Transcriptional regulation of the assT-dsbL-dsbI gene cluster in Salmonella enterica serovar Typhi IMSS-1 depends on LeuO, H-NS, and specific growth conditions.

The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ΔleuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions.

Improving random mutagenesis by purification of the oligonucleotide variants.

An oligonucleotide-based mutagenesis method is presented where, contrary to most classical mutagenic approaches, preselection of the variants is performed at the oligonucleotide level to avoid cloning of non-desired sequences. The method relies on the generation of differentially phosphate-protected oligonucleotides. Protection of the phosphates is accomplished by substoichiometric incorporation of an Fmoc-protected and n-propyl-protected trinucleotide phosphoramidite during ordinary oligonucleotide assembly. Instead of the alkali-labile beta-cyanoethyl group introduced in ordinary assembly, the trinucleotide introduces the alkali-stable n-propyl group. As a result, single mutants carry three ionic phosphates less than the wild-type sequence, double mutants carry six ionic phosphates less and so on. This difference in ionic ratio enables separation of the variants by conventional polyacrilamide gel electrophoresis. In the exemplified library described herein, two sub-populations containing mainly triple and quadruple mutants were selected out of five possible sub-populations.

GeConT: gene context analysis.

SUMMARY: The fact that adjacent genes in bacteria are often functionally related is widely known. GeConT (Gene Context Tool) is a web interface designed to visualize genome context of a gene or a group of genes and their orthologs in all the completely sequenced genomes. The graphical information of GeConT can be used to analyze genome annotation, functional ortholog identification or to verify the genomic context congruence of any set of genes that share a common property. AVAILABILITY: http://www.ibt.unam.mx/biocomputo/gecont.html

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Reciprocal domain evolution within a transactivator in a restricted sequence space.

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Suppression analysis of positive control mutants of NifA reveals two overlapping promoters for Klebsiella pneumoniae rpoN.

Activation of gene expression relies on direct molecular interactions between the RNA polymerase and transcription factors. Eubacterial enhancer-binding proteins (EBPs) activate transcription by binding to distant sites and, simultaneously, contacting the sigma(54)-holoenzyme form of the RNA polymerase (Esigma(54)). The interaction between the EBP and Esigma(54) is transient, such that it has been difficult to be studied biochemically. Therefore, the details of this molecular recognition event are not known. Genetic and physical evidences suggest that the highly conserved C3 region in the activation domain of the EBP has major determinants for positive control and for the interaction with Esigma(54). To further investigate the target of this region we searched for extragenic suppressors of some C3 region mutant derivatives of NifA. As a first step we mutagenized Klebsiella pneumoniae rpoN, the gene that codes for sigma(54). A mutant allele, rpoN1320, that suppressed two different NifA derivatives was obtained. Immunodetection of sigma(54) and transcriptional initiation studies demonstrated that the cause of the suppression was an enhanced expression of rpoN. A single point mutation was responsible for the phenotype. It mapped at the -10 region of an unidentified promoter, here denominated rpoNp1, and increased its similarity to the consensus. A second upstream promoter, denominated rpoNp2, was also identified. Its -10 region partially overlaps with the -35 region of rpoNp1. Interestingly, the promoter-up -10 mutation in rpoNp1 caused a reduction in the expression from rpoNp2, likely reflecting a stronger occupancy of the former promoter by the RNA polymerase at the expense of the latter. The presence of two overlapping promoters competing for the RNA polymerase implies a complex regulatory pattern that needs elucidation. The fact that increasing the concentration of sigma(54) in the cell can suppress positive control mutants of NifA adds further evidence for their direct interaction in the activation process.

Compilation and analysis of sigma(54)-dependent promoter sequences.

Promoters recognized by the RNA-polymerase with the alternative sigma factor sigma(54) (Esigma54) are unique in having conserved positions around -24 and -12 nucleotides upstream from the transcriptional start site, instead of the typical -35 and -10 boxes. Here we compile 186 -24/-12 promoter sequences reported in the literature and generate an updated and extended consensus sequence. The use of the extended consensus increases the probability of identifying genuine -24/-12 promoters. The effect of several reported mutations at the -24/-12 elements on RNA-polymerase binding and promoter strength is discussed in the light of the updated consensus.

Evolution of new protein function: recombinational enhancer Fis originated by horizontal gene transfer from the transcriptional regulator NtrC.

New protein function is thought to evolve mostly by gene duplication and divergence. Here we present phylogenetic evidence that the multifunctional protein Fis of the gamma proteobacterial species derived from the COOH-terminal domain of an ancestral alpha proteobacterial NtrC transcriptional regulatory protein. All of the known enterobacterial fis genes are preceded by an open reading frame, named yhdG, that is highly similar to nifR3, a gene that forms an operon with ntrC in several alpha proteobacterial species. Thus, we propose that yhdG and fis were acquired by a lineage ancestral to the gamma proteobacteria in a single horizontal gene transfer event, and later diverged to their present functions.

In vivo studies on the positive control function of NifA: a conserved hydrophobic amino acid patch at the central domain involved in transcriptional activation.

The eubacterial enhancer-binding proteins activate transcription by binding to distant sites and, simultaneously, contacting the RNA polymerase r54 promoter complex (Esigma54). The positive control function is located at the central domain of these proteins, but it is not know which specific region has the determinants for the interaction with Esigma54. Here, we present genetic evidence that a small region of hydrophobic amino acids, previously denominated C3, at the central domain of Bradyrhizobium japonicum NifA is involved in positive control. We obtained 26 missense mutants along this conserved region. Among these, only strains expressing the NifA(F307-->Y) and NifA(A310-->S) mutant proteins retained some of the transcriptional activity (<20%), whereas those carrying NifA(E298-->D) and NifA(T308-->S) had very low but detectable activity (< 1.0%). The rest of the NifA mutants did not induce any measurable transcriptional activity. When expressed in the presence of wild-type NifA, the great majority of the mutants displayed a dominant phenotype, suggesting that their oligomerization determinants were not altered. In vivo dimethyl-sulphate footprinting experiments for a subset of the NifA mutants showed that they were still able to bind specifically to DNA. Analysis of intragenic supressors highlight the functional role of a hydroxyl group at position 308 to activate transcription.

In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes.

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.

Did cyclodextrin glycosyltransferases evolve from alpha-amylases?

The hydrolytic enzymes, alpha-amylases, and the cyclodextrin glycosyltransferases (CGTases) are key enzymes in the depolymerization of starch. These two groups of enzymes are evolutionarily related. We propose that the transferase activity is likely to have evolved from an ancestral hydrolase. Sequence analysis provides support for this hypothesis. Consequently, we have conducted an experimental study to test the possible adaptive value for evolving a CGTase. We found that when an alpha-amylase and a CGTase are combined more glucose is generated from starch than would be expected from the independent action of either of these enzymes. Thus, we propose that the biological role of CGTases is to work in concert with alpha-amylases for the efficient saccharification of starch. This observation can be useful in industrial processes aimed at producing syrups with high contents of glucose or maltose.

A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition.

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor sigma 54 (E sigma 54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (@ontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alpha/beta topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain. ATPase activity of the E sigma 54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitution that alter the function of the E sigma 54 activators, leaving intact the Central domain ATPase activity, are mapped on region proposed to play an equivalent role as the effector region of the GTPase superfamily.

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes.

In Rhizobium etli the nitrogenase reductase genes are reiterated. Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog. Their sequences are identical up to 6 residues upstream from a sigma54-dependent promoter. A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ gene of Escherichia coli. Both genes were expressed at maximum levels under 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased. This expression was dependent on the integrity of nifA, and nifHc was expressed at higher levels than nifHa. The same pattern was observed with root nodule bacteroids. Expression of both genes in E. coli required sigma54 in addition to NifA bound to the upstream activator sequence. In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream. NifA protected an imperfect binding site upstream of nijHc at position 85 from the promoter. The integration host factor stimulated each gene differently, nifHa being more dependent on this protein. The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.

Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression.

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.

Influence of oxygen on DNA binding, positive control, and stability of the Bradyrhizobium japonicum NifA regulatory protein.

Central to the genetic regulatory circuit that controls Bradyrhizobium japonicum nif and fix gene expression is the NifA protein. NifA activates transcription of several nif and fix genes and autoregulates its expression during symbiosis in soybean root nodules or in free-living microaerobic conditions. High O2 tensions result in the lack of nif expression, possibly by inactivation of NifA through oxidation of an essential metal cofactor. Several B. japonicum nif and fix promoters have upstream activator sequences (UAS) required for optimal activation. The UAS are located more than 100 bp from the -24/-12 promoter and have been proposed to be binding sites for NifA. We investigated the interaction of NifA with the nifD promoter region by using in vivo dimethyl sulfate footprinting. NifA-dependent protection from methylation of the two UAS of this promoter was detected. Footprinting experiments in the presence of rifampin showed that UAS-bound NifA led to the formation of an open nifD promoter-RNA polymerase sigma 54 complex. Shift to aerobic growth resulted in a rapid loss of protection of both the UAS and the promoter, indicating that the DNA-binding and the activation functions of NifA were controlled by the O2 status of the cell. After an almost complete inactivation by oxygen, the NifA protein began to degrade. Furthermore, metal deprivation also caused degradation of NifA. In this case, however, the rates of NifA inactivation and NifA degradation were not clearly distinguishable. The results are discussed in the light of a previously proposed model, according to which the oxidation state of a NifA-metal complex influences the conformation of NifA for both DNA-binding and positive control functions.

The influence of the Klebsiella pneumoniae regulatory gene nifL upon the transcriptional activator protein NifA.

The influence of the Klebsiella pneumoniae nifL gene product upon the interaction of the transcriptional activator protein NifA with the nifH promoter has been examined using in vivo dimethylsulphate 'footprinting'. Binding of NifA to the upstream activator sequence (UAS) of the nifH promoter in the presence of the NifL protein was observed under nitrogen-limiting growth conditions. Growth in the presence of NH4+ or addition of NH4+ to nitrogen-limited cells diminished the interaction of NifA with the UAS when NifL was present. Repression of nif transcription by NifL may therefore involve an interaction between NifL and NifA which reduces the affinity of NifA for the UAS.

Activation of the Klebsiella pneumoniae nifU promoter: identification of multiple and overlapping upstream NifA binding sites.

The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1.

In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex.

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25. The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.