Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes

Hybrid 5' regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF'-'lacZ or nif'-'lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF5' flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the ntrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed in front of the nifD spacer plus promoter from B. japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH'-'lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product

The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.     

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.     

Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli

Through complementation of a trans-acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes. By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al. (1988). The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78,467. Analysis of the derived amino acid sequence showed that the carboxy-terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the 'regulators' of two-component regulatory systems. Carboxy-terminal truncation of, and introduction of amino-terminal deletions in, the fhlA gene delivered inactive gene products. When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presence of formate also under aerobic growth conditions. FHLA appears to bind to the upstream regulatory sequence (URS) in the 5' flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.     

Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.     

A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.     

Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E. coli: effects on in vivo function.

The selenocysteine-inserting tRNA (tRNA(Sec)) of E. coli differs in a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.     

Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.

Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons. The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation. It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNA(Sec). This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position. The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA. It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required. A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNA(Sec).     

A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium

The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.     

Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate,nitrite, and formate: roles for NarL and NarP

Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons. To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions. Complementary patterns of gene expression were seen. Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce fdnG-lacZ expression. However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.     

Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product.

The formate dehydrogenase (FDHF) of Escherichia coli is a selenocysteine-containing protein that occurs as a component of the formate-hydrogen lyase complex. The gene encoding this 80 kd polypeptide contains a TGA codon in the open reading frame. Several indirect lines of evidence showed earlier that the selenocysteine residue in the protein is inserted co-translationally in a TGA (UGA) dependent process. Direct proof that the selenocysteine is present in the polypeptide in the position corresponding to TGA as predicted from the gene sequence was obtained by automated amino acid sequence analysis of a 75Se-containing peptide isolated from the protein. Construction of a fusion gene comprising a small segment of the fdhF gene linked to the lacZ gene as reporter greatly facilitated isolation of the selenocysteine-containing protein. Subsequent cleavage of this isolated gene product with endoproteinase Asp-N gave rise to an easily purified small selenocysteine-containing peptide that was amenable to amino acid sequence analysis.     

Targeted insertion of selenocysteine into the alpha subunit of formate dehydrogenase from Methanobacterium formicicum.

Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side. To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacterium formicicum, which codes for the alpha subunit of the F420-reducing formate dehydrogenase. The M. formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E. coli fdhF mRNA hairpin structure. It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine. Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E. coli fdhF mRNA structure.     

The role of ntrA in the anaerobic metabolism of Salmonella typhimurium

The possible involvement of NtrA in the expression of several anaerobically induced genes in Salmonella typhimurium was investigated. Unlike Escherichia coli, where hydrogenase 3 is ntrA dependent, the introduction of a mutation in ntrA had virtually no effect on the hydrogenase activity, thought to be hydrogenase 3, of S. typhimurium LT7. Fumarate reductase and alcohol dehydrogenase activities were found to be diminished in ntrA mutant strains, but this may very well be indirect since fdhF mutant strains showed the same effect. These results suggest that in S. typhimurium NtrA is highly specific for the anaerobic expression of fdhF.     

同源过表达fnr、pncB和fdhF对克雷伯菌产氢代谢的影响

利用代谢工程手段改造克雷伯菌Klebsiella sp.HQ-3产氢途径中相关代谢调控因子及辅酶因子,以构建高效产氢工程菌。利用简并引物,以Klebsiella sp.总DNA为模板,克隆了甲酸-氢裂解酶系统的全局转录调控因子(FNR)fnr基因、编码甲酸脱氢酶(FDH-H)fdhF基因,以及NADH途径中编码烟酸转磷酸核糖激酶(NAPRTase)的pncB基因,构建了3种同源过表达重组菌株HQ-3-fnr、HQ-3-fdhF和HQ-3-pncB,以研究同源过表达产氢代谢调控因子及辅酶因子对克雷伯菌累积产氢、细胞生长、代谢终产物的影响。结果表明,过表达fnr、pncB和fdhF基因的克雷伯工程菌的产氢效率比携带空载体的克雷伯对照菌株分别提高12.26%、11.62%和7.28%;重组菌HQ-3-fnr、HQ-3-fdhF和HQ-3-pncB的葡萄糖利用率较克雷伯对照菌株HQ-3-C明显增加,过表达fnr、fdhF基因使代谢合成甲酸量增多;过表达pncB基因能促进NADH合成,使更多的NADH流入消耗NADH较多的乙醇与琥珀酸代谢路径,使得乙醇和琥珀酸含量增加,而乳酸含量降低。