Observations on the formation of clones containing araB-lacZ cistron fusions

Summary Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the begining of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 10¹⁰ cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32°C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Innoculation of pre-existing fusion clones at specific locations on the arabinose-lacteredselection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37° accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc ⁺ pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAP phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.     

Action of a Transposable Element in Coding Sequence Fusions

The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.     

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

Summary A new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.     

Identification and characterization of stationary phase inducible genes in Escherichia coli

During transition into stationary phase a large set of proteins is induced in Escherichia coli. Only a minority of the corresponding genes has been identified so far. Using the λplacMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated. In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase. csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma;^S (encoded by rpoS), cAMP/CRP and the relA gene product were investigated. The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes. All fusions were mapped to the E. coli chromosome. Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively. The other fusions seem to specify novel genes now designated csiA through to csiF. csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the Intricacies of gene-fusion technology when applied to the study of gene regulation.     

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12

Summary Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation

Summary The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products. The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF:: Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ:: Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ. The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome.     

The roles of starvation and selective substrates in the emergence of araB-lacZ fusion clones.

The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Effect of mutations in dnaK and dnaJ genes on cysteine operon expression in Escherichia coli

The effect of mutations indnaK anddnaJ genes on the expression of two operons that are part of cysteine regulon was determined usingEscherichia coli strains harboringcysPTWA::lacZ andcysJIH::lacZ fusions. NulldnaJ, anddnaKdnaJ mutants were impaired in β-galactosidase expression from both fusions. Effecient complementation of this defect by wild-type alleles present on a low-copy number plasmid was achieved. The presence of the pMH224 plasmid coding for CysB^* protein defective in DNA binding lowered β-galactosidase expression fromcysPTWA::lacZ fusion strain harboring wild-typednaKdnaJ alleles but did not diminish enzyme expression in ΔdnaJ and ΔdnaKdnaJ strains.     

Occurence of reiterated DNA sequences in strains of Streptomyces produced by an interspecific protoplast fusion

Summary DNA isolated from a Streptomyces strain produced by interspecific protoplast fusion of Streptomyces jumonjinensis and Streptomyces lipmanii contained a tandemly repeated six kilobase pair sequence which constituted 15%–18% of the total DNA. Examination of other strains produced by similar fusions showed that they also contained reiterated DNA sequences.     

The right end of MudI(Ap,lac)

Stable derivatives of the bacteriophage MudI(Ap,lac) were used to generate operon fusions in S. typhimurium which exhibit a sectoring phenotype with respect to lacZ expression. The Lac^- to Lac⁺ conversion was shown to be the result of small deletions involving the right end of the MudI element. DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage. A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac). In addition, this model explains the observed deletion formation and the Lac^- to Lac⁺ conversion in the sectoring fusions.This paper is dedicated to our padrinos, John and Marge Ingraham, whose love of truth has served us as constant inspiration     

Cloning and Sequence Analysis of cDNA encoding Rhizopus niveus Lipase

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of S. typhimurium. The DNA sequences was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence from the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.