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.     

Primary structure and expression of a gene homologous to nifH (nitrogenase Fe protein) from the archaebacterium Methanococcus voltae

Summary In Methanococcus voltae, a 3.0 kbp HindIII fragment carrying homology to nifH was recently cloned. In Escherichia coli maxicells, the fragment directed the synthesis of a 30 K polypeptide encoded by the region homologous to nifH. Plasmids carrying the fragment did not complement Klebsiella pneumoniae nifH mutants and did not inhibit the nitrogen fixation of a Nif⁺ strain. The complete nucleotide sequence of the nifH homologous region was determined. It contained an open reading frame (ORFnifH) of 834 bp encoding 278 amino acid residues (mol. wt. 30,362). The ORFnifH was surrounded by regions of very high A+T content as observed with other mc. voltae genes. The region upstream from ORFnifH contained potential prokaryotic-like promoters and a potential ribosome binding site located 5 bp preceding the translation initiation codon. Using a translational fusion to lacZ of a DNA fragment carrying the putative promoter region and the 5 end of ORFnifH, it was shown in E. coli that (i) a promoter activity was effectively carried by the cloned fragment and (ii) this activity was not significantly modified by the presence of nifA or ntrC products provided by multicopy plasmids. Though the codon usage was characteristic of Mc. voltae, ORFnifH was very similar to eubacterial nifH genes, in particular the position of the cysteine residues was highly conserved. These data confirmed the high conservation of nifH sequences. S_AB values (binary matching coefficients) of 0.5 were found with eubacterial nifH genes at the nucleotide or amino acid level suggesting that the mc. voltae ORFnifH sequence was distantly related to eubacterial nifH sequences.     

The regulatory protein Reg1 of Streptomyces lividans binds the promoter region of several genes repressed by glucose

The regulatory protein Reg1 of Streptomyces lividans (a member of the LacI family) was expressed in Escherichia coli as a translational fusion with the maltose-binding protein (MBP) of E. coli. The purified MBP-Reg1 binds the promoter region of genes of the maltose regulon (amylases, maltose utilization) and that of genes sensitive to catabolite repression (chitinase, xylanase, cellulase). Repeated sequences, in direct or inverted orientation, are involved in these DNA-protein interactions. They are present in all DNA fragments able to bind MBP-Reg1. The nucleotide sequence of the repeats and the variability of the spacing between them suggest a similarity in DNA-binding activity between Reg1 and CytR, another member of the LacI family.     

Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions

Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the -glucuronidase (gusA) and -galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.     

Transient expression of a Drosophila melanogaster hsp 70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae)

The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli -galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.     

Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a P, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

The Predicted Presence of Large Helical Structural Variation in Yeast HIS4 Upstream Region is Correlated with General Amino Acid Control on the CYC1 Gene

Abstract

A series of CYC1 constructions in which the upstream promoter portion has been replaced by a variety of HIS4 synthetic fragments has demonstrated that the 5′ TGACTC 3′ repeat is crucial for conferring amino acid general control. Efficient regulation, however, is obtained only with fragments containing both the repeat and flanking sequences. Analysis of the flanks shows the presence of a 16 nucleotide long sequence composed of alternations of two purines and two pyrimidines between the upstream and downstream repeats. Such a sequence has very large twist angle variations. Homologous sequences are observed in HIS1, HIS3, and in TRP5 upstream regions between copies of the repeat. Sequences which confer special structural characteristics may aid in protein recognition of the promoter region.