Transducing Phage Lambda Research Articles

Phosphotransferase-mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages lambda iex (crr) and lambda gsr (tgs).

The expression of genes adjacent to ptsI was investigated using a series of specialised transducing phages carrying different, overlapping, segments of the cysA-gsr-ptsI-ptsH- iex - cysZ -lig region of the genome of Escherichia coli. The polypeptides were synthesised following the infection of u.v.-irradiated lysogenic and non-lysogenic uvrA recA hosts or a uvrA recA host carrying the lambda cI+ plasmid pKB280 . The polypeptides were identified by SDS-polyacrylamide gel electrophoresis and fluorography. The gsr gene product had a mol. wt. of 23 000. The product of the iex gene was tentatively identified as a protein of mol. wt. of either 33 000 or 21 000. Hpr, the product of the gene ptsH, had a mol. wt. of 9000. The gsr gene appeared to be expressed at a higher level in a non-immune host, which suggests that it was transcribed from lambda promoters. A new lambda host strain, suitable for the detection of small polypeptides (mol. wt. less than 30 000) is described.

Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions

Six Escherichia coli che loci (cheA,-B,-R,-W,-Y, and Z) are located in two adjacent operons that map at minute 42 on the chromosome. Point mutants defective in any of these six functions have aberrant swimming patterns and are generally nonchemotactic. Deletions within the two major che gene operons were isolated in order to examine epistatic interactions among these genes. We first constructed a specialized transducing phage (lambda che22), which carries both of the che operons and their associated promoters. Deleted lambda che22 derivatives were selected by chelating agent inactivation, and these derivatives were characterized by mapping them against a series of host strains with point mutations. Representative nonpolar deletions were then transferred into the E. coli chromosome by homologous recombination. Although the phenotype of cheR mutants (smooth swimming) was expected to be epistatic to that of cheB mutants (tumbly swimming), we found that deletion mutants lacking both of these functions exhibited frequent directional changes or tumbling episodes as they swam. An examination of larger deletions indicated that both the cheA-cheW and cheY-cheZ functions were required for the anomalous tumbling behavior observed in these mutants. Loss of the cheB function was also correlated with an inverted behavioral response to sodium acetate, a strong repellent of wild-type cells. These findings indicate that an important component of the signal transducing machinery may be altered in cheB mutants.

Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac.

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.

Fusions of flagellar operons to lactose genes on a mu lac bacteriophage.

Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products.

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.

Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli.

The H+-ATPase (F1F0) of Escherichia coli was purified from cells labeled with either [35S]sulfate or [U-14C-D] glucose, and the molar ratio of subunits in the complex determined. The molar ratio was calculated from the radioactivity incorporated into each subunit, using either the subunit sulfur content or subunit molecular weight. These labeling experiments confirm an alpha 3 beta 3 gamma 1 delta 1 epsilon 1 ratio of subunits in F1, and indicate a chi 1 psi 2 omega 10 ratio of subunits in F0. The chi, psi, and omega designations used here refer to the subunits of F0 in order of decreasing molecular weight. Staining with Coomassie brilliant blue gave a reliable indication of the molar ratio of subunits in F1, but very erroneous values for each of the subunits of F0. We attempted to estimate the ratio of subunits in the native membrane, since the stoichiometry determined for the purified complex could be an anomaly of purification. These estimates were made after labeling cells with [35S]sulfate during amplification of the ATPase genes carried on a lambda transducing phage. The subunit ratios in the native membrane were reasonably close to those obtained with purified F1F0. We conclude that the stoichiometry determined reflects the composition of F1F0 in the native membrane. The most surprising conclusion from this study is that there are 10 +/- 1 omega ("proteolipid") subunits in each F1F0 complex. This is considerably more than had been assumed previously.

Cloning and sequence of the crp gene of Escherichia coli K 12.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.

Primary structure of Escherichia coli RNA polymerase nucleotide substitution in the beta subunit gene of the rifampicin resistant rpoB255 mutant.

The transducing phage lambda dsupM814 and the plasmid pIB1830 containing the wild-type rpoB gene have been constructed and the primary structure of the gene's central fragment has been established. In contrast with the wild-type, the gene of the rpoB255 mutant, whose primary structure has been published, was found to contain an A.T. leads to T.A. transversion entailing the substitution of a valine residue for the aspartic acid residue (516) of the wild-type beta subunit.

Cloning and the nucleotide sequence of the genes for Escherichia coli ribosomal proteins L28 (rpmB) and L33 (rpmG).

The specialized transducing bacteriophage lambda dpyrE DNA was used as a source of DNA to clone two ribosomal protein genes rpmB (L28) and rpmG (L33) on the cloning vehicle pACYC184. Using one of these plasmids, the nucleotide sequence of these two genes and their flanking regions were determined. The amino acid sequences of both proteins deduced from the nucleotide sequences match with the amino acid sequences previously determined, with one exception. The nucleotide sequences suggest that these two ribosomal protein genes are cotranstribed. There was no expression of the second gene of the operon, rpmG, in the absence of the 5' sequences adjacent to the first gene, rpmB. Observation of the structure of mRNA also strongly supports the idea that rpmB and rpmG are in a single transcription unit whose order is: rpmBp-rpmB-rpmG-rpmGt.

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli.

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca++, Mg++ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages lambda asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp1, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, delta), alpha, gamma, (epsilon, beta) which in the notation of Downie el al. (1981) reads atp B (EFH) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc-) mutations. We made use of the fact that specialized transducing phages lambda asn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn+, and that the resulting Asn+ cells grow slowly if the lambda asn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the lambda asn which either eliminated atp genes or affected their expression ("promoter" mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, is discussed.

Construction and characterization of guaB-lacZ fusions in Escherichia coli K12.

GuaB-lacZ fusion strains of Escherichia coli K12 have been constructed using the Casadaban (1976) gene-fusion technique. The major modification to the procedure was the removal of the guanine requirement of Mu-induced guaA auxotrophs by introduction of a ColEl-gua+ plasmid prior to selection of the fusion. Conversion to prototrophy was necessary to overcome problems associated with repression of the gua operon by guanine added to selection media. Induction of the lysogenic fusion strains yielded plaque-forming lambda transducing phages that carried the lac genes and either the complete guaB gene and the gua promoter or only a distal portion of guaB.

Lambda transducing phages carrying plasmid R100 replication genes

VAλ73, a λ bacteriophage carrying R100 replication genes, maintains itself as an independent plasmid under conditions in which the λ replication genes are inactive.

Strains of Escherichia coli carrying the structural gene for histidyl-tRNA synthetase on a high copy-number plasmid.

That portion of the Escherichia coli chromosome carried by a number of lambda transducing phages, all of which carry the gua operon, was mapped using restriction endonucleases. The DNA from one of these transducing phages was subcloned onto pBR322. We have identified two recombinant plasmids which carry the Escherichia coli gene hisS, the structural gene for histidyl-tRNA synthetase. The two plasmids, pSE301 and pSE401, have in common a 3,540 bp fragment of E. coli DNA which is bounded by BglII and SalI restriction endonuclease recognition sites. Strains carrying these plasmids overproduce histidyl-tRNA synthetase 20 to 30 fold. The growth rate of these strains is not affected although the histidine biosynthetic enzymes are derepressed. This derepression seems to be in addition to that caused by introduction of a hisT mutation on the chromosome.

Physical characterization of the ilvHI operon of Escherichia coli K-12.

The ilvHI and leu genes of Escherichia coli K-12 are contained on a single 10.9-kilobase EcoRI fragment of deoxyribonucleic acid derived from the leu transducing phage lambda G4. Since the expression of all of these genes is controlled by leucine, we investigated whether they are part of single operon or whether they constitute separate but adjacent operons controlled from a common site. Both cloning and hybridization studies indicated that ilvHI and leu are distinct operons. They are transcribed in opposite directions and are separated by approximately 1,500 base pairs of deoxyribonucleic acid. Hybridization experiments showed that the expression of ilvHI is regulated chiefly at the level of transcription. The size of the ilvHI messenger ribonucleic acid is estimated to be 2,550 bases.

Transcription of the E. coli tufB gene: cotranscription with four tRNA genes and inhibition by guanosine-5'-diphosphate-3'-diphosphate.

The transcription of the tufB gene by purified RNA polymerase holoenzyme was studied using the transducing phage lambda rifd 18 DNA and the hybrid plasmid pTUB1 DNA (Miyajima et al. 1979) as templates. The size of tufB mRNA synthesized in this system was about 1,700 nucleotides, and the same strand as for rrnB was transcribed. By electron microscopic examination of the R-loop formed between lambda fus3 DNA and tufB mRNA synthesized under the direction of pTUB1 DNA, it was found that the untranslated sequence of about 500 nucleotides is at the 5' end of tufB mRNA. The sequencing of the 5' region of tufB mRNA synthesized on the truncated template has revealed that the tufB gene is cotranscribed with its upstream genes for four tRNAs (thrU, tyrU, glyT, and thrT). The synthesis of this mRNA molecule is completely abolished by low concentrations of ppGpp. Neither pppGpp, ppGp, nor pGpp was effective as inhibitor in this cell-free system.

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae.

The lysA gene of Escherichia coli has been cloned from a lambda transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.

Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome.

The structures of several lambda dmet and related lambda darg transducing phage were studied by restriction fragment mapping and electron microscopic measurements of homoduplexes and heteroduplexes. A new transducing phage (lambda dmet141), in which metF is the only functional gene of the cluster, was isolated. In contrast, lambda dmet117, which expresses the entire metBJLF cluster, has only 3 kilobases more bacterial deoxyribonucleic acid (DNA) than lambda dmet141. An EcoRI restriction fragment of lambda dmet117, which carries the leftmost 6 kilobases of the bacterial DNA insert, was isolated and shown to contain a functional copy of metB. Small structural differences at the attachment sites of some of the phage were shown to result from different sites of lambda integration in the two parent insertion lysogens.

Isolation of fusions between the lac genes and several genes of the exu regulon: analysis of their regulation, determination of the transcription direction of the uxaC-uxaA operon, in Escherichia coli K-12.

Gene fusions between the lac structural genes and different genes of the hexuronate system were isolated by the two methods described by Casadaban (1976, 1979). Mud (Apr lac) mutants which have the lac genes fused to the regulatory region of exuT, uxaC, uxaA and uxaB genes were constructed. Separately, the lac genes carried by a lambda plac-Mu hybrid phage were placed into a Mucts prophage inserted into uxaC gene and fusions were obtained, by selection of deletions putting the lac genes under the control of the uxaC regulatory elements. Induction of the lysogen led to the isolation of a lambda transducing phage bearing this uxaC-lac fusion. In all the fusion strains, beta-galactosidase expression was shown to be inducible by the natural inducers of hexuronate system enzymes, sensitive to catabolite repression and under the negative control of the exuR regulatory gene. The mutants with Mu insertion were used to establish the direction of transcription of the uxaC-uxaA operon, which is counter-clockwise on the circular genetic map of Escherichia coli (from uxaC to uxaA).

Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.

By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.

Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1.

Transducing lambda phages have been isolated that carry segments of the Escherichia coli chromosome in the aspC region, 20.5 min on the E. coli map. One of these phages, lambda aspC2, carries rpsA, the structural gene for the ribosomal protein S1. A three kilobase fragment from this phage, cloned into either the plasmid pACYC184 or the plasmid pBR322, was found to express S1. In cells carrying the rpsA gene on the high copy number plasmid pBR322 the rate of rpsA mRNA synthesis was increased 40-fold, whereas the rate of protein S1 synthesis was doubled, in comparison with these rates in an rpsA haploid.