The Zur of Xanthomonas campestris functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters

It has been long considered that zinc homeostasis in bacteria is maintained by export systems and uptake systems, which are separately controlled by their own regulators and the uptake systems are negatively regulated by Zur which binds to an about 30-bp AT-rich sequence known as Zur-box present in its target promoters to block the entry of RNA polymerase. Here, we demonstrated in vivo and in vitro that in addition to act as a repressor of putative Zn(2+)-uptake systems, the Zur of the bacterial phytopathogen Xanthomonas campestris pathovar campestris (Xcc) acts as an activator of a Zn(2+) efflux pump. The Xcc Zur binds to a similar Zur-box with approximately 30-bp AT-rich sequence in the promoters of the genes encoding putative Zn(2+)-uptake systems but a 59-bp GC-rich sequence with a 20-bp inverted repeat overlapping the promoter's -35 to -10 sequence of the gene encoding a Zn(2+)-export system. Mutagenesis of the inverted repeat sequence resulted in abolishment of the in vitro binding and the in vivo and in vitro activation of the export gene's promoter by Zur. These results reveal that the Xcc Zur functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.     

The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites

The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent PI and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA-binding domain of the P7 auto-repressor lies in the amino-terminal end of the P7 ParA protein. This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.     

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.     

Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F

Summary The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cl repressor of bacteriophage very inefficiently or not at all. The functional implications of these results are discussed.     

Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and −10 and −35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, V11 and 69, were shown to be deletions in the control region. The V11 mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their −35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.     

Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence

Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels. In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box. An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence. We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur. The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog. Our analysis of the affinity and stoichiometry of DNA binding by B. subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix. The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR. Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of approximately 30 bp. By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes.     

Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS).     

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the -10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A37 for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A37-RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to -10 sequences, DNA sequence at -35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in -10 and -35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions.     

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae , multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.