In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.     

Phage-Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherichia coli Strain

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.     

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16-3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16-3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co-evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.     

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.     

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.     

Isolation of Bacteriophage T4 Mutants Defective in the Ability to Degrade Host Deoxyribonucleic Acid

A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4nd28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 x 10(8) daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media.     

A traC mutant that retains sensitivity to f1 bacteriophage but lacks F pili.

An F lac pro mutant which was temperature sensitive for infection by the filamentous bacteriophage f1 but resistant to the F-specific icosahedral RNA phage f2 was isolated. Cells carrying the F' mutation failed to elaborate F pili at all temperatures. Mutant cells were able to pair with recipient cells during bacterial conjugation, but transfer of conjugal DNA occurred at a greatly reduced frequency. Complementation analyses showed the F' mutation to be in the traC gene. When a plasmid carrying traC was introduced into hosts harboring the F' mutation, phage sensitivity, the ability to elaborate F pili, and conjugation efficiency were restored. The mutation was named traC1044. The F lac pro traC1044 mutant appears to be unique among traC mutants in retaining host sensitivity to the filamentous phage f1 in the absence of expression of extended F pili. Phage f1 attachment sites appeared to be present at the cell surface in traC1044 mutants. The reduced accessibility of these sites may account for the reduced efficiency of phage f1 infection of traC1044 hosts, although the possibility that a defect was present in the receptor site itself was not eliminated. Membranes of hosts carrying the F' mutation contained a full complement of mature F-pilin subunits, so the product of traC is presumably required for pilus assembly but not for pilin processing. This, together with the deficiency in conjugal DNA transfer, suggests that traC may be part of a membrane-spanning tra protein complex responsible for pilus assembly and disassembly and conjugal DNA transmission.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Host participation in bacteriophage lambda head assembly

Mutants of Escherichia coli, called groE, specifically block assembly of bacteriophage λ heads. When groE bacteria are infected by wild type λ, phage adsorption, DNA injection and replication, tail assembly, and cell lysis are all normal. No active heads are formed, however, and head related “monsters” are seen in lysates. These monsters are similar to the structures seen on infection of wild-type cells by phage defective in genes B or C.We have isolated mutants of λ which can overcome the block in groE hosts and have mapped these mutants. All groE mutations can be compensated for by mutation of phage gene E (hence the name groE). Gene E codes for the major structural subunit of the phage head. Some groE mutants, called groEB, can be compensated by mutation in either gene E or in gene B. Gene B is another head gene.During normal head assembly the protein encoded by phage head gene B or C appears to be converted to a lower molecular weight form, h3, which is found in phage. The appearance of h3 protein in fast sedimenting head related structures requires the host groE function.We suggest that the proteins encoded by phage genes E, B and C, and the bacterial component defined by groE mutations act together at an early stage in head assembly.     

fipB and fipC: two bacterial loci required for morphogenesis of the filamentous bacteriophage f1.

We describe the identification of two mutations in bacterial genes, designated as fipB and fipC, which resulted in temperature-sensitive morphogenesis of bacteriophage f1. These mutations mapped at separate loci but had to be present simultaneously to block f1 production at 41.5 degrees C. One mutation defined the locus fipB at 85.3 min on the Escherichia coli linkage map; the other defined the locus fipC, which mapped very close to rpsL at 73 min. Since these mutations did not appear to affect phage DNA replication, gene expression, or protein localization, they probably interfered with the its life cycle at the level of assembly. fipB mutants were partially deficient in adsorption of bacteriophage lambda, and fipB and fipC mutants leaked beta-lactamase into the medium, suggesting that the mutations affect outer-membrane structure or function.     

Genome rearrangements in host-range mutants of the polyvalent staphylococcal bacteriophage 812

Mutations extended the host range of the polyvalent bacteriophage 812 of the family Myoviridae in up to 95 % of Staphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negative Staphylococcus species. Mutational changes in the genome of several host-range mutants of phage 812 were identified. Host-range mutant 812F1 harbors a deletion in endolysin gene that arose together with intron excision. Four mutants (812i, 812b, 812p, 812F3) harbor deletion in the structural gene orf8 that results from a genome rearrangement associated with intron insertion. This rearrangement was also detected in the genome of the closely related phages U16 and phi131. Another intron was discovered in the recA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragment PstI-O of three mutants (812b, 812F3, 812g) and phages U16 and phi131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.     

Interaction of bacteriophage K10 with its receptor, the lamB protein of Escherichia coli.

The lamB protein of Escherichia coli was initially recognized as the receptor for bacteriophage lambda. It is now shown also to constitute the receptor for phage K10. The lamB protein interacts with phage K10 in vitro, but this interaction does not lead to phage inactivation. Most lambda-resistant labB mutants are also resistant to K10, and vice versa. However, a significant proportion of the mutants resistant to one of the phages is sensitive to the other. Nineteen K10-resistant lambda-sensitive mutants have been studied. Only six of them produce a lamB protein which seems totally unimpaired in its ihe same deletion interval of the lamB gene. The corresponding region of the lamB polypeptide must be specifically involved in the interaction with phage K10. An unusual pattern of K10 host range mutants has been obtained; two calsses of such mutants could be defined, growing on two distinct classes of K10-resistant lamB mutants.     

High frequency of mutations that expand the host range of an RNA virus

The ability of a virus population to colonize a novel host is predicted to depend on the equilibrium frequency of potential colonists (i.e., genotypes capable of infecting the novel host) in the source population. In this study, we investigated the determinants of the equilibrium frequency of potential colonists in the RNA bacteriophage 6. We isolated 40 spontaneous mutants capable of infecting a novel Pseudomonas syringae host and sequenced their host attachment genes to identify the responsible mutations. We observed 16 different mutations in the host attachment gene and used a new statistical approach to estimate that 39 additional mutations were missed by our screen. Phenotypic and fitness assays confirmed that the proximate mechanism underlying host range expansion was an increase in the ability to attach to the novel host and that acquisition of this ability most often imposed a cost for growth rate on two standard hosts. Considered in a population genetic framework, our data suggest that host range mutations should exist in phage populations at an equilibrium frequency (3 x 10(-4)) that exceeds the phage mutation rate by more than two orders of magnitude. Thus, colonization of novel hosts is unlikely to be limited by an inability to produce appropriate mutations.     

The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein.

T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.     

Genetic basis of infectivity evolution in a bacteriophage

Antagonistic coevolution between hosts and parasites is probably ubiquitous. However, very little is known of the genetic changes associated with parasite infectivity evolution during adaptation to a coevolving host. We followed the phenotypic and genetic changes in a lytic virus population (bacteriophage; phage Φ2) that coevolved with its bacterial host, Pseudomonas fluorescens SBW25. First, we show the rapid evolution of numerous unique phage infectivity phenotypes, and that both phage host range and bacterial resistance to individual phage increased over coevolutionary time. Second, each of the distinct phage phenotypes in our study had a unique genotype, and molecular evolution did not act uniformly across the phage genome during coevolution. In particular, we detected numerous substitutions on the tail fibre gene, which is involved in the first step of the host-parasite interaction: host adsorption. None of the observed mutations could be directly linked with infection against a particular host, suggesting that the phenotypic effects of infectivity mutations are probably epistatic. However, phage genotypes with the broadest host ranges had the largest number of nonsynonymous amino acid changes on genes implicated in infectivity evolution. An understanding of the molecular genetics of phage infectivity has helped to explain the complex phenotypic coevolutionary dynamics in this system.     

Identification of Tail Genes in the Temperate Phage 16-3 of Sinorhizobium meliloti 41

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.The Sinorhizobium meliloti-Medicago symbiosis is an important model for endosymbiotic nitrogen fixation. The genome sequence of S. meliloti (strain 1021) has been established (14), and the Medicago truncatula genome is under intensive investigation (3). Phage 16-3 is a temperate, double-stranded DNA phage of S. meliloti strain 41. It is by far the best-studied rhizobiophage and serves as a tool in analyses of rhizobium genetics, in the isolation of some symbiotic mutants, and in the construction of special vectors. Genetic determinants and molecular mechanisms of many aspects of the 16-3 life cycle, such as phage integration and excision (8, 26, 38), regulation of the lytic/lysogenic switch (5, 6, 9, 24, 28), immunity to superinfection (4), phage DNA packaging (15), and the role of gene h in the host range (32), have been examined in detail. Moreover, the complete 60-kb phage genome sequence (accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ500118","term_id":"111054886","term_text":"DQ500118"}}DQ500118) has been determined recently (P. P. Papp et al., unpublished results). However, little is known about the genes and structural elements involved in the interaction between the phage and its host, and furthermore, only one study of the 16-3 virion proteins has been reported (11).The initial interaction between a tailed phage and its bacterial host cell is mediated by the distal part of the phage tail, which specifically binds to the phage receptor located on the host surface. Earlier results demonstrated that phage 16-3 adsorption is connected to the strain-specific capsular polysaccharide of S. meliloti 41, the K_R5 antigen. So far, three bacterial gene clusters involved in K_R5 antigen production, including the rkp-1, rkp-2, and rkp-3 regions, have been described. rkp mutants are defective in the invasion of the host plant for symbiosis. In addition, they cannot adsorb phage 16-3, suggesting that the K_R5 antigen is required for both functions (19, 20, 30).In order to elucidate the molecular mechanism of phage 16-3 and S. meliloti 41 recognition, bacterial mutants carrying an altered phage receptor and host range phage mutants able to overcome the adsorption block have been characterized previously (32). It was shown that the RkpM protein, together with other yet uncharacterized elements, is a component of the phage receptor. With the use of rkpM mutants, host range mutations in phage gene h, which probably encodes the tail fiber protein, were identified. Interestingly, some mutations influencing phage-host recognition could not be localized in the rkpM and h genes, indicating that on both sides, additional components are important for bacteriophage-host recognition.The aim of this study was to identify additional genetic determinants involved in S. meliloti 41 and phage 16-3 recognition by characterizing new host range and receptor mutants. Furthermore, by using insertional mutagenesis, we examined a region of the phage chromosome supposed to be responsible for tail formation and identified six new genes essential for phage assembly.