A cos-mini-Mu vector for in vivo DNA cloning.

Construction of a mini-Mu plasmid vector containing a cosmid replicon is described. Upon derepression of mini-Mu transposition, bacterial DNA sequences can be flanked by the integrated mini-Mu. These sequences can then be packaged into lambda heads by superinfection with a lambda helper phage. Cosmid clones carrying particular bacterial genes can be recovered by selection after infection of appropriate strains with the cosmid transducing lambda lysate. We report here the successful in vivo cloning of several Escherichia coli genes using the transposoncosmid vector.

Expression of cloned genes by in vivo insertion of tac promoter using a mini-Mu bacteriophage.

The strong trp-lac(tac) promoter has been incorporated into the mini-Mu bacteriophage genome to form a mini-Mu-tac (mMu-tac) expression transposon. This mMu-tac element can transpose efficiently in Escherichia coli cells when derepressed and occasionally insert into a recombinant plasmid. When mMu-tac integration occurs upstream of a cloned gene in the orientation of its transcription, the tac promoter can direct the expression of the gene insert. mMu-tac contains translation stop codons downstream of the tac promoter. Thus, mMu-tac should be useful to express only those genes containing their own translational information. We report here the successful expression in E. coli of several prokaryotic genes using the transposon expression system.

Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes.

A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated.

Production of actinorhodin, a polyketide antibiotic made by Streptomyces coelicolor A3(2), normally occurs only in stationary-phase cultures. S1 nuclease protection experiments showed that transcription of actII-ORF4, the activator gene required for expression of the biosynthetic structural genes, increased dramatically during the transition from exponential to stationary phase. The increase in actII-ORF4 expression was followed by transcription of the biosynthetic structural genes actIII and actVI-ORF1, and by the production of actinorhodin. The presence of actII-ORF4 on a multicopy plasmid resulted in enhanced levels of actII-ORF4 mRNA, and transcription of actIII and actinorhodin production during exponential growth, suggesting that actinorhodin synthesis in rapidly growing cultures is normally limited only by the availability of enough of the activator protein. bldA, which encodes a tRNA(Leu)UUA that is required for the efficient translation of a single UUA codon in the actII-ORF4 mRNA, was transcribed throughout growth. Moreover, translational fusions of the 5' end of actII-ORF4 that included the UUA codon to the ermE reporter gene demonstrated the presence of functional bldA tRNA in young, exponentially growing cultures and no increase in the efficiency of translation of UUA codons, relative to UUG codons, was observed during growth. The normal growth-phase-dependent production of actinorhodin in the liquid culture conditions used in these experiments appears to be mediated at the transcriptional level through activation of the actII-ORF4 promoter.

In vivo cloning of DNA into multicopy cosmids by mini-Mu-cosduction.

A general in vivo procedure for cloning Escherichia coli genes into cosmids has been developed. The method we describe here uses a deleted Mu phage (a mini-Mu) to transpose E. coli genes into cosmids during mini-Mu replication. The resulting cosmids clones are packaged in vivo into lambda phage particles. Plasmids carrying a particular DNA sequence can be selectively recovered after infection of a new host with the in vivo constructed genomic cosmid library. This system was used successfully to clone several E. coli genes.

De novo fatty acid synthesis is required for establishment of cell type-specific gene transcription during sporulation in Bacillus subtilis.

A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric septum. The developmental programme of these two cells involves the compartmentalized activities of sigmaE in the larger mother cell and of sigmaF in the smaller prespore. A potential role of de novo lipid synthesis on development was investigated by treating B. subtilis cells with cerulenin, a specific inhibitor of fatty acid biosynthesis. These experiments demonstrated that spore formation requires de novo fatty acid synthesis at the onset of sporulation. The transcription of the sporulation genes that are induced before the formation of two cell types or that are under the exclusive control of sigmaF occurred in the absence of fatty acid synthesis, as monitored by spo-lacZ fusions. However, expression of lacZ fusions to genes that required activation of sigmaE for transcription was inhibited in the absence of fatty acid synthesis. The block in sigmaE-directed gene expression in cerulenin-treated cells was caused by an inability to process pro-sigmaE to its active form. Electron microscopy revealed that these fatty acid-starved cells initiate abnormal polar septation, suggesting that de novo fatty acid synthesis may be essential to couple the activation of the mother cell transcription factors with the formation of the differentiating cells.

Overproduction and localization of components of the polyketide synthase of Streptomyces glaucescens involved in the production of the antibiotic tetracenomycin C.

Three proteins, including the beta-keto acyl synthase and the acyl carrier protein, involved in the synthesis of the polyketide antibiotic tetracenomycin C by Streptomyces glaucescens GLA.0 were produced in Escherichia coli by using the T7 RNA polymerase-dependent pT7-7 expression vector. Changing the N-terminal codon usage of two of the genes greatly increased the level of protein produced without affecting mRNA levels, suggesting improvements in translational efficiency. Western immunoblot analysis of cytoplasmic and membrane fractions of S. glaucescens with antibodies raised to synthetic oligopeptides corresponding to the two presumed components of the beta-keto acyl synthase indicated that both proteins were membrane bound; one appears to be proteolytically cleaved before or during association with the membrane. The beta-keto acyl synthase could be detected in stationary-phase cultures but not in rapidly growing cultures, correlating with the time of appearance of tetracenomycin C in the medium.

Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2).

Transcription of redD, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redD expression was followed by transcription of redX, a biosynthetic structural gene, and the appearance of the antibiotic in the mycelium, and coincided with the intracellular appearance of ppGpp. However, ppGpp production elicited either by nutritional shift-down of, or addition of serine hydroxamate to, exponentially growing cultures had no stimulatory effect on redD transcription. The presence of redD on a multicopy plasmid resulted in elevated levels of the redD transcript and production of redX and undecylprodigiosin during exponential growth; the normal growth-phase-dependent production of undecylprodigiosin appeared to be mediated entirely through the redD promoter, which shows limited similarity to the consensus sequence for the major class of eubacterial promoters.

redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD.

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.