**5. Bacterial processes controlled by RNA thermometers**

The regulation of gene expression in response to changes in environmental temperature is important for survival of all bacteria and for virulence of pathogenic bacteria. RNATs have been found to confer efficient temperature-dependent regulation onto the expression of bacterial genes encoding factors involved in two critically important bacterial processes —heat shock response and virulence. In the following section, each of these two critical biological processes will be briefly introduced and the role that RNATs play in facilitating them will be discussed.

**Figure 7.** Key processes are controlled by RNA thermometers in bacteria. The influence of RNA thermometers on the bacterial heat shock response and on bacterial virulence is indicated by highlighting the different groups of genes whose expression is directly regulated by an RNA thermometer.

## **5.1. Heat shock response**

The primary effect of increased temperature on bacteria is the resulting denaturation of temperature-sensitive proteins. Similar to regulatory RNA molecules, the function of a protein is strictly dependent on its structure, a feature that can be impacted by environmental temperature. Increased environmental temperature can result in partial or complete denatu‐ ration of a protein, resulting in a stable but often non-functional molecule [50]. In addition to the denaturation of proteins, high temperature is also associated with disruption of the bacterial cell membrane as well as damage to DNA molecules [46,51]. As a result of these effects, increased environmental temperature can be lethal to bacterial life and thus represents a stress that must be overcome.

In order to facilitate responsive adaptation to a rise in environmental temperature, bacteria express several genes that encode for factors that function to protect the organism from the detrimental effects generated by heat, collectively termed the heat shock response [52]. The main components of the heat shock response include 1) alternative sigma factors that direct the transcription of other heat shock responding genes; 2) heat shock proteins (Hsps), such as protein chaperon machinery that facilitate the proper folding of other proteins; 3) small Hsps (sHsps) that have multiple functions including preventing the formation of protein aggregates and protecting the integrity of cellular membrane; and 4) enzymes that degrade denatured proteins, repair damaged DNA, and more*.*

Understanding the molecular mechanisms underlying the temperature-dependent regulation of factors that facilitate the bacterial heat shock response is a major focus of ongoing investi‐ gations; the discovery of RNATs is rooted in these important studies. Since the identification of an RNAT that regulates the expression of a small heat shock protein (HspA from *B. japonicum*) and the heat shock alternative sigma factor σ32 (RpoH from *E. coli*), many other players in the heat shock response have been found to be regulated by RNATs, including chaperon component (GroES from *Salmonella*), heat-induced protease (HtrA from *E. coli*), and other small heat shock proteins [7,8,29,35] (Figure 7). Regulation of heat shock response is a complex process that involves the regulation of multiple factors at different steps of gene expression. That said, it seems that temperature-dependent regulation by RNAT is a funda‐ mental regulatory mechanism that coordinately influences nearly all types of heat shock response factors. Given the high degree of conservation seen between heat shock responses factors produced by a wide variety of living organisms, temperature-dependent regulation mediated by RNATs is expected to be present in many organisms, including eukaryotic systems [52]. A finding that directly supports this prediction is that of a secondary structure within the 5′ UTR of *Drosophila* Hsps encoding mRNAs that functions to regulate translation from the transcript in response to environmental temperature [53]. The full extent of RNATs in controlling heat shock response in bacteria and beyond is yet to be revealed.

#### **5.2. Virulence-associated genes of pathogenic bacteria**

**5. Bacterial processes controlled by RNA thermometers**

170 Nucleic Acids - From Basic Aspects to Laboratory Tools

discussed.

The regulation of gene expression in response to changes in environmental temperature is important for survival of all bacteria and for virulence of pathogenic bacteria. RNATs have been found to confer efficient temperature-dependent regulation onto the expression of bacterial genes encoding factors involved in two critically important bacterial processes —heat shock response and virulence. In the following section, each of these two critical biological processes will be briefly introduced and the role that RNATs play in facilitating them will be

**Figure 7.** Key processes are controlled by RNA thermometers in bacteria. The influence of RNA thermometers on the bacterial heat shock response and on bacterial virulence is indicated by highlighting the different groups of genes

The primary effect of increased temperature on bacteria is the resulting denaturation of temperature-sensitive proteins. Similar to regulatory RNA molecules, the function of a protein is strictly dependent on its structure, a feature that can be impacted by environmental temperature. Increased environmental temperature can result in partial or complete denatu‐ ration of a protein, resulting in a stable but often non-functional molecule [50]. In addition to the denaturation of proteins, high temperature is also associated with disruption of the bacterial cell membrane as well as damage to DNA molecules [46,51]. As a result of these effects, increased environmental temperature can be lethal to bacterial life and thus represents

In order to facilitate responsive adaptation to a rise in environmental temperature, bacteria express several genes that encode for factors that function to protect the organism from the detrimental effects generated by heat, collectively termed the heat shock response [52]. The main components of the heat shock response include 1) alternative sigma factors that direct the transcription of other heat shock responding genes; 2) heat shock proteins (Hsps), such as protein chaperon machinery that facilitate the proper folding of other proteins; 3) small Hsps (sHsps) that have multiple functions including preventing the formation of protein aggregates and protecting the integrity of cellular membrane; and 4) enzymes that degrade denatured

whose expression is directly regulated by an RNA thermometer.

**5.1. Heat shock response**

a stress that must be overcome.

proteins, repair damaged DNA, and more*.*

Once within the body of the host, and throughout the course of a natural infection, pathogenic bacteria face several challenges, including but not limited to 1) the need to adhere to host cells, 2) the need to evade killing by the host immune system, and 3) the need to acquire essential nutrients. To overcome these challenges and progress of an infection, bacteria produce specific virulence factors. As the production of virulence factors is most beneficial to an invading bacterium when it is within the host, several levels of regulation are often employed to ensure that the production of these important factors occurs only when the bacteria is within an environment that resembles that encountered within the infected host. RNATs are involved in regulating the production of a variety of virulence factors in several species of pathogenic bacteria, ensuring that these factors are most efficiently produced at the relatively high temperatures encountered within the infected host (Figure 7).

The expression of many virulence-associated genes is controlled by protein-based regulation, specifically that carried out by transcriptional regulators. Interestingly, RNATs have been found to directly control the production of three transcriptional activators that, in turn, function to control the expression of virulence-associated genes: *prfA* from *L. monocytogenes*, *lcrF* from *Y. pestis*, and *toxT* from *V. cholera* [40,41,47]. Another regulatory system that controls the expression of multiple virulence factors is quorum sensing. To date, one gene whose product is involved in quorum sensing-dependent modulation of virulence gene expression has been found to be regulated by an RNAT; this gene is *lasI* from *P. aeruginosa* [37]. RNATs within *lcrF* and *toxT* are FourU RNATs, while the RNATs controlling the expression of *prfA* and *lasI* have currently unique structure.

RNATs have also been implicated in controlling the expression of virulence-associated genes that encode factors involved in adhesion and immune evasion. For example, three virulenceassociated genes in *N. meningitis* have been found to be regulated by RNATs: *cssA,* a gene encoding a factor involved in capsule production; *fHbp,* a gene encoding a factor H binding protein; and *lst,* a gene encoding a factor required for modifications of lipopolysacccharides [24]. In *L. interrogans, ligA* and *ligB*, two genes encoding putative lipoprotein, are also regulated by RNATs [43]. Additionally *P. aeruginosa rhlA,* a gene encoding an enzyme required for the synthesis of rhamnolipid, a compound that can prevent killing of the bacteria by host immune system, is regulated by an RNAT [37]. Except for *rhlA* RNAT, which is a member of the RSOElike family, these other RNATs mentioned above have unique structures and thus are not members of the ROSE-like or FourU families of regulators.

To date, two genes involved in the acquisition of essential nutrients have been shown to be regulated by RNATs: *S. dysenteriae shuA*, a gene encoding an outer membrane heme-binding protein, and its homologous gene *chuA* in pathogenic *E. coli* [11]. Translation of *shuA/chuA* is controlled by a FourU RNAT located in the relative large 5′ UTR of the corresponding gene. Production of ShuA or ChuA facilitates the utilization of iron from heme, a potential source of essential iron found only within the relatively warm environment of the infected host [54].

For many pathogenic bacteria, the transmission from one host to the next involves exposure to different environments with different temperatures. The expression of many virulenceassociated genes is influenced by environmental temperature, a signal that varies between the host and non-host environments. With an increasing number of virulence-associated genes that are now known to be regulated by the activity of RNATs, it is possible that temperaturedependent regulation mediated by RNATs will emerge as one of the basic regulatory strategies utilized by pathogenic bacteria. The full and potentially expansive role that RNATs play in controlling virulence of pathogenic bacteria is yet to be revealed.
