Specificity and topology of the Escherichia coli xanthosine permease, a representative of the NHS subfamily of the major facilitator superfamily.

The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits saturation kinetics with an apparent K(m) of 136 microM. A 12-transmembrane-segment model was confirmed by translational fusions to alkaline phosphatase and the alpha fragment of beta-galactosidase.

Isolation and characterization of mutations in the Escherichia coli regulatory protein XapR.

In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in Escherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes.

From a genomic library of Sulfolobus solfataricus DSM1617 we have isolated and identified the purEK locus. Two open reading frames are identified as homologs of the purE and purK purine biosynthetic genes in Escherichia coli. The C-terminus of purE overlaps with the N-terminus of purK. When either of the genes is expressed from an E. coli promoter they can complement the corresponding purE and purK mutations in E. coli. PurE seems to be more closely related to eubacteria than to other archaea and to eukaryotes. Also the purK gene, which has not yet been found in other archaea, is more closely related to eubacteria than to eukaryotes.

Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli.

We have characterized four genes from the 52-min region on the Escherichia coli linkage map. Three of these genes are directly involved in the metabolism of xanthosine, whereas the function of the fourth gene is unknown. One of the genes (xapA) encodes xanthosine phosphorylase. The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG. The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB. The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer xanthosine. Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins. In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of xanthosine.

Gene-regulatory modules in Escherichia coli: nucleoprotein complexes formed by cAMP-CRP and CytR at the nupG promoter.

Repression by CytR depends on the formation of nucleoprotein complexes in which the CytR repressor and the cAMP-CRP activator complex bind co-operatively to the DNA. Transcription initiation from CytR-regulated promoters requires cAMP-CRP; therefore, the cAMP-CRP complex functions both as an activator and as a co-repressor in these promoters. Another interesting aspect of the CytR regulon is that each promoter appears to have individual features. Therefore, structural and functional rules governing the formation of repression and activation complexes in one promoter may not be valid for other promoters of the CytR regulon. Here we show that the Escherichia coli nupG gene contains one CytR- and four CRP-binding sites in the control region. Notably, the architecture of the CytR binding site is different from previously described targets. In addition, the CytR repressor triggers a DNA repositioning of a cAMP-CRP complex in the -35 region upon binding to its operator. Thus, formation of the repression and activation complexes at the nupG promoter involves different subsets of CRP-binding sites. These findings show that the bacterium uses positive and negative regulatory modules to differentially control the expression of CytR- and cAMP-CRP-regulated genes.

DNA specificity of Escherichia coli deoP1 operator-DeoR repressor recognition.

We have studied the importance of the specific DNA sequence of the deo operator site for DeoR repressor binding by introducing symmetrical, single basepair substitutions at all positions in the deo operator and tested the ability of these variants to titrate DeoR in vivo. Our results show that a 16 bp palindromic sequence constitutes the deo operator. Positions outside this palindrome (positions +/- 9, +/- 10) can be changed without any major effect on DeoR binding. Most of the central 6-8 bp of the palindrome (positions +/- 1, +/- 2, +/- 3) can be substituted with other nucleotides with no or only minor effects on DeoR binding, while changes at position +/- 4 and +/- 5 give a more heterogeneous response. Finally, changes at positions +/- 6, +/- 7 and +/- 8 severely disrupt DeoR binding.

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology.

The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist.

deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization.

Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected. The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.

Purification and characterization of the deoR repressor of Escherichia coli.

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.

Long-range cooperativity between gene regulatory sequences in a prokaryote.

Regulation of transcription initiation by proteins binding at DNA sequences some distance from the promoter region itself seems to be a general phenomenon in both eukaryotes and prokaryotes. Proteins bound to an enhancer site in eukaryotes can turn on a distant gene, whereas efficient repression of some prokaryotic genes such as the gal, ara and deo operons of Escherichia coli, requires the presence of two operator sites, separated by 110, 200 and 600 base pairs (bp) respectively. In the deo operon, which encodes nucleoside catabolizing enzymes, we have shown that efficient and cooperative repression can be obtained when the distance between the two sites ranges from 224 to 997 bp. Here, we report that transcription initiation can be regulated from an operator site placed 1 to 5 kilobases (kb) downstream of the deoP2 promoter (and downstream of the transcribed gene), and present the first experimental data for prokaryotic regulation at distances greater than 1 kb. Our results support the model of DNA loop formation as a common regulatory mechanism explaining both some prokaryotic regulation and the action of eukaryotic enhancers.

Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli.

DeoP1 and deoP2 promoter fragments from the deo operon of Escherichia coli have been transcriptionally fused to the galactokinase gene. From single-copy expression of these fusions it is shown that the deoR binding site of both deoP1 and deoP2 are necessary to achieve full repression of the deo operon by the deoR repressor. Repression of the promoters can be achieved either by supplying extra deoR repressor in trans or by introduction of an extra deoR binding site at a position between 224 and 997 bp upstream of the promoter. Furthermore, the deoP2 promoter is shown to be regulated in a cumulative way by both the deoR and the cytR repressors, while deoP1 is only regulated by the deoR repressor. DeoP2 is a strong promoter being 20 times stronger than araPBAD and four times stronger than deoP1.