Melvin G. Sunshine

Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

SummaryWe show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr-dnaK-dnaJ-leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

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 nontypable 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 -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°C and were able to grow at 37°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.

A new host gene (groPC) necessary for lambda DNA replication

SummaryThe isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda π mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.

Identification of a mutation within the structural gene for the a subunit of DNA-dependent RNA polymerase of E. coli

An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered alpha subunit. Histidine is substituted for leucine in one tryptic peptide from the mutant alpha subunit. The existence of only one rpoA gene within the E. coli chromosome is indicated.

Bacteriophage P2 and P4.

Publisher Summary Bacteriophage P4 is a small temperate bacteriophage that grows lytically only in the presence of a helper bacteriophage, such as P2. Each phage carries the genes necessary to assure its own DNA replication and integration into the host chromosome, but only P2 has genes that encode proteins needed to produce phage particles and lyse the cell. P4 activates the expression of the P2 structural proteins and is encapsidated within a phage particle that is similar to the P2 particle with the exception that the capsid is smaller. P4 can also exist as an integrated prophage or as a plasmid, and exploiting this dexterity leads to many strategies for manipulating DNA in vitro and in vivo. The major advantage of P4-derived vehicles is that they permit plasmids to be transferred efficiently from cell to cell without DNA isolation and with few constraints on the genotype of bacterial strains. P2 forms plaques on many enteric bacteria, including isolates of Klebsiella , Salmonella , Serratia , Shigella , and Yersinia , and it can be used to introduce encapsidated plasmid DNA into other gram-negative bacteria such as Rhizobium and Pseudomonas .

Relief of P2 bacteriophage amber mutant polarity by the satellite bacteriophage P4

Abstract Amber mutations in four of the late genes of bacteriophage P2 ( P , O , V and F ) exert strong polar effects on genes distal to the mutant genes. These effects can be relieved by the host polarity suppressor, suA (rho) . We now show that the satellite phage P4, which depends on all 18 known late genes of P2 for lytic multiplication, is able to relieve the polar effects of P2 amber mutants. Three types of experiments demonstrate this effect: (1) P2 Oam71 , which is polar onto the lysis gene K , lyses the cell only when the host is co-infected with P4. (2) Complementation between polar and distal P2 amber mutants in the same transcription unit occurs only in the presence of P4. (3) In extracts from cells infected with a polar P2 mutant, a P2 protein corresponding to a distal gene in the same transcription unit can be detected on polyacrylamide gels only if the cells are co-infected with P4. P4 mutants deficient in gene α do not relieve P2 polarity.

Interaction of satellite phage P4 with phage 186 helper

Abstract Satellite phage P4 can utilize a derepressed 186 prophage as a helper for lytic growth. P4 requires all the 186 head, tail, and lysis genes, but none of the known 186 early genes. The P4 transactivation gene can replace the 186 B gene as a positive regulator of late gene expression. The ogr gene of P2 helper phage is the functional equivalent of the 186 B gene. A repressed 186 prophage does not support P4 growth, and thus P4 does not form plaques on a 186-lysogenic strain. Although P4 is able to derepress a P2 prophage helper ( E. W. Six and B. H. Lindquist, 1978 , Virology 87, 217–230), it is unable to derepress a 186 prophage. Thus some repressible helper function may be required for P4 growth. Alternatively, 186 prophage may produce a P4 inhibitor.

Sub, a host mutation that specifically allows growth of replication-deficient gene B mutants of coliphage P2

SummaryBacteriophage P2 normally requires the products of its early genes A and B for lytic growth in its host, Escherichia coli C. A host mutation, sub-1, which allows P2 to grow without a functional B gene product is described. The sub-1 mutation is recessive and maps at approximately 10 min on the E. coli genetic map.

The P 2–P 4 Transactivation System

The interactions between bacteriophage P 2 and its satellite phage P 4 comprise a promising model system for studying the control of transcription because the two phage have relatively small and therefore tractable genomes and yet exhibit a surprisingly complex set of effects upon one another. Each phage possesses the ability to drastically alter the expression of the other’s genome.