Capsid size determination in the P2-P4 bacteriophage system: suppression of sir mutations in P2's capsid gene N by supersid mutations in P4's external scaffold gene sid.

The sid gene of the P2-dependent phage P4 provides an external scaffold so P2 N gene encoded protomers assemble as T = 4 capsids rather than as P2's T = 7 capsids. Mutations (sir) in the middle of N interfere with Sid's function. We describe a new P4 mutant class, nms ("supersid") mutations, which direct also P2 sir to provide small capsids. Three different nms mutations were located near the sid end, commingled with sid(-) mutations. Suppression of sir by nms is not allele-specific. Our results favor this interpretation of capsid size control: (i) sir mutations reduce pN protomer flexibility and thereby interfere with the generation of T = 4 compatible hexons; (ii) the C-termini of Sid molecules link up when forming the scaffold; nms mutations strengthen these Sid-Sid contacts and thus allow the scaffold to force even sir-type protomers to form T = 4 compatible hexons. Some related findings concern suppression of N ts mutations by P4.

Study of the role of the htrB gene in Salmonella typhimurium virulence.

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.

Mutational analysis of the bacteriophage P4 capsid-size-determining gene.

Satellite phage P4 (11,624 bp) depends on the morphopoietic genes (capsid, tail) and lysis genes of its helper phage P2 (33.5 kb) for its lytic development. In the morphopoietic process, P4 redirects the assembly pathway of large, P2 size, capsids (diameter = 60 nm) to yield smaller, P4 size, capsids (diameter = 45 nm), 1/3 in volume of that of its helper. The P4-specified capsid size determination is dependent on the function of the 27-kDa gpSid. To study the capsid size-determining function, we carried out a mutational analysis of the P4 sid gene. Use of a P4-derived genome of 29.1 kb (P461), which can be packaged only into large, P2 size, capsids allowed us to select P4 Sid- mutants. By DNA sequencing we characterized 25 P4 Sid- mutants, of which 10 contain base pair substitutions and 15 contain deletions. Both types of mutations are clustered in separate locations within the sid gene. Our results suggest that the Sid polypeptide contains three distinct functional domains.

Mutation of the htrB locus of Haemophilus influenzae nontypable strain 2019 is associated with modifications of lipid A and phosphorylation of the lipo-oligosaccharide.

The HtrB protein was first identified in Escherichia coli as a protein required for cell viability at high temperature, but its expression was not regulated by temperature. We isolated an htrB homologue from non-typable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E. coli htrB mutation. The promoter for the NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two genes are divergently transcribed. The deduced amino acid sequence of NTHi 2019 HtrB had 56% homology to E. coli HtrB. In vitro transcription-translation analysis confirmed production of a protein with an apparent molecular mass of 32-33 kDa. Primer extension analysis revealed that htrB was transcribed from a sigma 70-dependent consensus promoter and its expression was not affected by temperature. The expression of htrB and rfaE was 2.5-4 times higher in the NTHi htrB mutant B29 than in the parental strain. In order to study the function of the HtrB protein in Haemophilus, we generated two isogenic htrB mutants by shuttle mutagenesis using a mini-Tn3. The htrB mutants initially showed temperature sensitivity, but they lost the sensitivity after a few passages at 30 degrees C and were able to grow at 37 degrees C. They also showed hypersensitivity to deoxycholate and kanamycin, which persisted on passage. SDS-polyacrylamide gel electrophoresis analysis revealed that the lipo-oligosaccharide (LOS) isolated from these mutants migrated faster than the wild type LOS and its color changed from black to brown as has been described for E. coli htrB mutants. Immunoblotting analysis also showed that the LOS from the htrB mutants lost reactivity to a monoclonal antibody, 6E4, which binds to the wild type NTHi 2019 LOS. Electrospray ionization-mass spectrometry analysis of the O-deacylated LOS oligosaccharide indicated a modification of the core structure characterized in part by a net loss in phosphoethanolamine. Mass spectrometric analysis of the lipid A of the htrB mutant indicated a loss of one or both myristic acid substitutions. These data suggest that HtrB is a multifunctional protein and may play a controlling role in regulating cell responses to various environmental changes.

Bacteriophage P2: genes involved in baseplate assembly.

The sequences of two previously defined tail genes, V and J, of the temperate bacteriophage P2, and those of two new essential tail genes, W and I, were determined. Their order is the late gene promoter, VWJI, followed by the tail fiber genes H and G, and a transcription terminator. The V gene product is the small spike at the tip of the tail, and the J gene product lies at the edge of the baseplate. The W gene product may be homologous to the product of gene 25 of T4 phage, which is part of the T4 baseplate. A temperature-sensitive mutation in gene V affects satellite phage P4 production more than it affects the production of P2 helper phage. P4 mutations that partially compensate for this defect of gene V lie in the P4 capsid size determination gene, sid.

Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis.

The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein.

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.

Retronphage phi R73: an E. coli phage that contains a retroelement and integrates into a tRNA gene.

Some strains of Escherichia coli contain retroelements (retrons) that encode genes for reverse transcriptase and branched, multicopy, single-stranded DNA (msDNA) linked to RNA. However, the origin of retrons is unknown. A P4-like cryptic prophage was found that contains a retroelement (retron Ec73) for msDNA-Ec73 in an E. coli clinical strain. The entire genome of this prophage, named phi R73, is 12.7 kilobase pairs and is flanked by 29-base pair direct repeats derived from the 3' end of the selenocystyl transfer RNA gene (selC). P2 bacteriophage caused excision of the phi R73 prophage and acted as a helper to package phi R73 DNA into an infectious virion. The newly formed phi R73 closely resembled P4 as a virion and in its lytic growth. Retronphage phi R73 lysogenized a new host strain, reintegrating its genome into the selC gene of the host chromosome and enabling the newly formed lysogens to produce msDNA-Ec73. Hence, retron Ec73 can be transferred intercellularly as part of the genome of a helper-dependent retronphage.

Morphopoietic switch mutations of bacteriophage P2.

During the growth of bacteriophage P4, for which the genome of bacteriophage P2 is needed as helper, the decision whether to make large, P2 size, heads or small, P4 size, heads depends on the size-directing function of P4's sid gene and on P2's "sid responsiveness." P2 mutants (=P2 sir) impaired in their response to P4's sid function are readily obtainable as one class of P2 plaque formers selected on certain P4 cl plasmid lysogens. We describe nine P2 sir mutants of independent origin. For eight we could assign their sir mutation to P2 gene N, which encodes the major capsid protein. DNA sequencing indicated an open reading frame of 357 codons for gene N and showed these sir mutations to affect only four codons within a 38-codon segment in the middle of N. Seven mutations are missense mutations (three of them identical); one is a deletion of one codon. There seems to be a correlation between the phenotypic "strength" of the sir mutations and the type of amino acid replacement by missense mutations. Although the weakest mutation, sir7, could not yet be assigned to any P2 gene, it appears clear from this work that P2's N gene product is the major (or only) target of P4's Sid gene function.

A mutation of the transactivation gene of satellite bacteriophage P4 that suppresses the rpoA109 mutation of Escherichia coli.

Satellite bacteriophage P4 requires the products of the late genes of a helper such as P2 in order to grow lytically. The Escherichia coli rpoA109 mutation, which alters the alpha subunit of RNA polymerase, prevents transcription of the late genes of bacteriophage P2. Suppressor mutations that define the P2 ogr gene overcome this block. We found that P4 lytic growth using a P2 ogr+ prophage helper was prevented by the rpoA109 mutation but that this block was overcome when the P2 helper carried the suppressor mutation in the ogr gene. Furthermore, we isolated and characterized four independent mutations in P4, called org, that suppress the E. coli rpoA109 mutation by allowing P4 lytic growth using a P2 ogr+ helper. DNA sequence analysis revealed that the four independent org mutations are identical and that they occur in the P4 delta gene, which codes for a factor that positively regulates the transcription of the P2 and P4 late genes. delta is predicted to code for a basic 166-amino-acid residue protein. Each 83-residue half of the predicted delta gene product is similar to the predicted 72-residue proteins encoded by the ogr gene of P2 and the B gene of phage 186.

A bacterial mutation blocking P2 phage late gene expression.

A mutant of Escherichia coli strain C has been isolated, called gro109, that blocks bacteriophage P2 propagation by interfering with late gene expression. DNA replication proceeds normally in P2+-infected gro109 cells, but late phage proteins are not made. Early P2 mRNA is made in normal amounts, but very little late mRNA can be detected. P2 mutants (P2 ogr) able to overcome the gro109 block have been isolated in which synthesis of late P2 mRNA and phage proteins Is restored. The gro109 mutation is closely linked to the cluster of ribosomal genes at 64 min and is recessive to the wild-type (gro+) allele. A P2 ogr mutation has been mapped on the left arm of the P2 genome, between the right-most known late gene (D) and the phage attachment site. P2 ogr can complement P2+ in gro109 cells, indicating that ogr codes for a diffusible product.

Extent of host deletions associated with bacteriophage P2-mediated eduction.

A series of independent Escherichia coli K eductants has been isolated and tested to determine the extent of their deletions. The deletions cover the P2 prophage in location H, the his operon, a suppressor of the recBC phenotype (sbcB), the gene for gluconate-6-phosphate dehydrogenase (gnd), a locus involved in cell wall synthesis (rfb), and in some cases all or part of genes involved in methylgalactoside uptake (mglP). One end of the deletion, the P2 prophage end, appears to be the same for all eductants. The other end, however, can be located before, within, and after the mglP locus.

Studies on P2 prophage-host relationships. I. Alteration of P2 prophage localization patterns in Escherichia coli by interstrain transduction.

The linkage of P2 prophage location I in Escherichia coli C and location H in E. coli K with the histidine operon has been confirmed. Evidence is presented that alteration of the histidine region of the chromosome of E. coli K and E. coli C strains can result in alteration of the pattern of localization of P2 prophage.