Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes.

Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene. Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 [or Pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes. Induction was not seen in transformed Escherichia coli cells or in a P. putida mutant lacking chromosomally encoded benzoate catabolic functions.     

Involvement and necessity of the Cpx regulon in the event of aberrant β‐barrel outer membrane protein assembly

The Cpx and σ^E regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σ^E pathway monitors the biogenesis of β‐barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β‐barrel OMP mis‐assembly, by utilizing mutants expressing either a defective β‐barrel OMP assembly machinery (Bam) or assembly defective β‐barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σ^E pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β‐barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β‐barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly‐defective β‐barrel OMP species. Together, these results showed that both the Cpx and σ^E regulons are required to reduce envelope stress caused by aberrant β‐barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.     

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Isolation of a mutant TOL plasmid with increased activity and transmissibility from Pseudomonas putida (arvilla) mt-2.

Strains with greater ability to dissimilate m-toluate were obtained from the wild-type Pseudomonas putida (arvilla) mt-2 that harbors the TOL plasmid. Increased growth of a mutant strain on aromatic substrates was coupled with simultaneous increase in the activity of metapyrocatechase, an enzyme coded by the TOL plasmid, without changing its catalytic properties. In the mutant and the wild-type strains, the inducer specificity and the induction kinetics of metapyrocatechase synthesis were the same, and a half-maximal effect of m-toluate on the enzyme synthesis was observed at 0.25 mM. Thus, the increased utilizability seen in a mutant strain appeared to be due to an increased quantity of the enzymes coded by the TOL plasmid. The properties of the mutant strain were dependent upon the mutation on the TOL plasmid but not on the chromosome mutation. Transfer experiments with a strain carrying the mutant TOL (TOL-H) or the wild-type TOL plasmid revealed that the TOL-H transfer was 1,000 times greater than that of the wild type.     

Regulatory mutations of the Pseudomonas plasmid alk regulon.

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk+ strains. These revertants showed altered responses to normal inducers of alkA+, alkB+, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 X 10(-8). Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity, and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.     

Apparent fusion of the TOL plasmid with the R91 drug resistance plasmid in Pseudomonas aeruginosa.

The TOL catabolic plasmid was shown to be compatible with the R91 drug resistance plasmid. However, the TOL plasmid was extremely unstable in mutant PA03 of P. aeruginosa. By selecting for stabilization of the TOL plasmid in PA03 harbouring R91, it was possible to isolate a strain in which markers from both R91 and TOL appeared to exist in a single recombinant plasmid. This plasmid, pND3, encoded resistance to carbenicillin, was able to transfer at the same frequency as the R91 plasmid and encoded the ability to grow on m-toluate, p-toluate, m-xylene, p-xylene and toluene. In addition, it was shown to be incompatible with the NAH catabolic plasmid and it could be transferred by transduction. The TOL plasmid could stabilize in PA03 harbouring R91 without fusion with R91, and could stabilize in PA03 in the absence of R91. PA03 harbouring either the recombinant plasmid or the stable TOL plasmid in the absence of R91 could promote bacterial chromosome transfer between mutant derivatives of P. aeruginosa strain PA0.     

Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWW0

Mutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of approximately 37 kDa.     

Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway.

Mutant derivatives of the TOL plasmid pWW0-161, containing Tn5 insertions in the xylS and xylR regulatory genes of the catabolic pathway, have been identified and characterized. The two genes are located together on a 1.5- to 3.0-kilobase segment of TOL, just downstream of genes of the enzymes of the meta-cleavage pathway. As predicted by a current model for regulation of the TOL catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxylic acid) pathway, was induced by m-methylbenzyl alcohol in xylS mutant bacteria but not in a xylR mutant, whereas catechol 2,3-oxygenase, a representative enzyme of the lower (meta-cleavage) pathway, was induced by m-toluate in a xylR mutant but not in the xylS mutants. Unexpectedly, however, catechol 2,3-oxygenase was not induced by m-methylbenzyl alcohol in xylS mutants but was induced by benzyl alcohol and benzoate. These results indicate that expression of the TOL plasmid-encoded catabolic pathway is regulated by at least three control elements, two of which (the products of the xylS and xylR genes) interact in the induction of the lower pathway by methylated hydrocarbons and alcohols and one of which responds only to nonmethylated substrates.     

Absence of the outer membrane phospholipase A suppresses the temperature-sensitive phenotype of Escherichia coli degP mutants and induces the Cpx and sigma(E) extracytoplasmic stress responses

DegP is a periplasmic protease that is a member of both the sigma(E) and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42 degrees C. [U-(14)C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, beta-lactamase, and beta-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42 degrees C, displayed increased viability at 44 degrees C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and sigma(E) regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and sigma(E) regulons rather than by inactivating the phospholipase per se.     

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.     

The Streptococcus sanguinis competence regulon is not required for infective endocarditis virulence in a rabbit model

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.     

Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.     

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.     

Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent

The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase. The ratio of whole plasmid loss to deletion was substrate and pH dependent. Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0. It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage.