The DAN/TIR genes encode nine cell wall mannoproteins in Saccharomyces cerevisiae which are expressed during anaerobiosis (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, TIR4, and TIP1). Most are expressed within an hour of an anaerobic shift, but DAN2 and DAN3 are expressed after about 3 h. At the same time, CWP1 and CWP2, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts. TIP1, TIR1, TIR2, and TIR4 are also induced during cold shock. Correspondingly, CWP1 is downregulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the DAN/TIR genes (but not of TIP1). We show that CWP2 (but not CWP1) is controlled by the same factors, but in reverse fashion-primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of TIR1, TIR3, or TIR4 prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.The cell wall of Saccharomyces cerevisiae is a rigid structure which determines cell morphology and also serves as a protective barrier, providing mechanical protection and enabling selective uptake of macromolecules (1a, 4, 7). A major component of the cell wall is mannoprotein, comprising about 40% of its mass. Mannoproteins are believed to be determinants of cell wall permeability (31), and certain ones are also essential for developmental events such as mating and transition to hyphal growth (18,24). Some mannoproteins can be extracted from the cell wall with detergent; others are covalently bound but can be released with glucanase (7). Proteins in the latter category have common features, including a region rich in serine and threonine, a glycosylphosphatidylinositol (GPI) anchor attachment signal at C termini (3,8), and an endoplasmic reticulum localization sequence; several also have a PAU domain. This segment of about 100 amino acids is shared among a group of proteins known as "seripauperins" (29).The Cwp2 mannoprotein (28) is one of the most abundant proteins of the cell wall and is believed to play a role in its stabilization, along with another homologous constituent, Cwp1 (23, 27, 28). While Cwp1 and Cwp2 are expressed under normal growth conditions, other mannoproteins are expressed in response to environmental stress. The most extensive response is the induction of several homologou...

Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Delta or mot3Delta cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1-Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present-at much lower levels-demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.

The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors.

e Streptococcus pneumoniae is a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. While S. pneumoniae produces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of the cps locus in the type 2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the ؊25 nucleotide (G) upstream of cps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferasebased transcriptional reporter, we showed that the full transcription of the cps genes not only depends on the core promoter immediately upstream of cps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, common cps gene (cps2ABCD) mutants did not show significant abnormality in cps transcription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation of S. pneumoniae in a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of the cps locus in S. pneumoniae. Streptococcus pneumoniae (pneumococcus) is a major cause of bacterial infections in humans, including otitis media, pneumonia, bacteremia, and meningitis (1). As the outermost structure, the capsule is the major virulence factor of S. pneumoniae and protects the bacterium by interfering with host phagocytic killing (2). Virtually all of the clinical isolates are encapsulated, and mutations in the cps locus result in the loss of virulence (3). The capsule of S. pneumoniae is composed of capsular polysaccharides (CPSs). The CPSs are immunogenic and thus serve as the target antigens for the current pneumococcal vaccines (4). Due to host immune selection that targets the capsule, the CPSs have undergone extensive chemical and antigenic variation via genetic diversity in the CPS biosynthesis genes. At least 94 antigenically distinct capsular serotypes have been identified in S. pneumoniae (5, 6). While the CPSs of types 3 and 37 are produced as relatively simple repeats of polysaccharides by the synthase pathway (7, 8), all other capsule types are much more complex and are synthesized by WZY-dependent polymerization, a mechanism of capsule production that is widely found in other bacteria (9-11).Except for type 37, all of the CPS types in S. pneumoniae are determined by a set of capsule biosynthesis genes, which are historically designated cps or cap (12). Bentley et al. recently renamed the pneumococcal capsule genes on the basis of their overall...

Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of "anaerobic" genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation analysis showed an Rpd3-dependent reduction in DAN1 promoter-associated nucleosomes upon induction. Taken together, these data provide evidence that during anaerobiosis, the Rpd3 complex acts at the DAN1 promoter to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and that chromatin remodeling by Swi/Snf is necessary for normal expression.The Saccharomyces cerevisiae DAN/TIR genes are among a large group of genes that are upregulated during adaptation to anaerobic growth (37,59,65,66). These genes code for cell wall mannoproteins, which play a significant role in cell wall permeability. The kinetics of expression of these genes ranges from 30 minutes to 3 hours following the onset of anaerobiosis (1,59). This suggests that as the cells descend towards anaerobiosis, certain requirements that are necessary for survival in that milieu are incrementally satisfied by specific alterations in gene expression. The importance of the DAN/TIR genes is further underlined by the fact that disruption of some of them, such as TIR1, TIR3, and TIR4, abrogates anaerobic growth (1), indicating that the corresponding proteins play essential functions during anaerobic adaptation. Moreover, it appears that a complex programmed cell wall remodeling occurs during adaptation to anaerobiosis, as shown by the fact that the major aerobic cell wall mannoproteins encoded by CWP1 and CWP2 are replaced by their anaerobic counterparts, encoded by the DAN/TIR genes, under those conditions (1).The precise mechanisms by which the DAN/TIR genes are regulated are still being elucidated. We showed earlier that these genes are regulated by heme, which is synthesized only in the presence of oxygen, and by three DNA binding transcription factors (2, 13, 59). The activator Upc2 acts through a consensus site termed AR1 to induce the expression of these genes in anaerobiosis. Upc2 was also identified as a regulator of the anaerobic sterol transport system (2, 75). It shares extensive homology with Ecm22 and with a Candida albicans protein (CaUpc2) (71). The repressors Ro...