As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P450 steroid hydroxylase genes, including the gene encoding steroid NGFI-B (also called nur77) is a nuclear receptor encoded by an early-response gene that is rapidly induced in cells stimulated with growth factors and/or other extracellular ligands (7, 11). As with many other early-response (or immediate-early) genes that encode transcription factors (e.g., c-Fos, c-Jun, and NGFI-A), the NGFI-B protein presumably regulates the expression of other genes, ultimately culminating in phenotypic changes (reviewed in reference 8). To begin to understand the contribution of these proteins to cellular responses, we must identify target gene(s) whose expression is regulated by the early-response proteins. Toward this end, we recently used a genetic selection approach to identify the nucleotide sequence bound by NGFI-B: AAAGGTCA. This NGFI-B response element (NBRE) contains an estrogen receptor half-site (AGGTCA) preceded by two adenines (21, 22). The identification of this site allowed us to establish that NGFI-B activates transcription of reporter genes in a variety of mammalian cell lines in the absence of exogenously added ligand (13, 21).Steroid 21-hydroxylase (21-OHase, Cyp2l) is one of a group of related cytochrome P-450 enzymes that are required for steroid hormone biosynthesis (reviewed in reference 12). It is expressed only in the adrenal cortex, where it is essential for the production of both glucocorticoids and mineralocorticoids. Within adrenocortical cells, the transcription of 21-OHase is primarily regulated by corticotropin (ACTH), the predominant regulator of glucocorticoid bio-* Corresponding author.synthesis. This ACITH-dependent increase in 21-OHase transcription is mimicked by treatment of adrenocortical cells with cyclic AMP (cAMP) analogs, leading to the hypothesis that cAMP mediates this transcriptional induction (9). In contrast to most other cAMP-regulated genes (17), the ACTH-mediated increase in 21-OHase transcription is manifest only after several hours and requires ongoing protein synthesis, suggesting that ACTIH induces early-response genes which then increase 21-OHase transcription (9,19). Previous analysis of the promoter region of the mouse 21-OHase gene established that only 330 nucleotides (nt) of the 5'-flanking region were required for cell-specific and hormonally inducible expression (6). Consistent with the distinctive characteristics of 21-OHase induction by cAMP, none of the regulatory elements that were subsequently identified closely matched the consensus of the cAMPresponsive element found in most other cAMP-responsive genes (15, 17). However, essential elements at -210 and -65 contain nuclear receptor half-sites (4), implicating nuclear hormone receptor proteins in 21-OHase gene regulation.Specifically, the element at nt -65 contains 8 nt that correspond to the recognition sequence bound by NGFI-B (NBRE [21]).In this...

A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at the ureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivarius could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.Bacterial ureases are multisubunit enzymes that require Ni 2ϩ for catalytic activity (9). Several bacterial urease gene clusters have been isolated, and high degrees of homology between species have been observed (5, 9). Most bacterial ureases consist of three subunits, ␣, ␤, and ␥, encoded by ureC, -B, and -A, respectively. Other genes are present in urease clusters, i.e., ureDEFG, which encode proteins that are required for incorporation of Ni 2ϩ into the metallocenter of the catalytic site (9). Additional gene products involved in urease biogenesis and urea metabolism include nickel and urea transporters (10,14).Urea is present in saliva and crevicular fluids at 3 to 10 mM in healthy individuals (7,8), and it is hydrolyzed by ureases to generate two molecules of ammonia and one molecule of CO 2 . Ammonia can neutralize acids generated from bacterial glycolysis, inhibiting the initiation and progression of tooth decay. Ureolysis also creates a less acidic environment, enhancing the survival of acid-sensitive species and promoting the stability of a healthy oral flora (1). Despite the abundance of urea and ureolytic activity in the oral cavity, and the impact of ureolysis on oral health and ecology, the benefits for oral microorganisms of possessing ureases are not established.In general, urease expression in enteric organisms is positively regulated and transcription is activated either in the absence of an assimilable nitrogen source or in the presence of urea (5, 9). Unlike in enteric bacteria, Streptococcus salivarius 57.I urease expression is derepressed at low pH and is further enhanced in the presence of excess carbohydrate (2). Based on this mode of regulation, ureolysis by S. salivarius may function primarily to protect the organisms against acid damage or the bacteria may use ureolysis to acquire nitrogen when carbohydrates are present in excess. To test these hypotheses, an otherwise isogenic, urease-deficient derivative of S. salivarius 57.I was constructed, and the behavior of this mutant and that of the wild-type strain under different growth conditions were compared.Construction of a urease-deficient S. salivarius strain. To construct a urease-deficient S. salivarius strain, a gene specifying erythromycin resistance (erm) was cloned within ureC, the gene encoding the ␣ subunit of urease, to generate plasmid pMC81 (Fig. 1A). Plasmid pMC81 contains an Escherichia coli replicon and thus can be used as a suicide vector in streptococcal hosts. Plasmid pMC81 was introduced into wild-type 57.I by electroporation as previously described (4), and erythromycin-resistant (Em r ) transformants were selected and subjected to S...

The Streptococcus salivarius 57.I urecluster was organized as an operon, beginning withureI, followed by ureABC (structural genes) andureEFGD (accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A ς70-like promoter could be mapped 5′ to ureI (PureI) by primer extension analysis. The intensity of the signal increased when cells were grown at an acidic pH and was further enhanced by excess carbohydrate. To determine the function(s) of two inverted repeats located 5′ toPureI, transcriptional fusions of the full-length promoter region (PureI), or a deletion derivative (PureIΔ100), and a promoterless chloramphenicol acetyltransferase (CAT) gene were constructed and integrated into the chromosome to generate strains PureICAT andPureIΔ100CAT, respectively. CAT specific activities ofPureICAT were repressed at pH 7.0 and induced at pH 5.5 and by excess carbohydrate. In PureIΔ100CAT, CAT activity was 60-fold higher than in PureICAT at pH 7.0 and pH induction was nearly eliminated, indicating that expression was negatively regulated. Thus, it was concluded that PureI was the predominant, regulated promoter and that regulation was governed by a mechanism differing markedly from other known mechanisms for bacterial urease expression.

A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs). Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa. Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells. Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway. Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1. Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage. This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.

The urease genes of Streptococcus salivarius 57.I are tightly repressed in cells growing at neutral pH. When cells are cultivated at acidic pH values, the urease genes become derepressed and transcription is enhanced when cells are growing under carbohydrate-excess conditions. Previously, the authors proposed that the bacterial sugar :phosphotransferase system (PTS) modulated the DNA-binding activity by phosphorylation of the urease repressor when carbohydrate was limiting. The purpose of this study was to assess whether enzyme I (EI) of the PTS could be involved in modulating urease expression in response to carbohydrate availability. An EI-deficient strain (ptsI18-3) of S. salivarius 57.I was constructed by insertional inactivation of the ptsI gene. The mutant had no measurable PTS activity and lacked EI, as assessed by Western analysis. The mutant grew as well as the wild-type strain on the non-PTS sugar lactose, and grew better than the parent when another non-PTS sugar, galactose, was the sole carbohydrate. The mutant was able to grow with glucose as the sole carbohydrate, but displayed a 24 h lag time and had a generation time some threefold longer than strain 57.I. The mean OD 600 attained after 48 h by ptsI18-3 supplied with fructose was 016, with no additional growth observed even after 3 d. Urease expression in the wild-type and mutant strains was assessed in continuous chemostat culture. Repression of urease at neutral pH was seen in both strains under all conditions tested. Growth of wild-type cells on limiting concentrations of lactose resulted in very low levels of urease expression compared with growth on PTS sugars. In contrast, under similar conditions, urease expression in ptsI18-3 was restored to levels seen in the parent growing on PTS sugars. Growth under conditions of lactose excess resulted in further derepression of urease, but ptsI18-3 expressed about threefold higher urease activity than 57.I. The results support a role for EI in urease regulation, but also indicate that additional factors may be important in regulating urease gene expression.