FAMILIES OF BACTERIAL SIGMA FACTORS

            Bacterial s factors belong to two large, and apparently unrelated, protein families: the s⁷⁰ and the s⁵⁴ families (Gross et al., 1998; 1992; Helmann, 1994; Helmann and Chamberlin, 1988). Within the s⁷⁰ family, there are several phylogenetic groups that often, but not always, correlate with function. Lonetto et al. (1992) originally distinguished between the primary (group 1) s factors, a group of closely related but non-essential paralogs (group 2), and the more divergent alternative s factors (group 3). To this classification, we can now add the ECF s factors (group 4) and the newly emerging TxeR family (group 5).

            The nomenclature of s factors and their genes has generated considerable confusion. In general, most s factors in E. coli and other Gram negative bacteria are given the designation of RNA polymerase subunits, rpo. Examples include the primary s, encoded by the rpoD gene, and the heat shock s factor, encoded by the rpoH gene. The s factors themselves are often identified by a superscript to reflect their molecular mass (in kDa): RpoD is s⁷⁰, RpoH is s³². For many of those s factors identified genetically, the s is still identified by the original gene name: examples include s^FecI in E. coli and s^AlgU in P. aeruginosa (also known now as s^E). In B. subtilis, and most other Gram positive organisms, an alternative scheme has been adopted in which each alternative s factor is given a letter designation and the corresponding genes are given sig designations. By convention, the primary s factor is s^A and is encoded by the sigA gene, and the alternative s factors are identified by other letters. In some cases, other nomenclature is still in place: for example in some species group 2 s factors (see below) carry hrd (homolog of rpoD) designations. It remains to be seen how the nomenclature will evolve now that at least one species (S. coelicolor) has more s factors then there are letters in the alphabet.

Group 1. The primary s factors

The Group 1 s factors include E. coli s⁷⁰ and its orthologs (Lonetto et al., 1992). These s factors are essential proteins responsible for most transcription in rapidly growing bacterial cells and are thus often referred to as the "primary" s factors. As a group, the primary s factors are usually between 40 and 70 kDa in size and have four characteristic conserved sequence regions: regions 1 through 4 (reviewed in Gross et al., 1998; Helmann and Chamberlin, 1988).  In addition, in most species where promoter selectivity is well understood, primary s factors recognize promoters of similar sequence: TTGaca near -35 and TAtaaT near -10 (where uppercase refers to more highly conserved bases).

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Group 2. Non-essential proteins highly similar to primary s factors

In some species there are s factors that are closely related to the primary s but dispensible for growth. These group 2 s factors include the E. coli s^S (RpoS) protein and three of the four Hrd (Homolog of RpoD) proteins in Streptomyces coelicolor: only HrdB is essential and is, by this criterion, a group 1 s (Buttner and Lewis, 1992). Like the group 1 s factors, the group 2 proteins contain all four of the conserved sequence regions characteristic of primary s factors (Lonetto et al., 1992). Moreover, the regions of s factor that determine promoter selectivity are often nearly identical between the group 1 and 2 s factors. Thus, it is likely that the group 1 and 2 proteins have extensive overlap in promoter recognition.

The most extensively studied group 2 s is the E. coli RpoS (s^S) stationary phase s factor (Hengge-Aronis, 1999; 2000). Many promoters transcribed by the s⁷⁰-containing holoenzyme are also recognized by s^S and only a few truly s^S-specific promoters have been described. Indeed, it has been quite difficult to discern those features of the DNA sequence that allow selective recognition by s^S. This was dramatically illustrated when SELEX methods were used to determine the optimal binding sequence for the s^S holoenzyme: the resulting "consensus" was identical to that already documented for s⁷⁰ (T. Gaal and R. Gourse, personal communication). This has led to a model in which consensus promoters, which are extremely rare, can be recognized by both s factors and the key to selectivity is the differential tolerance of non-consensus bases. For example, s^S transcribes efficiently from promoters lacking a consensus -35 element (Wise et al., 1996) or having a C adjacent to the upstream T of the -10 element whereas these changes can greatly reduce recognition by s⁷⁰ holoenzyme (Becker and Hengge-Aronis, 2001; Lee and Gralla, 2001).

            In the cyanobacteria and in S. coelicolor the situation is made more complex by the presence of three or more group 2 s factors. The functions of these s factors have remained elusive. They are clearly dispensible and even multiply mutant strains do not display obvious phenotypes (Buttner and Lewis, 1992). In several cases, these group 2 s factors have been found to be preferentially expressed during nutrient stress conditions (Caslake et al., 1997; Muro-Pastor et al., 2001). One interpretation of these data is that activation of one or more group 2 s factors can alter, perhaps in subtle ways, the precise set of genes that are expressed while maintaining expression of most housekeeping functions normally dependent on the primary s.

Group 3.  Secondary s factors

In 1992, Lonetto et al. assigned the remaining known alternative s factors to group 3. These proteins could all be clearly recognized as s factors based on the presence of the conserved amino acid sequences of regions 2 and 4. However, in many cases conserved region 1 and often region 3 was absent. These group 3 proteins are significantly smaller in size than their group 1 and 2 paralogs (typically 25 to 35 kDa in molecular mass).

            While the majority of the RNA polymerase (RNAP) core enzyme in rapidly growing cells is associated with the primary s factor (e.g. E. coli s⁷⁰ or B. subtilis s^A), the fraction associated with group 3 s factors can be greatly increased under conditions of stress or during developmental processes (Hecker and Volker, 1998; Price, 2000). By reprogramming RNAP, these s factors function as global regulators allowing the coordinate activation of numerous unlinked operons. As a class, the group 3 s factors are regulated in diverse ways: some at the level of synthesis, others by proteolysis, and others by the reversible interaction with an anti-s factor (Haldenwang, 1995; Helmann, 1999; Hughes and Mathee, 1998; Kroos et al., 1999).

The group 3 s factors can be divided into several clusters of evolutionarily related proteins, often with conserved or related functions. Thus, there is a heat shock cluster, a flagellar biosynthesis cluster, and a sporulation cluster (Lonetto et al., 1992). In some cases, these clusters of s factors are associated with conserved promoter sequences. For example, the promoter selectivity of the flagellar (s²⁸) sub-family is conserved between diverse bacteria (Helmann, 1991) and the B. subtilis s can partially complement the corresponding E. coli mutant (Chen and Helmann, 1992). Within the sporulation sub-family, different paralogs within B. subtilis display overlapping promoter selectivity such that some (but not all) target promoters can be recognized by more than one s factor allowing transcription initiation from coincident start points (Helmann and Moran, 2002).

Group 4: The Extracytoplasmic Function (ECF) sub-family

            In 1994, a convergence of several lines of research led to the initial designation of the extracytoplasmic function (ECF) sub-family of s factors (Lonetto et al., 1994). Mark Buttner identified the gene for the alternative s factor s^E in S.  coelicolor and noted that it had only distant similarity to known s factors. At about the same time, Mike Lonetto in Carol Gross' lab noted that S. coelicolor s^E, the recently identified E. coli s^E, and several other known regulatory proteins formed a distinct sub-family within the s⁷⁰ family of regulators.

Prior to the seminal paper of Lonetto et al. (1994), many of the ECF s factors were known to function as positive activators of gene expression, but were assumed to act as classical transcription activators functioning in conjunction with one or more forms of holoenzyme. This assumption was challenged by Lonetto et al. (1994) who predicted that these diverse regulators would all function as s factors. This prediction has been confirmed for all tested examples. Interestingly, the sequence similarity between one family member, Pseudomonas aeruginosa AlgU(AlgT), and B. subtilis s^H had been noted prior to the Lonetto study (Martin et al., 1993), but there was no experimental confirmation of the role of this protein as a s factor.

In keeping with the classification scheme introduced by Lonetto et al. (1992), I propose that the ECF s factors be referred to as "group 4." Note that previously Wosten (1998) assigned these s factors as sub-group 3.2 of the group 3 s factors. However, ECF s factors are significantly more divergent in sequence, and in many organisms they equal or exceed in numbers, the group 3 s factors. Therefore, it seems fitting that they define their own group with the s⁷⁰ family. This view is further supported by the assignment of ECF s factors as a unique group within the conserved orthologous groups (COG) database (Tatusov et al., 2000).

            As a class, the ECF s factors share several common features (Figure 1). First, they often recognize promoter elements with an “AAC” motif in the –35 region. Second, in many cases the ECF s factor is cotranscribed with a transmembrane anti-s factor with an extracytoplasmic sensory domain and an intracellular inhibitory domain. Third, they often control functions associated with some aspect of the cell surface or transport.

            The designation "extracytoplasmic function" (or ECF) evolved from an analysis of the functions of the known examples of group 4 factors (Lonetto et al., 1994). This phylogenetic cluster included regulators of a periplasmic stress and heat shock response (E. coli s^E), iron transport (FecI in E. coli), a metal ion efflux system (CnrH in Alcaligenes), alginate secretion (AlgU/T in P. aeruginosa), and synthesis of membrane-localized carotenoids in Myxococcus xanthus (CarQ). The only unifying feature of these diverse physiological processes is that they all involve cell envelope functions (transport, secretion, extracytoplasmic stress). Hence, the name extracytoplasmic function (or ECF) was suggested for this family of s factors. Even this broad generalization may be an oversimplification for this complex and rapidly growing family of regulators: at least one of the recently characterized ECF s factors (S. coelicolor s^R) controls a cytoplasmic stress response (see below).

            In the last several years the complete genome sequences of dozens of bacteria have been determined. A survey of currently available genome sequences reveals a wide range in the numbers of ECF s factors (Table 1): 2 in E. coli, 7 in B. subtilis, 10 in Mycobacterium tuberculosis, and ~50 in Streptomyces coelicolor!

Group 5:  The TxeR sub-family

The discovery of the ECF sub-family of s factors taught us that the biochemical identification of one or two regulators as s factors can provide insight into the mechanism of action of a large family of related proteins. A similar story appears to be unfolding with the recent description of TxeR as a s factor controlling toxin gene expression in Clostridium difficile. This regulatory protein functions biochemically as a s factor despite the fact that the sequence of the protein bears little discernable resemblance to other members of the s⁷⁰ family. Addition of purified TxeR protein is sufficient for recognition of the tox promoter by either E. coli or B. subtilis core RNA polymerase (Mani and Dupuy, 2001). Since several other positive regulators of toxin genes, including C. tetani TetR, C. botulinum BotR, and C. perfingens UviA, are related to TxeR, it seems reasonable to suggest that these proteins are yet another distantly related group (herein designated group 5) of the s⁷⁰ family. Promoter mapping studies confirm that the UV-inducible promoters of the bcn and uviAB genes are similar in sequence to the toxA and toxB promoters. Moreover, the UviA protein can activate transcription of a P_­toxA fusion and, conversely, the TxeR protein can activate transcription of the uviA and bcn promoters when they are both present in the heterologous host, B. subtilis (N. Mani, personal communication). Thus, the UviA and TxeR s factors appear to have similar promoter recognition properties. Recent biochemical studies have confirmed that UviA has the predicted s factor activity (N. Mani, personal communication).