The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions.

A protein mixture containing two major components able to catalyze a beta-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract. N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant. The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence. The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified. Their activities were examined in vitro in beta-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis. HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins. This was reflected in the maintenance of in vivo activity of the final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN strains. However, hupB/hupN double mutants were not viable. It is suggested that the requirement for protein-facilitated DNA bending is met in P. putida by two independent proteins that ensure an adequate supply of an essential cellular activity.

Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida.

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter.

Structure and function of the Pseudomonas putida integration host factor.

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.

sigma54-dependent transcription of the Pseudomonas putida xylS operon is influenced by the IIANtr protein of the phosphotransferase system in Escherichia coli.

IIANtr, encoded within the rpoN operons of many Gram-negative bacteria, is a homologue of a class of phosphoryl transfer proteins of the phosphoenolpyruvate: sugar phosphotransferase system. We have used a xylS operon-lacZ fusion from the TOL plasmid of Pseudomonas putida to show that IIANtr influences sigma 54-dependent transcription when the xylS operon is expressed in Escherichia coli. Loss of IIANtr influences, but does not abolish cyclic AMP-independent carbon catabolite repression.

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.

Transcriptional induction kinetics from the promoters of the catabolic pathways of TOL plasmid pWW0 of Pseudomonas putida for metabolism of aromatics.

We determined, under several growth conditions, the kinetics of mRNA synthesis from the four Pseudomonas putida pWW0 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 sigma 54-dependent upper-pathway operon promoter (Pu) and the xylS gene promoter (Ps) by effector-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 effectors, 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 sigma 54-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 sigma 54-dependent promoters. Expression from xylR gene tandem promoters occurred independently of the growth medium used.

Carbon source-dependent inhibition of xyl operon expression of the Pseudomonas putida TOL plasmid.

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.

Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid.

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.

Promoter-upstream activator sequences are required for expression of the xylS gene and upper-pathway operon on the Pseudomonas TOL plasmid.

Stimulation of transcription from the Pseudomonas TOL plasmid xylS gene promoter (Ps) and the upper-pathway operon promoter (Pu) is dependent on the positive regulator protein XylR activated by an effector molecule such as 3-cholorotoluene, and on RpoN, an RNA polymerase sigma factor. Mutational analysis of the Ps and Pu promoters showed that upstream activator sequences located between -110 and -218bp upstream of the main transcription initiation point are required for regulated expression from these promoters. A search for homologous nucleotide sequences in the -110 to -218bp region in Pu and Ps revealed conserved sequences that may act as putative recognition sequences for the XylR protein. Ps and Pu exhibit another well-conserved region at around -50bp, which is homologous to corresponding sites in other RpoN-dependent promoters and may constitute a binding site for integration host factor (IHF).

The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation.

The role of the Klebsiella pneumoniae PII protein (encoded by glnB) in nitrogen regulation has been studied using two classes of glnB mutants. In Class I mutants PII appears not to be uridylylated in nitrogen-limiting conditions and in Class II mutants PII is not synthesised. The effects of these mutations on expression from nitrogen-regulated promoters indicate that PII is not absolutely required for nitrogen control. Furthermore the uridylylated form of PII (PII-UMP) plays a significant role in the response to changes in nitrogen status by counteracting the effect of PII on NtrB-mediated dephosphorylation of NtrC. PII is not involved in the nif-specific response to changes in nitrogen status mediated by NifL.

Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles.

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.