Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

The PatB protein of Bacillus subtilis is a C-S-lyase.

The PatB protein of Bacillus subtilis had both cystathionine beta-lyase and cysteine desulfhydrase activities in vitro. The apparent K(m) value of the PatB protein for cystathionine was threefold higher than that of the MetC protein, the previously characterized cystathionine beta-lyase of B. subtilis. In the presence of cystathionine as sole sulfur source, the patB gene present on a multicopy plasmid restored the growth of a metC mutant. In addition, the patB metC double mutant was unable to grow in the presence of sulfate or cystine while the patB or metC single mutants grew similarly to the wild-type strains in the presence of the same sulfur sources. In a metC mutant, the PatB protein can replace the MetC enzyme in the methionine biosynthetic pathway.

CotA of Bacillus subtilis is a copper-dependent laccase.

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.

Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene.

In Bacillus subtilis, carbon catabolite repression (CCR) is mediated by the pleiotropic repressor CcpA and by ATP-dependent phosphorylation of the HPr protein of the phosphotransferase system (PTS). In this study, we attempted to identify novel genes that are involved in the signal transduction pathway that ultimately results in CCR in the presence of repressing carbon sources such as glucose. Seven mutants resistant to glucose repression of the levanase operon were isolated and characterized. All mutations were trans-acting and pleiotropic as determined by analyzing CCR of beta-xylosidase and of the sacPA and bglPH operon. Moreover, all mutations specifically affected repression exerted by glucose but not by other sugars. The mutations were mapped to three different loci on the genetic map, ptsG, glcR, and pgi. These three genes encode proteins involved in glucose metabolism. A novel repressor gene, glcR (ywpI), defined by two mutations, was studied in more detail. The glcR mutants exhibit loss of glucose repression of catabolic operons, a deficiency in glucose transport, and absence of expression of the ptsG gene. The mutant GlcR proteins act as super-repressors of ptsG expression.

Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.

The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.

S-adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts.

Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine. Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dAdoMet). In Gram-negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual 'pyruvoyl' AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self-cleavage of the protein at the upstream peptide bond. Neither the Gram-positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known. We have identified the corresponding B. subtilis speD gene (formely ytcF). Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B. subtilis SpeD, indicating that spermidine biosynthesis in Gram-positive bacteria and in archaea follows a pathway very similar to that of Gram-negatives and eukarya. In B. subtilis, transcription of speD is modulated by spermidine and methionine. Its expression is high under usual growth conditions. In contrast, the SpeD protein self-cleaves slowly in vitro, a noticeable difference with its archaeal counterpart. Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co-transcribed with gapB, the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme required for gluconeogenesis. This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis.

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.

Phosphorylation of HPr and Crh by HprK, early steps in the catabolite repression signalling pathway for the Bacillus subtilis levanase operon.

Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser-HPr. For certain operons, Crh, an HPr-like protein, is also implicated in CCR. In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser-HPr and P-Ser-Crh stimulated the binding of CcpA to the cre sequence of the lev operon.

Phosphorylation of either crh or HPr mediates binding of CcpA to the bacillus subtilis xyn cre and catabolite repression of the xyn operon.

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of Ser46 of the histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. A recently discovered HPr-like protein of B. subtilis, Crh, cannot be phosphorylated by PEP and enzyme I but becomes phosphorylated at Ser46 by the ATP-dependent, metabolite-activated HPr kinase. Genetic data suggested that Crh is also implicated in CCR. We here demonstrate that in a ptsH1 crh1 mutant, in which Ser46 of both HPr and Crh is replaced with an alanyl residue, expression of the beta-xylosidase-encoding xynB gene was completely relieved from CCR. No effect on CCR could be observed in strains carrying the crh1 allele, suggesting that under the experimental conditions P-Ser-HPr can substitute for P-Ser-Crh in CCR. By contrast, a ptsH1 mutant was slightly relieved from CCR of xynB, indicating that P-Ser-Crh can substitute only partly for P-Ser-HPr. Mapping experiments allowed us to identify the xyn promoter and a catabolite responsive element (cre) located 229 bp downstream of the transcription start point. Using DNase I footprinting experiments, we could demonstrate that similar to P-Ser-HPr, P-Ser-Crh stimulates binding of CcpA to the xyn cre. Fructose 1,6-bisphosphate was found to strongly enhance binding of the P-Ser-HPr/CcpA and P-Ser-Crh/CcpA complexes to the xyn cre, but had no effect on binding of CcpA alone.

The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis.

Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.

PRD--a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.

Several operon-specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD-containing regulators are very common in Gram-positive bacteria. In contrast, antiterminators controlling beta-glucoside utilization are the only functionally characterized members of this family from gram-negative bacteria. PRD-containing regulators are controlled by PTS-dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In gram-positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD-containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His-15. This form of HPr can, in turn, phosphorylate PRD-containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP-dependent phosphorylation of HPr at Ser-46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD-containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR.

LevR, which controls the expression of the levoperon of Bacillus subtilis, is a regulatory protein containing an N-terminal domain similar to the NifA/NtrC transcriptional activator family and a C-terminal domain similar to the regulatory part of bacterial anti-terminators, such as BgIG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)-dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar-specific proteins of the lev-PTS, LevD and LevE, form a signal transduction chain allowing the PEP-dependent phosphorylation of LevR, presumably at His-869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His-869 of LevR has been replaced with a non-phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP-dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His-585. Mutants in which His-585 has been replaced with an alanine had lost stimulation of LevR activity and PEP-dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His-585 is presumed to play a role in carbon catabolite repression.

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression.

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of beta-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of beta-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsl form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

Protein phosphorylation chain of a Bacillus subtilis fructose-specific phosphotransferase system and its participation in regulation of the expression of the lev operon.

The proteins encoded by the fructose-inducible lev operon of Bacillus subtilis are components of a phosphotransferase system. They transport fructose by a mechanism which couples sugar uptake and phosphoenolpyruvate-dependent sugar phosphorylation. The complex transport system consists of two integral membrane proteins (LevF and LevG) and two soluble, hydrophilic proteins (LevD and LevE). The two soluble proteins from together with the general proteins of the phosphotransferase system, enzyme I and HPr, a protein phosphorylation chain which serves to phosphorylate fructose transported by LevF and LevG. We have synthesized modified LevD and LevE by fusing a His-tag to the N-terminus of each protein allowing rapid and efficient purification of the proteins. We determined His-9 in LevD and His-15 in LevE as the sites of PEP-dependent phosphorylation by isolating single, labeled peptides derived from 32P-labeled LevD, LevD(His)6, and LevE(His)6. The labeled peptides were subsequently analyzed by amino acid sequencing and mass spectroscopy. Mutations replacing the phosphorylatable histidyl residue in LevD with an alanyl residue and in LevE with a glutamate or aspartate were introduced in the levD and levE genes. These mutations caused strongly reduced fructose uptake via the lev-PTS. The mutant proteins were synthesized with a N-terminal His-tag and purified. Mutant LevD(His)6 was very slowly phosphorylated, whereas mutant LevE(His)6 was not phosphorylated at all. The corresponding levD and levE alleles were incorporated into the chromosome of a B. subtilis strain expressing the lacZ gene under control of the lev promoter. The mutations affecting the site of phosphorylation in either LevD or LevE were found to cause constitutive expression from the lev promoter of B. subtilis.

The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.

Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon.

There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

Interactions of wild-type and truncated LevR of Bacillus subtilis with the upstream activating sequence of the levanase operon.

Transcription of the levanase operon of Bacillus subtilis is controlled by LevR, an activator of the NifA/NtrC family of regulators. An upstream activating sequence (UAS) located in a 16 bp palindromic structure has previously been characterized. LevR was overproduced in B. subtilis and interaction between the activator and the UAS was demonstrated by gel shift and footprint experiments. The LevR protein specifically binds to the two-halves of the palindromic structure centered at -125 bases upstream from the transcriptional start site. In addition, footprint analysis suggests that LevR interacts with a third DNA region located at positions -90 to -80. To investigate the function of the different domains of the LevR activator, stop codons were introduced at various positions in the levR gene. The ability of the truncated LevR polypeptides to activate transcription, to respond to the inducer or to interact with the UAS was tested. The results obtained suggest that LevR is a multidomain protein. The amino-terminal part of the protein is required for DNA binding whereas the central domain allows the activation of transcription. The carboxy-terminal region is involved in the modulation of the LevR activity by the inducer.

Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence.

The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified. This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.