V. de Lorenzo

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy

SummaryA new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.

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 β-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 MTβ 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).

An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid.

Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL‐encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)‐containing form of RNA polymerase. This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons. In vivo monitoring of the activity of a Pu‐lacZ fusion in E. coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA‐binding domain of the XylR does not impair its inhibitory effect when hyperproduced. One discrete IHF binding site and two potential XylR sites (consensus sequence 5′‐TTGANCAAATC‐3′), bracketted by short stretches of DNase I‐hypersensitive bonds, were detected upstream of the transcription initiation site. A model accounting for the features found is proposed which includes the IHF‐promoted looping of upstream XylR‐DNA complexes so that they contact the sigma 54(NtrA)‐RNA polymerase bound at ‐12/‐24 positions.

The IIANtr (PtsN) Protein of Pseudomonas putida Mediates the C Source Inhibition of the ς54-dependent Pu Promoter of the TOL Plasmid

The gene cluster adjacent to the sequence of rpoN (encoding sigma factor ς54) ofPseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of theupper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsNand ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss ofptsN, which encodes the IIANtr protein, madePu unresponsive to repression by glucose. TheptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directedptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some ς54 promoters in which IIANtr is a key protein intermediate.

The Behavior of Bacteria Designed for Biodegradation

Mineralization of organic molecules by microbes is essential for the carbon cycle to operate. The massive mobilization of compounds stored in natural resources, or the introduction of xenobiotics into the biosphere, leads to unidirectional fluxes, which result in the persistance of a number of chemicals in the biosphere, and thus constitute a source of pollution. Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of “new” activities, and construct totally “new” pathways through the assemblage of catabolic segments from different microbes. Although the number of genetically engineered microbes (GEMs) for potential use in biodegradation is not large, these recombinant microbes function in microcosms according to their design. The survival and fate of recombinant microbes in different ecological niches under laboratory conditions is similar to what has been observed for the unmodified parental strains. rDNA, both on plasmids and on the host chromosome, is usually stably inherited by GEMs. The potential lateral transfer of rDNA from the GEMs to other microbes is significantly diminished, though not totally inhibited, when rDNA is incorporated on the host chromosome. The behavior and fate of GEMs can be predicted more accurately through the coupling of regulatory circuits that control the expression of catabolic pathways to killing genes, so that the GEMs survive in polluted environments, but die when the target chemical is eliminated.

Modulation of gene expression through chromosomal positioning in Escherichia coli

Variations in expression of the nah genes of the NAH7 (naphthalene biodegradation) plasmid of Pseudomonas putida when placed in different chromosomal locations in Escherichia coli have been studied by employing a collection of hybrid mini-T5 transposons bearing lacZ fusions to the Psal promoter, along with the cognate regulatory gene nahR. Insertions of Psal-lacZ reporters in the proximity of the chromosomal origin of replication, oriC, increased accumulation of beta-galactosidase in vivo. Position-dependent changes in expression of the reporter product could not be associated with local variations of the supercoiling in the DNA region, as revealed by probing the chromosome with mobile gyrB-lacZ elements. Such variations in beta-galactosidase activity (and, therefore, the expression of catabolic genes) seemed, instead, to be linked to the increase in gene dosage associated with regions close to oriC, and not to local variations in chromosome structure. The tolerance of strains to the selection markers borne by the transposons also varied in parallel with the changes in LacZ levels. The role of chromosomal positioning as a mechanism for the outcome of adaptation phenotypes is discussed.

Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reaction-based saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent Ks′ values 5-100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met116, Arg132, Asn169, and Arg248 are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoters.

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the σ54‐containing RNA polymerase (σ54‐RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the −12/−24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C‐terminal domain of the α subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in σ70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of σ54‐RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of σ54‐RNAP for the −12/−24 site.

In vivo and in vitro effects of (p)ppGpp on the sigma(54) promoter Pu of the TOL plasmid of Pseudomonas putida.

The connection between the physiological control of the sigma(54)-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA. Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the relA gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in Pu activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in Pu performance through a range of sigma(54)-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of sigma(54)-systems.

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. IMPORTANCE This study demonstrates that EIIANtr influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIANtr protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks. This study demonstrates that EIIANtr influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIANtr protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.