TOL plasmid-encoded degradation of benzyl alcohol by Pseudomonas putida is inhibited by glucose and other compounds related to the main carbohydrate metabolism in Pseudomonas species. We report here that this effect is exerted at the level of expression of the xyl catabolic operons, and two xyl promoters, Pu and Ps, were identified as the primary targets of this inhibition. xyl promoter activation was also inhibited by glucose in the heterologous Escherichia coli system, apparently not however by the classical mechanism of enteric catabolite repression.

We determined, under several growth conditions, the kinetics of mRNA synthesis from the four Pseudomonas putida pWWO plasmid promoters involved in degradation of xylenes and methylbenzyl alcohols via toluates. Transcription by XylS of the meta-cleavage pathway operon promoter (Pm) for the metabolism of alkylbenzoates was stimulated immediately after the addition of an effector, both in Luria-Bertani (LB) medium and in minimal medium. Activation of the fr54-dependent upper-pathway operon promoter (Pu) and the xylS gene promoter (Ps) by eflector-activated XylR was dependent on the growth medium used: on minimal medium, activation of transcription from Pu and Ps occurred immediately after the addition of a XylR effector; in contrast, activation appeared only after several hours when cells were growing on LB medium. When Pm was induced through the physiological overexpression of XylS, mediated by XylR when this regulator was activated by upper-pathway eflectors, the kinetics of transcription from Pm was similar to that of Pu and Ps: maximum values were reached after delays of several hours in rich medium and after several minutes in minimal medium. The delay in the induction of transcription of zr54-dependent promoters reflects catabolite inhibition exerted by LB components, since the addition of yeast extracts, Casamino Acids, or several combinations of amino acids dramatically inhibited the synthesis of XylR-controlled f54-dependent promoters. Expression from xylR gene tandem promoters occurred independently of the growth medium used.The TOL plasmid pWWO (36) of Pseudomonasputida, which specifies enzymes for the oxidative catabolism of toluene, xylenes, and related hydrocarbons, contains two catabolic operons (12,14,15,28,32). The enzymes that successively oxidize the hydrocarbons to their corresponding alcohol, aldehyde, and carboxylic acid derivatives-the so-called upper pathway-are encoded by the "upper" operon (see Fig. 1). The meta-cleavage pathway operon encodes enzymes for the conversion of the carboxylic acids to catechols, whose aromatic rings are then cleaved (meta fission) to produce the corresponding semialdehydes, which are further metabolized to Krebs cycle intermediates (Fig. 1). Transcription of the upperpathway operon from its single promoter (Pu) is positively regulated by the XylR protein activated by toluene or xylenes or their alcohol catabolic products (2, 13, 22), in combination with sigma-54 factor, also called NtrA and RpoN (10,27,33). The maximal transcription level is dependent on integration host factor (1, 9) (Fig. 1). Transcription of the meta-pathway operon from its promoter (Pm) is regulated by the XylS protein, which is activated in turn by meta-pathway substrates and is independent of (54 protein (33) (Fig. 1). Expression of the meta operon is also induced by toluene-or xylene-activated XylR protein via a cascade regulatory system in which this protein, in combination with U54, stimulates transcription from the xylS gene promoter (Ps). Overproduction of the XylS protein in turn in...

The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein PII, has been cloned and sequenced. The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene. The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln+ suppressor of this mutation were also determined. The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of PII, and the suppression was shown to be caused by Tn5 insertion in glnB. The 3' end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB. Potential homologues of glnB encoding polypeptides extremely similar in sequence to PII were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.

The xylR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters. Transcription from the xylS gene promoter, Ps, is positively regulated by effector-activated XylR protein and requires the specific RNA polymerase sigma 54 subunit (RpoN). Deletions and point mutations in the Ps upstream region localized the site of XylR interaction to the region between -133 bp and -207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XylR in the XylR-regulated 'upper' catabolic pathway promoter, Pu. Gel retardation experiments showed binding of IHF to the Ps promoter region. Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between -35 bp and -47 bp, Ps proximal site) and further upstream overlapping the XylR recognition sequence (Ps distal site). In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between -141 bp and -153 bp on the lower strand). Expression from Ps, either under inducing or non-inducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma 54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P U promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.Integration host factor (IHF) is a sequence-specific DNAbinding and -bending heterodimeric protein that is abundant in the Escherichia coli cell. Its two subunits are coded by the ihfA gene (previously termed himA) and by the ihfB gene (previously termed hip or himD) (33). IHF belongs to the family of bacterial histone-like proteins that share amino acid sequence homology with HU proteins from a variety of bacteria (3,5,7,23). One member of the family, HU of Bacillus stearothermophilus, was crystallized (28, 35), and its three-dimensional structure serves as a model for the whole family. According to the model, the N-terminal half of the protein is involved in dimer formation, and the long arms, consisting of antiparallel double-stranded ␤ ribbons of the two subunits, wrap around the DNA. Recent solution structure studies of HU showed that its arms are highly flexible (32).IHF participates in a number of cellular processes such as site-specific recombination, transposition and inversion, phage DNA packaging, plasmid and phage DNA replication, and the positive and negative control of gene expression (for reviews, see references 6 and 7). Yang and Nash (37) proposed a model for IHF interaction with DNA in which the "arms" of the IHF protein contact DNA through the minor groove; the C termini provide for additional contacts made possible by the bending of the DNA. Although the two subunits of IHF are similar in structure, experimental evidence indicates that the two subunits contribute differently to the function of IHF (34, 38).IHF was found to participate in gene regulation in a number of gram-negative bacteria. A large number of these genes are transcribed by ...