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 lacI^q 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 pWWO, 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.     

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.

The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate. In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene [corrected], also at a high rate. A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate. No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found. Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway. A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate. By transfer of a TOL plasmid into Pseudomonas sp. strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained.     

Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid.

Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes. The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively. A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr. DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF). IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF. In contrast, no alteration in the synthesis of XylR protein in the E. coli IHF-deficient mutant was detected.     

Changes in ionic selectivity with changes in density of water in gels and cells

Gels equilibrated with aqueous solutions of impermeant solutes reached a steady state in which, in the absence of a pressure difference, the activity of water in the pores of the gel was higher than that of water in the external solution. The chemical potential of water in the gel/polymer solution slurry was higher than that in the supernatant polymer solution removed from the gel. Water in the pores of the gel decreased in density to 0.96 as increasing osmotic stress was applied. It is argued that at constant temperature and pressure water can equilibrate between two compartments of unequal osmolality only by adjusting its molar volume. Experiments showed that when gel water had a higher activity than external water it was K⁺ selective; when it had a lower activity it was Na⁺ selective. It is proposed that a continuous spectrum of water structures can exist in these two compartment systems from dense, reactive, weakly-bonded water which selects highly hydrated ions, to expanded, stretched, unreactive, viscous water which is strongly hydrogen bonded and selects K⁺ and univalent anions. These findings are related to the state and properties of cytoplasmic water which is probably held under osmotic stress by the activity of the sodium pump.     

Brassinolide alleviated the adverse effect of water deficits on photosynthesis and the antioxidant of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.)

Brassinolide (BR) is a relatively new plant growth regulator. To test whether BR could be used to increase tolerance to water deficits in soybean, the effects of BR application on photosynthesis, assimilate distribution, antioxidant enzymes and seed yield were studied. BR at 0.1 mg l⁻¹ was foliar applied at the beginning of bloom. Two levels of soil moisture (80% field capacity for well-watered control and 35% for drought-stressed treatment) were applied at pod initiation. BR treatment increased biomass accumulation and seed yield for both treatments. Drought stress inhibited translocation of assimilated ¹⁴C from the labeled leaf, but BR increased the translocation for both treatments. Drought stress depressed chlorophyll content and assimilation rate (A), while chlorophyll content and A of BR-treated plants were greater than that of drought-stressed plants. BR treatment increased maximum quantum yield of PS II, the activity of ribulose-1,5-bisphosphate carboxylase, and the leaf water potential of drought-stressed plants. Treatment with BR also increased the concentration of soluble sugars and proline, and the activities of peroxidase and superoxide dismutase of soybean leaves when drought-stressed. However, it decreased the malondialdehyde concentration and electrical conductivity of leaves under drought stress. This study show that BR can be used as a plant growth regulator to enhance drought tolerance and minimize the yield loss of soybean caused by water deficits.