral/genética , Genômica , Listeria/virologia , Prófagos/genética , Sequência de Aminoácidos , Bacteriófagos/classificação , Bacteriófagos/metabolismo , Sequência de Bases , Microscopia Eletrônica , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.
Assuntos
Vacinas contra Antraz/imunologia , Antraz/imunologia , Anticorpos Antibacterianos/sangue , Bacillus anthracis , Vacinas de Produtos Inativados/imunologia , Animais , Antraz/microbiologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Antígenos de Bactérias/imunologia , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Bacillus anthracis/efeitos da radiação , Feminino , Furocumarinas , Cobaias , Imunidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutação , Coelhos , Esporos Bacterianos/genética , Raios Ultravioleta , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , VirulênciaRESUMO
Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (approximately 6 kb) and K (approximately 20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.
Assuntos
Genoma Viral , Bactérias Gram-Positivas/virologia , Myoviridae/genética , DNA Viral/química , DNA Viral/genética , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Microscopia Eletrônica , Modelos Genéticos , Dados de Sequência Molecular , Myoviridae/ultraestrutura , Fases de Leitura Aberta/genética , Análise de Sequência de DNARESUMO
P2 is the original member of a highly successful family of temperate phages that are frequently found in the genomes of gram-negative bacteria. This article focuses on the organization of the P2 genome and reviews current knowledge about the function of each open reading frame.
RESUMO
Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail.
Assuntos
Bacteriófagos/fisiologia , Listeria monocytogenes/virologia , Receptores Virais/metabolismo , Sorogrupo , Ácidos Teicoicos/metabolismo , Ligação Viral , Bacteriófagos/ultraestrutura , Especificidade de Hospedeiro , Listeria monocytogenes/classificação , Listeria monocytogenes/ultraestrutura , Microscopia Imunoeletrônica , Siphoviridae/fisiologia , Siphoviridae/ultraestrutura , Proteínas Estruturais Virais/metabolismoRESUMO
Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.
Assuntos
Bacillus anthracis/virologia , Equidae/microbiologia , Filogenia , Siphoviridae/genética , Microbiologia do Solo , Animais , Sequência de Bases , Teorema de Bayes , Demografia , Endopeptidases/genética , Endopeptidases/metabolismo , Genoma Viral/genética , Modelos Genéticos , Dados de Sequência Molecular , Namíbia , Análise de Sequência de DNA , Siphoviridae/patogenicidade , Siphoviridae/ultraestrutura , Especificidade da EspécieRESUMO
The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies - illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens.
Assuntos
Fagos Bacilares/isolamento & purificação , Bacillus anthracis/virologia , Bacillus cereus/virologia , Bacillus thuringiensis/virologia , Fagos Bacilares/classificação , Fagos Bacilares/genética , Fagos Bacilares/ultraestrutura , DNA Viral/química , DNA Viral/genética , Ordem dos Genes , Genoma Viral , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Myoviridae/ultraestrutura , Análise de Sequência de DNA , Sintenia , Transdução Genética , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/genéticaRESUMO
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).
Assuntos
Bacteriófagos/fisiologia , Parede Celular/química , Listeria monocytogenes/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Ácidos Teicoicos/análise , Acetilglucosamina/análise , Bacteriófagos/crescimento & desenvolvimento , Deleção de Genes , Genes Bacterianos , Teste de Complementação Genética , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeria monocytogenes/virologia , Proteínas Tirosina Fosfatases/genética , Ligação ViralRESUMO
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.
RESUMO
Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.
Assuntos
Vacinas Bacterianas/genética , Expressão Gênica , Vetores Genéticos , Listeria monocytogenes/genética , Proteínas Recombinantes/biossíntese , Recombinação Genética , Seleção Genética , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Alanina Racemase/genética , Alanina Racemase/metabolismo , Animais , Vacinas Bacterianas/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Deleção de Genes , Genes Bacterianos , Teste de Complementação Genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Humanos , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Proteínas Recombinantes/genética , Baço/microbiologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
Our analysis of the plague diagnostic phage L-413C genome sequence and structure reveals that L-413C is highly similar and collinear with enterobacteriophage P2, though important differences were found. Of special interest was the mosaic nature of the tail fiber protein H in L-413C, given the differentiating specificity of this phage for Yersinia pestis vs. Yersinia pseudotuberculosis. While the N-terminal 207 and C-terminal 137 amino acids of L-413C display significant homology with the P2 H protein, a large (465 amino acid) middle section appears to be derived from a T4-related H protein, with highest similarity to the T6 and RB32 distal tail fibers. This finding along with appropriate preadsorption experiments suggest that the unique H protein of L-413C may be responsible for the specificity of this phage for Y. pestis, and that the Y. pestis receptors that are recognized and bound by L-413C either do not exist in Y. pseudotuberculosis or have a different structure.
Assuntos
Bacteriófago P2/classificação , Bacteriófago P2/genética , Yersinia pestis/virologia , Bacteriófago P2/isolamento & purificação , Bacteriófago P2/fisiologia , Enzimas de Restrição do DNA , Escherichia coli/metabolismo , Escherichia coli/virologia , Genoma Viral , Lisogenia , Dados de Sequência Molecular , Peste/microbiologia , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas Virais , Yersinia pseudotuberculosis/virologiaRESUMO
PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3'-protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty-seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle-specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram-positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N-termini but different C-termini. Matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) of tryptic peptide fragments was used to identify the modified C-termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3' ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis-acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3' pseudoknot to stimulate such an event.
Assuntos
Bacteriófagos/genética , Bacteriófagos/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico , Genoma Viral , Listeria monocytogenes/virologia , Proteoma , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Bacteriófagos/química , Sequência de Bases , DNA , DNA Viral/química , Ordem dos Genes , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento de Peptídeos , Biossíntese de Proteínas , Proteômica , Homologia de Sequência de Aminoácidos , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/químicaRESUMO
Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of approximately 10(-4) per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA(Arg) gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.
Assuntos
Bacteriófagos/genética , Vetores Genéticos , Listeria monocytogenes/genética , Integração Viral/genética , Animais , Sítios de Ligação Microbiológicos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano , Humanos , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano , RNA de Transferência/genética , RNA Viral , Alinhamento de SequênciaRESUMO
The origin-binding domain of the gpalpha protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gpalpha activity by the protein Cnr. We have determined the crystal structure of P4-OBD at 2.95 A resolution. The structure of P4-OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4-OBD subunit is the DNA-binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4-OBD dimer is suggested. P4-OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gpalpha dimer. P4-OBD dimerization is mediated by helices alpha1 and alpha3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA-binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo- or heterodimerization scaffold.