In Streptococcus pneumoniae, the type 1 pilus is involved in many steps of pathogenesis, including adherence to epithelial cells, mediation of inflammation, escape from macrophages, and the formation of biofilms. The type 1 pilus genes are expressed in a bistable fashion with cells switching between "on" and "off" expression states. Bistable expression of these genes is due to their control by RlrA, a positive regulator subject to control by a positive-feedback loop. The type 1 pilus genes are also thought to be negatively regulated by a large number of repressors. Here we show that expression of the type 1 pilus genes is thermosensitive and switched off at growth temperatures below 31°C. We also report that the on expression state of the type 1 pilus genes is highly stable, a phenomenon which we show likely contributed to the erroneous identification of many proteins as negative regulators of these genes. Finally, we exploited the effect of low temperature on pilus gene expression to help identify SP_1523, an Snf2-type protein, as a novel negative regulator of the pilus genes. Our findings establish that the type 1 pilus genes are thermoregulated and are repressed by a member of the Snf2 protein family. They also refute the notion that these genes are controlled by 8 previously described negative regulators.IMPORTANCE Streptococcus pneumoniae is the leading cause of death from respiratory infections in children. Many bacterial factors contribute to pneumococcal virulence and nasopharyngeal colonization. The type 1 pneumococcal pilus plays an important role in mouse models and in epithelial adherence and is expressed in a bistable fashion. Here we show that the "on" state is highly stable, which may explain the prior misidentification of negative regulators of pilus expression. We also show that expression of pilus genes is thermosensitive: virtually no expression can be detected at temperatures found in the anterior nares of humans. We took advantage of this property to identify a negative regulator of pilus expression, a member of a family of proteins widely conserved across Gram-positive bacteria.KEYWORDS coagulase-negative staphylococcal regulators, gene expression, pilus, pneumococcus, thermosensitivity S treptococcus pneumoniae (pneumococcus) accounts for more than 860,000 childhood deaths annually (1). The bacterium is a frequent colonizer of the nasopharynx in children; most are colonized at some point during the first 2 years of life. Colonization is the first, and critical, step in pathogenesis (2); the ability of pneumococcal conjugate vaccines to protect against colonization and transmission is a major component of their success (3).Over the past several decades, there has been a concerted effort directed at the identification of pneumococcal proteins that play important roles in colonization, with the thought that these may represent interesting vaccine targets. Of the many antigens

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully.