Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification.

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins.     

Nucleotide sequence of the O gene and of the origin of replication in bacteriophage lambda DNA.

The nucleotide sequence of the O gene in bacteriophage lambda DNA is presented. According to two possible initiator codons, the primary structure of the O protein deduced from the DNA sequence consists of 278 or 299 amino acid residues. Structure and function of the O protein--one of the two phage initiator proteins for lambda DNA replication--are discussed in the light of a secondary structure model for the O protein. The central part of the O gene contains a cluster of symmetrical sequences extending over 160 base pairs. The point mutation of the cis-dominant replication mutant ti12 is located in this region.     

Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus.

The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage lambda gt11 and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.     

Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.     

Genetic and molecular analyses of the C-terminal region of the recE gene from the Rac prophage of Escherichia coli K-12 reveal the recT gene.

The nucleotide sequence of the C-terminal region of the recE gene of the Rac prophage of Escherichia coli K-12 reveals the presence of a partially overlapping reading frame we call recT. Deletion mutations show that recT is required for the RecE pathway of conjugational recombination. By cloning recT with a plasmid vector compatible with pBR322, we showed by cis-trans tests that the portion of the recE gene encoding ExoVIII DNA nuclease activity is also required for RecE pathway conjugational recombination. The recT gene can replace the redB gene of lambda for recA-independent plasmid recombination. A Tn10 insertion mutation previously thought to be in recE is located in recT and is renamed recT101::Tn10. Discrepancies between the molecular mass estimates of wild-type ExoVIII protein determined from mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and calculated from the predicted amino acid sequence are discussed. The hypothesis that wild-type ExoVIII protein results from fusion of RecE and RecT proteins is disproved genetically, thus supporting a previous hypothesis that the discrepancies are due to abnormal protein mobility in SDS-PAGE. A computer-performed scan of the bacteriophage nucleotide sequence data base of GenBank revealed substantial similarity between most of recE and a 2.5-kb portion of the b2 region of lambda. This suggests interesting speculations concerning the evolutionary relationship of lambda and Rac prophages.     

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.     

Sequence of the bacteriophage SP01 gene 30.

The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.     

Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA.

Recombination of plasmid DNAs and recombination of bacteriophage lambda red mutants in recB recC sbcA Escherichia coli mutants, in which the recE region is expressed, do not require recA. The recE gene is known to encode exonuclease VIII (exoVIII), which is an ATP-independent exonuclease involved in the RecE pathway of recombination. A 33,000-molecular-weight (MW) protein was observed to be coexpressed with both exoVIII and a truncated version of exoVIII, pRac3 exo, when they were overproduced under the control of strong promoters. We have purified this 33,000-MW protein (p33) and demonstrated by protein sequence analysis that it is encoded by the same coding sequence that encodes the C-terminal 33,000-MW portion of exoVIII. p33 is expressed independently of exoVIII but is probably translated from the same mRNA. p33 was found to bind to single-stranded DNA and also to promote the renaturation of complementary single-stranded DNA. It appears that p33 is functionally analogous to the bacteriophage lambda beta protein, which may explain why RecE pathway recombination does not require recA.     

Purification and properties of the Escherichia coli dnaK replication protein

The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).     

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.     

The Nu1 subunit of bacteriophage lambda terminase

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.     

The complete DNA sequence of the O antigen gene region of Plesiomonas shigelloides serotype O17 which is identical to Shigella sonnei form I antigen

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.     

High-level expression of a semisynthetic dam gene in Escherichia coli

We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.