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.     

The araC regulatory gene mRNA contains a leader sequence

Summary An estimation of the size of the araC gene in Escherichia coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.     

The araC gene of Citrobacter freundii

The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.     

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Summary Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara⁺, plasmid containing strains. These studies demonstrated that strains containing (araO1BA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.     

In vivo association of protein fragments giving active AraC

Frameshift mutations in a restricted portion of the arabinose operon regulatory gene araC from Escherichia coli give rise to active AraC protein, likely from the in vivo synthesis of two Incomplete fragments that are active together. Synthesis of corresponding fragments, each separately inactive, from two plasmids within cells also resulted in complementation. © 1996 Wiley-Liss, Inc.     

Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).     

Paucity of sites mutable to constitutivity in the araC activator gene of the l-arabinose operon of Escherichia coli

A large number of high-level and low-level constitutive mutations in the araC gene of Escherichia coli were shown by deletion mapping to lie almost exclusively in two regions of the araC gene. Recombination data show that the high-level constitutive mutations are located within two very small regions, each probably less than ten base-pairs, while the low-level constitutive mutations are spread over two broader areas, each centered at the same two regions. All constitutive mutations isolated in either the presence or absence of d-fucose, an analog of l-arabinose which antagonizes induction by arabinose, are altered from the wild type in their response to this analog. A nonsense mutation that maps in one of the constitutive regions can be suppressed to wild type, “low-level” constitutive, or “high-level” constitutive phenotypes, depending on the amino acid inserted at the site of the mutation. This demonstrates that changing a single amino acid can cause dramatic alterations in the regulatory properties of the araC activator protein.     

Nucleotide sequence of the thioredoxin gene fromEscherichia coli

The nucleotide sequence of the thioredoxin gene fromEscherichia coli was determined. The structural gene was identified on a cloned 3-kbPvuII Iragment by hybridization with a synthetic oligodeoxyribonucleotide corresponding to a part of the amino acid sequence of thioredoxin. Restriction-enzyme fragments were used as templates in the dideoxy sequence method, directly and after subcloning into M13mp8. A segment of 450 nucleotides was determined using both strands7 alternatively, without extensive overlaps. The sequence contains the thioredoxin coding region, a potential ribosome-binding site, and a putative promotor region. The predicted amino acid sequence differs by two inversions from the previously given thioredoxin sequence. The revised sequence is presented and the results further show that thioredoxins fromE. coli B and K12 are identical.     

Genetic analysis of bacteriophage lambda strains which transduce the genes for leucine biosynthesis

Summary By means of two specific genetic tests as well as additional transduction studies, the genetic compositions of four different leucine-transducing lambda-phages and the two E. coli lysogens from which these phages originated were analyzed. Three of the phages, No. 267, 517 and 889, are of the bio-type, e.g. carry bacterial genes adjacent to prophage attachment element P. The former two contain a large portion of the E. coli leucine operon (genes leu B through leu D, see Fig. 2), the latter carries only gene leu B and part of leu C. Phage No. 518 is of the gal-type and carries at least part of the leu A gene. The two lysogens, No. 73 and 75, from which these phages arose, contain the prophage between two mutation sites in gene leu A and leu B, in an orientation that is opposite to the normal one for lambda.     

Protein folding in the periplasm of Escherichia coli

With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right. Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E. coli is very recent and is often speculative. This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E. coli proteins involved in this process. After an introduction to the specific physiological situation in the periplasm of E. coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation — disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones — and briefly cover the guided assembly of multi-subunit structures. Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena.     

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis- responsive activators in H. influenzae gene expression.     

Ectopic Gene Conversions in Four <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Genomes: Increased Recombination in Pathogenic Strains

We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli. Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent. Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain. Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12. This higher recombination level might be due to mutations in some of their mismatch-repair genes. In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E. coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes. In E. coli, gene conversions are not more frequent near the origin of replication. However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant. This suggests that, in E. coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E. coli cells.     

Stabilization and size of araC protein

Summary AraC protein from Escherichia coli has been further stabilized and characterized. pH is a critical variable in conferring stability. araC protein has a sedimentation coefficient of 4.0±0.2 s on standardized 5%–20% glycerol gradients. Its isoelectric point is at a pH of 7.1.     

Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu

A general procedure is described for transposing the lac genes to selected locations on the Escherichia coli chromosome. These transpositions were designed so that the lac² genes could be fused to nearby promoters. In particular, the lac genes were fused to the promoters for the araBAD, araC and leu genes. In these fusions the lac genes are regulated by the controls of the genes to which they are fused. These fusions are therefore useful in discovering new types of regulation of gene expression, as was found in the case of the araC gene. λ transducing phage carrying the fusion as well as nearby genes can easily be isolated. Some of these fusions may result in the formation of hybrid proteins.     

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae

Several species of Enterobacteriaceae were investigated for their ability to synthesise selenium-containing macromolecules. Selenated tRNA species as well as selenated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobicaly induced formate dehydrogenase isoenzymes of E coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunollogical and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hydridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of convervation of the selenating system throughout the enterobacteria.Abbreviations DTT dithiothreitol - SDS sodium dodecyl sulfate - Lac lactose operon gene(s) - amp ampicillin - IPTG isopropyl-thio--d-galactopyranoside