Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli

The OCT plasmid encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. Structural components are encoded on the 7.5-kilobase pair alkBAC operon, whereas positive regulatory components are encoded by alkR. We have constructed plasmids containing fusions of cloned alkBAC and alkR DNA and used these fusion plasmids to study the functional expression of the alkBAC operon and the regulatory locus alkR in Pseudomonas putida and in Escherichia coli. Growth on alkanes requires a functional chromosomally encoded fatty acid degradation system in addition to the plasmid-borne alk system. While such a system is active in P. putida, it is active in E. coli only in fadR mutants in which fatty acid degradation enzymes are expressed constitutively. Using such mutants, we found that E. coli as well as P. putida grew on octane as the sole source of carbon and energy when they were supplied with the cloned complete alk system. The alkR locus was strictly necessary in E. coli as well as in P. putida for expression of the alkBAC operon. The alkBAC operon could, however, be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent. Although with this reduction the plasmid-encoded alkanol dehydrogenase activity was lost, chromosomally encoded alkanol dehydrogenases in P. putida and E. coli compensated for this loss. The induction kinetics of the alk system was studied in detail in P. putida and E. coli. We used specific antibodies raised against alkane hydroxylase to follow the appearance of this protein following induction with octane. We found the induction kinetics of alkane hydroxylase to be similar in both species. A steady-state level was reached after about 2 h of induction in which time the alkane hydroxylase accounted for about 1.5% of total newly synthesized protein. Thus, alkBAC expression is very efficient and strictly regulated to both P. putida and E. coli.     

Transcriptome analysis of Pseudomonas putida in response to nitrogen availability

This work describes a regulatory network of Pseudomonas putida controlled in response to nitrogen availability. We define NtrC as the master nitrogen regulator and suggest that it not only activates pathways for the assimilation of alternative nitrogen sources but also represses carbon catabolism under nitrogen-limited conditions, possibly to prevent excessive carbon and energy flow in the cell.     

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium

The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.     

Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes

Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as proteins, nucleic acids and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this review, recent advances in our knowledge of ammonium uptake, its assimilation, and related regulatory systems in Corynebacterium glutamicum, a Gram-positive soil bacterium used for the industrial production of amino acids, are summarized and discussed with respect to the situation in the bacterial model organisms, Escherichia coli and Bacillus subtilis, and in comparison to the situation in other actinomycetes, namely in mycobacteria and streptomycetes. The regulatory network of nitrogen control in C. glutamicum seems to be a patchwork of different elements. It includes proteins similar to the UTase/GlnK pathway of E. coli and expression regulation by a repressor protein as in B. subtilis, but it lacks an NtrB/NtrC two-component signal transduction system. Furthermore, the C. glutamicum regulation network has unique features, such as a new sensing mechanism. Based on its extremely well-investigated central metabolism, well-established molecular biology tools, a public genome sequence and a newly-established proteome project, C. glutamicum seems to be a suitable model organism for other corynebacteria, such as Corynebacterium diphtheriae and Corynebacterium efficiens.     

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli

Beta-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to beta-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.     

Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.     

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.