Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors

The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.

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Pseudomonas putida—a versatile host for the production of natural products

The gene encoding green fluorescent protein (GFP) has recently become an important visual marker of gene expression in eukaryotic organisms, as it is more sensitive than other reporter genes, requires no special cofactors for detection (7), and can be quantitated with a spectrofluorimeter (24). GFP has not been as widely applied to prokaryotic organisms because of a lack of constructs useful for diverse groups of bacteria, although GFP vectors are available for specialized bacterial systems (13, 24, 33, 41, 42). The wild-type gfp gene has been mutated to improve detection and expression of the fluorescent protein in prokaryotes (10, 18, 30), and both the wild-type and mutated forms have been used to construct less specialized bacterial GFP vectors.

A broad-host-range plasmid expressing the improved gfp(mut2) (10) gene from either a lac or an npt-2 promoter has been used successfully to tag gram-negative soil bacteria with GFP (27). Escherichia coli-Pseudomonas spp. shuttle vectors containing gfp(mut2) expressed from lac and tac promoters have been constructed (5), and a GFP cloning cassette containing a similarly improved gfp gene is available for creating transcriptional fusions in prokaryotes (30). A suicide plasmid containing a promoterless gfp that recombines wholly with the bacterial chromosome has been constructed to create genomic gfp fusions in a diverse range of gram-negative bacteria (22). Transposons provide an alternative method to insert reporter genes directly into the genomic DNAs of target strains. Several Tn5-based transposons containing either a promoterless gfp gene or a gfp gene expressed from a broad-host-range promoter have been generated for use in tagging diverse bacterial species (6, 9, 27, 40). Tn5 reporter gene systems, however, are not effective in all gram-negative bacteria (2, 36).

GFP-tagged bacteria have been used in ecological studies to monitor single cells or cell populations in activated sludge communities (14) during symbiosis with plant cells (15), during infection of macrophages (24), during plasmid conjugation on semisolid surfaces (9), and in survival studies of E. coli in aquatic environments (26). There have been recent advances in detecting the presence of specific genes in single cells, thereby enabling identification of specific cells in mixtures by in situ PCR (20, 21), as well as in detecting the expression of genes in single cells by in situ PCR after an initial reverse transcription step targeting the mRNA in the cell (8, 39). Although these methods are useful approaches for many experiments, they all involve killing the cells because of the fixation step in the procedure and the heating steps in the PCR regimen.

We are interested in studying regulation of gene expression (37, 38), surface colonization behavior (11), and plasmid transfer (3) in several diverse marine bacterial species in situ. To investigate these processes in living microbial communities, it is necessary to find methods of tagging the different marine species so that cells may be visualized in situ and in real time. For this purpose we compared levels of expression of gfp(mut2) from two different bacterial promoters on a broad-host-range plasmid (27) in three gram-negative marine bacterial species. As Tn5-based transposons do not work with the marine bacteria we have tested and Tn10-based transposons do (2), we also constructed a Tn10-based reporter transposon containing promoterless gfp(mut2) (hereafter referred to as gfp) to create transcriptional fusions in order to investigate gene expression in marine bacteria further.

Here we show the differences in levels of expression of gfp in three vector-transposon constructs in three marine species: Pseudoalteromonas sp. strain S91, Vibrio sp. strain S141, and Psychrobacter sp. strain SW5H. GFP-tagged S91 cells were used to investigate initial biofilm formation on a natural biodegradable substratum, squid pen, and laser scanning confocal microscopy (LSCM) was used to visualize the hydrated structure of the biofilm at the squid pen surface.

Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria

Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: a practical approach for a critical review.

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.

Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems.

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.

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Expression of the Lactose Transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The lac operon introduced into Proteus mirabilis shows two anomalies of expression: the maximal induced level is reduced by about an order of magnitude, and the basal level becomes about 100 times higher than in E. coli, as a result of which the induction ratio appears very small, around 2-5 in contrast to as much as 1000 in E. coli. It was suggested by Baumberg and Dennison (1975) that the two anomalies might be manifestations of a single effect whereby some exogenous promoters give poor expression in this host, since they could result from poor expression of the lacZYA and lacI promoters respectively. We show here that when the lacP class II promoter mutation L305 was transferred on an F-prime into P. mirabilis, its effect on lac expression was much as in E. coli. However, when Flac bearing the IQ1 up-promoter mutation was introduced into P. mirabilis, the basal level decreased by three orders of magnitude, in accord with poor expression of lacI+ being responsible for the usual low induction ratio. These results are consistent with the hypothesis of Baumberg and Dennison (1975) but do not prove it: poor expression of lacI and/or lacZYA could also result from weak translation initiation, problems of continuation of transcription or translation (e.g. due to pause sequences or differences in codon use), or diminished mRNA stability.

Anomalous expression of the E. coli lac operon in Proteus mirabilis. II. Effects of lacI and lacP mutations.

The lac operon shows anomalous expression in Proteus mirabilis: the maximal induced level is 10% or less of that in E. coli, while repression reduces this by a factor of only 2-5. We have sought to determine whether this effect relates in any way to CRP-mediated activation of expression, by comparing expression in P. mirabilis of lac operons (introduced for technical reasons on IncP1 plasmids) either regulatorily wild-type or bearing L8 or L8UV5. Derivatives of RP1 bearing L8UV5 were obtained by homogenotisation of pGC9114 (RP1::Tn951) in a L8UV5 background; while derivatives of RP4 bearing lac+, L8 or L8UV5 were obtained by Mu-mediated translocation of chromosomal regions bearing these alleles, following partial heat-induction of Mucts62 on pGM14 (RP4::Mucts62) in the appropriate hosts. These plasmids could be readily transferred to, and stably maintained in, the P. mirabilis strains employed. It was found that L8 reduced the maximal level of beta-galactosidase activity, and L8UV5 restored this activity to around wild-type, in P. mirabilis quantitatively very much as in E. coli. Nevertheless, the low maximal level of expression and high basal level characteristic of the former host were unchanged. The simplest explanation of these results is that P. mirabilis contains a protein that mimics the E. coli CRP protein in interacting with the appropriate DNA binding site and thereby stimulating transcription; and that the anomalous regulation of lac in this host is unconnected with the CRP system.

Anomalous expression of the E. coli lac operon in Proteus mirabilis. I. Effects of L8 and L8 UV5.

The lac operon shows anomalous expression in Proteus mirabilis: the maximal induced level is 10% or less of that in E. coli, while repression reduces this by a factor of only 2–5. We have sought to determine whether this effect relates in any way to CRP-mediated activation of expression, by comparing expression in P. mirabilis of lac operons (introduced for technical reasons on IncP1 plasmids) either regulatorily wild-type or bearing L8 or L8UV5. Derivatives of RP1 bearing L8UV5 were obtained by homogenotisation of pGC9114 (RP1:: Tn951) in a L8UV5 background; while derivatives of RP4 bearing lac+, L8 or L8UV5 were obtained by Mu-mediated translocation of chromosomal regions bearing these alleles, following partial heat-induction of Mucts62 on pGM14 (RP4:: Mucts62) in the appropriate hosts. These plasmids could be readily transferred to, and stably maintained in, the P. mirabilis strains employed. It was found that L8 reduced the maximal level of β-galactosidase activity, and L8UV5 restored this activity to around wild-type, in P. mirabilis quantitatively very much as in E. coli. Nevertheless, the low maximal level of expression and high basal level characteristic of the former host were unchanged. The simplest explanation of these results is that P. mirabilis contains a protein that mimics the E. coli CRP protein in interacting with the appropriate DNA binding site and thereby stimulating transcription; and that the anomalous regulation of lac in this host is unconnected with the CRP system.

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Anomalous expression of the E. coli lac operon in Proteus mirabilis

coli K12 (lac deletion) strains (JC3272, X5097 and GL1490) containing either F128 or pGC9114 were grown with glycerol, glucose or glucose 6-phosphate as carbon/ energy source with or without inducer IPTG and their P-galactosidase activities were measured (Table 1). Clearly lac expression was quantitatively different between pGC9 1 14 and Flac. Induced levels for the former were always lower, by factors

Margo Roberts

Papers

Expression of the Lactose Transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

Anomalous expression of the E. coli lac operon in Proteus mirabilis. II. Effects of lacI and lacP mutations.

Anomalous expression of the E. coli lac operon in Proteus mirabilis. I. Effects of L8 and L8 UV5.

Anomalous expression of the E. coli lac operon in Proteus mirabilis

SHORT COMMUNICATION Expression of the Lactose Transposon Tn951 in Escherichia coli, Proteus and Pseudomonas