Enhanced metalloadsorption of bacterial cells displaying poly-His peptides.

The properties of Escherichia coli cells, acquired by cell surface presentation of one or two hexahistidine (His) clusters carried by the outer membrane LamB protein, have been examined. Strains producing LamB hybrids with the His chains accumulated greater than 11-fold more Cd2+ than E. coli cells expressing the protein without the His insert. Furthermore, the hexa-His chains on the cell surface caused cells to adhere reversibly to a Ni(2+)-containing solid matrix in a metal-dependent fashion. Thus, expression of poly-His peptides enables bacteria to act as a metalloaffinity adsorbent. These results open up the possibility for biosorption of heavy ions using engineered microorganisms.

An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA.

The export of Escherichia coli hemolysin across the cytoplasmic and the outer membranes requires the COOH-terminal signal sequence of HlyA, the two specific translocator proteins HlyB and HlyD, and the outer membrane protein TolC. We have developed an export cloning system that is composed of two vectors: one in which the fusion of the desired gene with the 3'-end of hlyA is generated, and a second in which the sequences containing the fusion are combined with the accessory genes hlyB and hlyD, thereby reconstructing the natural organization of the hly locus. In the second vector the fusion and the accessory genes are flanked by Notl sites, allowing subcloning of the whole cluster into a variety of minitransposons to achieve the stable integration of the constructs into the chromosome of Gram-negative bacteria. Since some applications may require the production of transcriptional fusions, an alternative version of the system provides the efficient translation initiation region of T7 phage gene 10 upstream of the fusion protein coding sequence. The usefulness of the system was assessed by constructing a fusion between the gene encoding the B subunit of Shiga-like toxin lle and the 3'-end of hlyA. An attenuated Salmonella typhimurium vaccine strain harboring the resulting construct, either in multicopy or monocopy, efficiently expressed and exported the chimeric protein. We anticipate that this system will lead to a higher stability of the engineered function and permit a faithful monitoring of the export of the recombinant peptide under physiologic single-copy conditions.

Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil.

Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions. DNA sequences encoding MT were fused to the autotransporter beta-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane. The translocation, surface display, and functionality of the chimeric MTbeta protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R. eutropha. The resulting bacterial strain, named R. eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd2+-polluted soil with R. eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana).

Expression systems and physiological control of promoter activity in bacteria.

Promoter activity in vivo is not just dependent on the performance of the regulator/promoter pair which may predominantly control transcription initiation in response to a given signal, it also relies on overimposed mechanisms that connect the activity of individual promoters to the metabolic and energetic status of the bacterial cells. Such mechanisms - which frequently become limiting for biotechnological applications involving regulated promoters - include classic (i.e. cAMP/CRP-mediated) or alternative catabolite control checks, recruitment of protein intermediates of the phosphotransferase sugar transport system, coregulation through protein-induced DNA bending and the interplay of sigma factors during various growth stages.

Physical and functional analysis of the prokaryotic enhancer of the sigma 54-promoters of the TOL plasmid of Pseudomonas putida.

The physical and the functional organization of the upstream cis-acting sequence that controls at a distance the transcriptional activity of Pu and Ps, the two sigma 54-dependent promoters of the TOL (toluene/xylene biodegradation) operons of Pseudomonas putida, have been determined. DNase I and hydroxyl radical footprinting of the promoters with the purified and pre-activated enhancer-binding protein XylR clearly indicated the presence of two distinct binding sites (proximal and distal) that were occupied independently and did not share an evident sequence similarity. However, alignment of the sequence on the basis of the cleavage protection patterns, along with those produced on Po, a third XylR-responsive promoter of a phenol degradation operon, generated a consensus sequence 5'-TTGATCAATTGATCAA-3' having greater similarity to the proximal than to the distal boxes. To verify that this consensus was the sequence recognized by XylR, we footprinted in vitro a synthetic site, the results indicating that it was strongly bound by the activator with the predicted pattern of interactions. The mode of protection indicated that XylR recognized the sequence as a palindrome and not as a tandem repeat, interacting with it on one side of the DNA helix. In vivo experiments involving directed deletions through the entire 5' region of the Pu promoter confirmed that the proximal XylR-binding sequence suffices for promoter activity. In vivo data also suggested that XylR binding to the upstream sequences promoted the assembly of the oligomeric form of the activator that is competent for transcription initiation.

In vitro activities of an N-terminal truncated form of XylR, a sigma 54-dependent transcriptional activator of Pseudomonas putida.

A truncated derivative of the XylR protein, which is able to constitutively activate the sigma 54-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to homogeneity and its various activities have been separately examined, in vitro. The truncated regulator XylR delta A was deleted of the signal reception N-terminal module present in wild-type XylR, but retained its central activation domain and the DNA binding segment, located at its C terminus. XylR delta A bound to the region -120 to -190 bp upstream of the transcription initiation site of the Pu promoter, where previous analyses have located the XylR target site. XylR delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA containing the native upstream activation sequences of Pu. Both ATPase activity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPase activity but retained the nucleotide-binding ability of the parental protein. XylR delta A was able to activate transcription in vitro with sigma 54-RNA polymerase alone, although its activity was enhanced up to 20-fold in the presence of the integration host factor protein. The requirements for activation of the Pu promoter in vitro are consistent with the view that DNA-facilitated oligomerization of the regulator for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma 54-dependent promoters. Furthermore, additional co-regulation elements seem to adjust promoter activity in vivo to the physiological status of the cells.

The organization of intercistronic regions of the aerobactin operon of pColV-K30 may account for the differential expression of the iucABCD iutA genes.

The complete nucleotide sequence of the 8.3 kilobase operon of the enterobacterial virulence plasmid pColV-K30, which encodes a high-affinity iron transport system mediated by the hydroxamate siderophore aerobactin, has been determined. The region includes five open reading frames which correspond to the genes iucA, iucB, iucC and iucD, encoding the enzymes of the biosynthetic pathway for aerobactin, and iutA for the outer membrane receptor of ferri-aerobactin complexes. The sequences of the iucABCD genes are tightly coupled without any intervening non-coding sequence. The predicted secondary mRNA structures at the gene junctions within the iucABCD cluster, along with their codon usage, may account for the differential expression of each of the protein products, as observed in vivo with minicells. The genes iucA and iucC, which determine the two subunits of the aerobactin synthetase complex, showed a considerable homology within three stretches of their amino acid sequence. A potential operator sequence (iron box) for the binding of the iron(II)-responsive Fur repressor protein was found within the iucA coding region, suggesting that the operon is subjected to an additional level of transcriptional repression by iron (II).

Binding of the fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence.

The mode of DNA binding of the Fur (ferric uptake regulator) repressor which controls transcription of iron-responsive genes in Escherichia coli, has been re-examined. Using as a reference the known sites at the promoter of the aerobactin operon of Escherichia coli, we have compared in detail the patterns of interaction between the purified Fur protein and natural or synthetic DNA targets. DNase I and hydroxyl radical footprinting, as well as missing-T assays, consistently revealed that functional Fur sites are composed of a minimum of three repeats of the hexameric motif GATAAT rather than by a palindromic 19 bp target sequence. Extended binding sites, constructed by stepwise addition of one or two direct repeats of the same sequence, were occupied co-operatively by Fur with the same pattern of interactions as those observed with the core of three repeats. This indicated that functional sites with a range of affinities can be formed by the addition of discrete GATAAT extensions to a minimal recognition sequence. The fashion in which Fur binds its target, virtually unknown in prokaryotic transcriptional regulators, accounts for the observed helical wrapping of the protein around the DNA helix.

Identification of a cis-acting sequence within the Pm promoter of the TOL plasmid which confers XylS-mediated responsiveness to substituted benzoates.

The DNA sequences within the Pm promoter/operator region of the meta operon of the TOL plasmid of Pseudomonas putida, which confer XylS-mediated responsiveness to substituted benzoates, have been identified through deletion analysis and mutagenesis of the region. Integrity and proper phasing of two homologous tandem sequences 5'-TGCAAPuAAPu-PyGGNTA-3', separated by six base-pairs and overlapping with the -35 region of the Pm promoter, was essential for m-toluate activation of a Pm-lacZ fusion in xylS+ strains. The spacing between equivalent bases in each of the half-sites is 21 base-pairs, i.e. two turns of DNA helix. The activity of Pm varieties containing identical half-sites suggested that the XylS-responsive element is arranged as a direct repeat, the distal sequence being the one which provided the most stringent regulation when duplicated. The role of the repeats as the target for XylS binding to Pm is discussed.

Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30.

Transcription of the iron-controlled aerobactin operon of the enterobacterial plasmid pColV-K30 is negatively regulated through the interaction of a Fe2+-binding repressor (the Fur protein) with operator sequences within the promoter region of the operon. The DNA sequences essential for interaction with the repressor were located by site-directed mutagenesis of specific regions within the 31 base-pair protected by the repressor from DNase I nicking. Occupation of two contiguous repressor-binding sites appears to be required for the complete repression of the system. Contacts of the Fur repressor with the corresponding operator sequences were analyzed with hydroxyl radical footprint and methylation protection experiments. These indicate that DNA-protein contacts approach a symmetrical mode and take place at all sides of the DNA helix.

Genetic engineering strategies for environmental applications.

Environmental applications of genetically engineered microorganisms are currently hampered not only by legal regulations restricting their release, but also by the frequent dearth of adequate genetic tools for their construction in the laboratory. Recent approaches to strain development include the use of non-antibiotic markers as selection determinants, the use of transposon-vectors for the permanent acquisition of recombinant genes, and the utilization of expression devices based on promoters from promiscuous plasmids and biodegradative pathway genes.

Interplay of the PtsN (EIIA(Ntr)) protein of Pseudomonas putida with its target sensor kinase KdpD.

The nitrogen phosphotransferase system (PTS(Ntr) ) of Pseudomonas putida is a multi-component regulatory device that participates in controlling a variety of physiological processes in a post-translational fashion. A general survey of genes regulated by PtsN exposed transcription of the kdpFABC operon is most conspicuously affected. Measurements of kdpFp promoter activity in different pts mutants showed that PtsN is responsible for repression of kdpFABC transcription. This effect could be assigned mainly to PtsN∼P, depending on the external K(+) concentration. Bacterial two-hybrid assays demonstrated that kdpFp regulation is implemented through direct interaction of the PtsN protein with the sensor kinase KdpD of the KdpD/KdpE two-component system. Interaction between KdpD and PtsN was detectable with a PtsN variant that imitates the non-phosphorylated form as well as with a PtsN type mimicking the phosphorylated form of PtsN. These results raise a regulatory scenario in which the Kdp system is regulated by the action of PtsN through direct interaction with the sensor kinase KdpD, and the outcome of such an interaction depends on the phosphorylation state of PtsN as well as on the external K(+) concentration.

Designing microbial systems for gene expression in the field.

Unlike bacteria grown in the laboratory, genetically modified microorganisms destined for deliberate release as agents of bioremediation, or as live vaccines must be able to express their phenotype under the control of external signals that are present in the environment into which they are released. This is a major difference from other biotechnological processes (for example, in a bioreactor) in which the working conditions can be fixed at the will of the operator. In the field, operating conditions are determined by the external environment. The main problem is, therefore, how to programme bacteria physiologically and genetically to express the desired phenotype at the correct level and the right time, under physicochemical circumstances over which we have little or no control. This challenge has encouraged the development of new broad-host-range expression systems specifically tailored for bacteria, particularly Pseudomonas, but also various other Gram-negative organisms, for use in the field.

VTR expression cassettes for engineering conditional phenotypes in Pseudomonas: activity of the Pu promoter of the TOL plasmid under limiting concentrations of the XylR activator protein.

A simplified procedure to construct recombinant Pseudomonas putida (Pp) and related bacteria, which transcribe conditionally specific genes inserted into their chromosome in response to lac inducers such as IPTG, has been developed. The method is based on the so-called VTR expression cassettes. These are three small (1.98-kb) DNA segments engineered as NotI restriction fragments that include a lacIq gene along with the hybrid trp/lac promoter, Ptrc, followed by an optimised translation initiation region with a leading ATG and a multiple cloning site in each of the three reading frames. This arrangement allows the chromosomal insertion of the conditionally expressed genes of interest through its transfer to any of the mini-Tn5 transposon vectors available. VTR cassettes permit construction of specialized strains that are instrumental to address, by genetic means, otherwise intractable regulatory problems observed in biodegradative pathways of Pp. In this context, the VTR system was employed to examine the effect of the intracellular concentration of XylR, the main regulator of the TOL (toluene biodegradation) plasmid pWW0, on the exponential silencing of the promoter of the upper operon, Pu. Increasing concentrations of XylR resulted in more intense induction of the system that, however, remained silent during fast cell growth regardless of activator levels.

A T7 RNA polymerase-based system for the construction of Pseudomonas strains with phenotypes dependent on TOL-meta pathway effectors.

A general method to construct recombinant Pseudomonas putida (and related bacteria), which transcribe specific genes inserted into their chromosome in response to the presence of alkyl- and halobenzoates, has been developed. The system is based on the ability of the T7 RNA polymerase (T7pol) to initiate transcription from cognate promoter sequences located upstream from cloned genes. A specialized transposon, mini-Tn5 xylS/Pm::T7pol, was constructed which contains the structural T7 gene 1 downstream from the XylS protein/benzoate-regulated Pm promoter of the meta-operon of the TOL catabolic plasmid. This transposon was stably inserted into the chromosome of a P. putida target strain so that gene 1 is transcribed upon exposure of the bacteria to benzoate effectors of the XylS regulator. Genes whose expression is to be mediated by T7pol are cloned in mini-Tn5 transposons containing T7 promoter sequences upstream from the cloning site and then the hybrid transposons are inserted into different positions in the same chromosome. In this way, expression of the genes cloned within the mini-Tn5 vectors is dependent on the T7pol/XylS/Pm system. This expression device is particularly well suited for applications in which the expression of two or more genes is to take place in response to a single chemical signal, i.e., exposure to certain aromatic compounds.

Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes.

Novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of Pseudomonas and related Gram- bacteria. The properties of lacIq/Ptrp-lac were used to construct mini-Tn5 expression vector transposons and RSF1010-derived plasmids for controlled expression and generation of conditional phenotypes. These plasmids were used to hyper-express the XylS regulator of the meta operon of the TOL plasmid of P. putida or the bphB and bphC genes of the polychlorobiphenyl-degrading pathway of Pseudomonas sp. LB400 in different strains of Pseudomonas instead of in Escherichia coli. Specific activity of 2.3 dihydroxybiphenyl dioxygenase (bphC gene product) was increased tenfold when hyperproduced in its native host as compared to E. coli, but under the same in vivo conditions, the XylS regulator formed protein aggregates. The other lacIq/Ptrp-lac-based expression vector presented here, transposon mini-Tn5 lacIq/Ptrc, facilitates the insertion of genetic cassettes containing heterologous genes under the control of lac inducers in the chromosome of target bacteria, as shown by monitoring expression of a lacZ reporter cloned in mini-Tn5 lacIq/Ptrc and inserted in the chromosome of P. putida.

Engineering of alkyl- and haloaromatic-responsive gene expression with mini-transposons containing regulated promoters of biodegradative pathways of Pseudomonas.

Four recombinant mini-Tn5 transposons are described which contain outward-facing Pm, Pu or Psal promoters from the catabolic plasmids TOL and NAH of Pseudomonas putida, along with their cognate wild-type regulatory genes (xylS, xylR, nahR) or mutant varieties (xylS2). Transcription from such promoters is activated when the host bacteria encounters certain aromatic compounds, such as alkyl- and halobenzoates (XylS, XylS2), alkyl- and halotoluenes (XylR) or salicylates (NahR). These transposons enable the generation of conditional phenotypes dependent on the presence of specific effectors, as well as the engineering of strains expressing heterologous genes that are regulated by aromatic inducers. A mini-Tn5 xylS/Pm::luxAB, was used to construct Pseudomonas strains emitting light upon exposure to concentrations of m-toluate as low as 5-10 microM. The broad-host-range transposition system of Tn5 and the stability of the inserted genes due to the loss of the transposase-encoding gene during delivery of the mobile element make these transposons particularly well suited for the construction of stable strains exhibiting halo/alkyl aromatic-regulated conditional phenotypes in the absence of antibiotic selection, as is required for some uncontained bioremediation and biomonitoring applications.

Bioaccumulation of heavy metals with protein fusions of metallothionein to bacterial OMPs.

In view of potential biotechnological applications, eukaryotic metallothioneins (MTs) have been expressed in Escherichia coli as fusions to membrane or membrane-associated proteins such as LamB, the peptidoglycan-associated lipoprotein protein (PAL) or a hybrid Lpp/OmpA carrier sequence. The use of different anchors enables the MT moiety to be targeted into various cell compartments thus bringing the metal-binding ability of the resulting hybrids to specific sites of the cell structure. To this end, both full-size and partial sequences of the human or mouse MTs have been genetically fused to: i) the permissive site 153 of the LamB sequence, which loops out the MT to the external medium; ii) the N-terminus of a PAL variant devoid of its N-terminal cystein, which targets expression of the fusion into the periplasm; and iii) the C-terminus of Lpp-OmpA, for anchoring the MT to the outer membrane protein as an N-terminal fusion. Each type of fusion presented a distinct behavior in terms of expression, stability and ability to endow E. coli cells an enhanced accumulation of Cd2+, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions. The expression in vivo of metalloproteins bound to bacterial envelope structures opens a way to design biomass with specific metal-binding properties.

Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene.

In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.

Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption.

Metallothioneins (MTs) are small, cysteine-rich proteins with a strong metal-binding capacity that are ubiquitous in the animal kingdom. Recombinant expression of MT fused to outer-membrane components of gram-negative bacteria may provide new methods to treat heavy-metal pollution in industrial sewage. In this work, we have engineered Pseudomonas putida, a per se highly robust microorganism able to grow in highly contaminated habitats in order to further increase its metal-chelating ability. We report the expression of a hybrid protein between mouse MT and the beta domain of the IgA protease of Neisseria in the outer membrane of Pseudomonas cells. The metal-binding capacity of such cells was increased three-fold. The autotranslocating capacity of the beta domain of the IgA protease of Neisseria, as well as the correct anchoring of the transported protein into the outer membrane, have been demonstrated for the first time in a member of the Pseudomonas genus.