The rcsA Promoter of Pantoea stewartii subsp. stewartii Features a Low-Level Constitutive Promoter and an EsaR Quorum-Sensing-Regulated Promoter

ABSTRACT The upstream region of the Pantoea stewartii rcsA gene features two promoters, one for constitutive basal-level expression and a second autoregulated promoter for induced expression. The EsaR quorum-sensing repressor binds to a site centered between the two promoters, blocking transcription elongation from the regulated promoter under noninducing conditions.

Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system

Natural competence for genetic transformation in Streptococcus pneumoniae is controlled by the ComCDE signal-transduction pathway. Together, ComD, a membrane histidine kinase, and ComE, its cognate response regulator, constitute a typical two-component regulatory system involved in sensing the comC-encoded competence-stimulating peptide (CSP). The comCDE operon is strongly upregulated when CSP reaches a critical threshold, probably to coordinate competence induction throughout the population. During a study of the early regulation of the comCDE operon, a mutation which resulted in increased β-galactosidase production from a comC : : lacZ fusion was isolated. This mutation, which was characterized as a G→T change in the transcription terminator of the tRNAArg located immediately upstream of comCDE, is suggested to destabilize the terminator and to allow transcriptional readthrough of comCDE. Here, it is shown that, quite unexpectedly, the mutation confers reduced transformability. A series of experiments undertaken with the aim of understanding this surprising phenotype is described. Evidence is presented that increased basal-level expression of comDE impedes both spontaneous and CSP-induced competence in S. pneumoniae. There is a discussion of how an increased concentration of ComD and/or ComE could affect competence development.

Restoration of Defective Cross Talk in ssi2 Mutants: Role of Salicylic Acid, Jasmonic Acid, and Fatty Acids in SSI2-Mediated Signaling

The Arabidopsis mutants ssi2 and fab2 are defective in stearoyl ACP desaturase, which causes altered salicylic acid (SA)- and jasmonic acid (JA)-mediated defense signaling. Both ssi2 and fab2 plants show spontaneous cell death, express PR genes constitutively, accumulate high levels of SA, and exhibit enhanced resistance to bacterial and oomycete pathogens. In contrast to constitutive activation of the SA pathway, ssi2 and fab2 plants are repressed in JA-mediated induction of the PDF1.2 gene, which suggests that the SSI2-mediated signaling pathway modulates cross talk between the SA and JA pathways. In this study, we have characterized two recessive nonallelic mutants in the ssi2 background, designated as rdc (restorer of defective cross talk) 2 and rdc8. Both ssi2 rdc mutants are suppressed in constitutive SA signaling, show basal level expression of PR-1 gene, and induce high levels of PDF1.2 in response to exogenous application of JA. Interestingly, while the rdc8 mutation completely abolishes spontaneous cell death in ssi2 rdc8 plants, the ssi2 rdc2 plants continue to show some albeit reduced cell death. Fatty acid (FA) analysis showed a reduction in 16:3 levels in ssi2 rdc8 plants, which suggests that this mutation may limit the flux of FAs into the pro-karyotic pathway of glycerolipid biosynthesis. Both rdc2 and rdc8 continue to accumulate high levels of 18:0, which suggests that 18:0 levels were responsible for neither constitutive SA signaling nor repression of JA-induced expression of the PDF1.2 gene in ssi2 plants. We also analyzed SA and JA responses of the fab2-derived shs1 mutant, which accumulates levels of 18:0 over 50% lower than those in the fab2 plants. Even though fab2 shs1 plants were morphologically bigger than fab2 plants, they expressed PR genes constitutively, showed HR-like cell death, and accumulated elevated levels of SA. However, unlike the ssi2 rdc plants, fab2 shs1 plants were unable to induce high levels of PDF1.2 expression in response to exogenous application of JA. Together, these results show that defective cross talk in ssi2 can be restored by second site mutations and is independent of morphological size of the plants, cell death, and elevated levels of 18:0.

Expression of the Nitroarene Dioxygenase Genes in Comamonas sp. Strain JS765 and Acidovorax sp. Strain JS42 Is Induced by Multiple Aromatic Compounds

ABSTRACT This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.

Role of Ribosome Release in Regulation oftna Operon Expression in Escherichia coli

ABSTRACT Expression of the degradative tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the tna operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region, tnaC, that contains a single, crucial, Trp codon. Studies with a lacZ reporter construct lacking the spacer region between tnaC and the first major structural gene,tnaA, suggested that tryptophan induction might involvecis action by the TnaC leader peptide on the ribosome translating the tnaC coding region. The leader peptide was hypothesized to inhibit ribosome release at thetnaC stop codon, thereby blocking Rho’s access to the transcript. Regulatory studies with deletion constructs of thetna operon of Proteus vulgaris supported this interpretation. In the present study the putative role of thetnaC stop codon in tna operon regulation inE. coli was examined further by replacing the naturaltnaC stop codon, UGA, with UAG or UAA in atnaC-stop codon-tnaA′-′lacZ reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in E. coli translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the tnaC stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1, prfA1, increased basal level expression 60-fold when the tnaC stop codon was UAG and 3-fold when this stop codon was UAA; basal level expression was reduced by 50% in the construct with the natural stop codon, UGA. In strains with any of the three stop codons and the prfA1mutation, the induced levels of tna operon expression were virtually identical. The effects of tnaC stop codon identity on expression were also examined in the absence of Rho action, using tnaC-stop codon-′lacZ constructs that lack the tnaC-tnaA spacer region. Expression was low in the absence of tnaC stop codon suppression. In most cases, tryptophan addition resulted in about 50% inhibition of expression when UGA was replaced by UAG or UAA and the appropriate suppressor was present. Introduction of the prfA1 mutant allele increased expression of the suppressed construct with the UAG stop codon; tryptophan addition also resulted in ca. 50% inhibition. These findings provide additional evidence implicating the behavior of the ribosome translating tnaC in the regulation of tna operon expression.

A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site

The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis. Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression. This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present. Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS. The element has homology to the consensus REB1-binding sequence CGGGTARNNR. Gel retardation assays showed that REB1 binds specifically to this element. We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site. Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression. Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site. A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site. This suggests that ABF1 and REB1 may have related functions within the cell. Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.

A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site.

The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis. Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression. This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present. Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS. The element has homology to the consensus REB1-binding sequence CGGGTARNNR. Gel retardation assays showed that REB1 binds specifically to this element. We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site. Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression. Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site. A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site. This suggests that ABF1 and REB1 may have related functions within the cell. Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.