ECF SIGMA FACTORS IN GRAM POSITIVE BACTERIA.doc

The Extracytoplasmic Function ECF Sigma Factors NOTE as submitted some additional changes were made during editing for Advances in Microbial Physiology vol 46 John D Helmann Department of Microbiology Wing Hall Cornell University Ithaca NY 14853 8101 phone 607 255 6570 FAX 607 255 3904 e mail jdh9 cornell edu 1 INTRODUCTION 2 FAMILIES OF BACTERIAL SIGMA FACTORS 2 1 Group 1 The primary factors 2 2 Group 2 Non essential proteins highly similar to primary factors 2 3Group 3 Secondary factors of the 70 family 2 4Group 4 The ECF sub family 2 5 Group 5 The TxeR sub family 3 FUNCTIONS OF ECF FACTORS 3 1Strategies to assigning function 3 2 Escherichia coli 3 2 1 E 3 2 2 FecI 3 3 Bacillus subtilis 3 3 1 X 3 3 2 W 3 3 3 M 3 3 4Other ECF factors 3 4Streptomyces coelicolor 3 4 1 E 3 4 2 R 3 4 3 BldN 3 4 4Other ECF factors 3 5Mycobacterium tuberculosis 3 5 1 E 3 5 2 H 3 6Pseudomonas aeruginosa 3 6 1 E 3 6 2 PvdS and its relatives 3 7ECF factors in other organisms 4 RECURRING THEMES IN THE STUDY OF ECF FACTORS ACKNOWLEDGEMENTS REFERENCES 2 ABSTRACT Bacterial sigma factors are an essential component of RNA polymerase and determine promoter selectivity The substitution of one factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent As a class alternative factors play key roles in the coordinating gene transcription during various stress responses and during morphological development The extracytoplasmic function ECF factors are small regulatory proteins that are quite divergent in sequence relative to most other factors Many bacteria particularly those with more complex genomes contain multiple ECF factors and these regulators often outnumber all other types of factor combined Examples include Bacillus subtilis 7 ECF factors Mycobacterium tuberculosis 10 Caulobacter crescentus 13 Pseudomonas aeruginosa 19 and Streptomyces coelicolor 50 The roles and mechanisms of regulation for these various ECF factors are largely unknown but significant progress has been made in selected systems As a general trend most ECF factors are cotranscribed with one or more negative regulators Often these include a transmembrane protein functioning as an anti factor that binds and inhibits the cognate factor Upon receiving a stimulus from the environment the factor is released and can bind to RNA polymerase to stimulate transcription In many ways these anti pairs are analogous to the more familiar two component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA binding response regulator Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane Here I review current knowledge of some of the better characterized ECF factors discuss the variety of experimental approaches that have proven productive in defining the roles of ECF factors and present some unifying themes that are beginning to emerge as more systems are studied INTRODUCTION The latter half of the twentieth century has witnessed an information explosion More than ever we now appreciate that the utility of information rests both with its content and its accessibility On the cellular level the vast storehouse of genetic information embodied in the DNA must be retrieved in a highly selective and timely manner This requires the coordinated effort of sensory proteins that function to monitor the prevailing environmental conditions and regulatory proteins that control the flow of genetic information from DNA to RNA to protein In bacteria the primary checkpoint for controlling gene expression is the transcription of DNA into RNA by RNA polymerase RNA polymerase is a complex multisubunit enzyme that contains a dissociable sigma subunit responsible for promoter recognition Typically the vast majority of transcription in rapidly growing bacteria requires a single primary subunit similar to 70 of Escherichia coli Control of genes transcribed by this dominant RNA polymerase often rests with DNA binding repressor and activator proteins In other cases however transcription is regulated by a switching mechanism in which the primary subunit is replaced by an alternative factor with a distinct promoter selectivity Alternative also called secondary factors function in place of the primary factor by binding to core RNA polymerase to allow promoter recognition In general alternative factors control specialized regulons active during growth transitions in stationary phase in response to stress conditions or during morphological differentiation With the exception of the 54 subfamily of regulators alternative factors are related in sequence and presumably in structure to 70 Lonetto et al 1992 Historically most alternative factors were discovered either through biochemical studies of transcription selectivity e g their initial discovery in Bacillus subtilis and its phages 3 Haldenwang 1995 Kroos et al 1999 or by the sequencing of regulatory genes e g the characterization of E coli htpR later designated 32 Grossman et al 1984 However with the exponential growth in DNA sequence information including the availability of dozens of complete genome sequences many putative factor genes have now been identified for which we do not yet have functional confirmation In this review I focus on one rapidly growing group of factors the ECF sub family Lonetto et al 1994 Related reviews focus more specifically on Escherichia coli E Missiakas and Raina 1998 Ravio and Silhavy 2001 FecI Braun 1997 the Streptomyces coelicolor ECF factors Paget et al 2002 and the roles of anti factors Hughes and Mathee 1998 Herein I summarize these systems and also review recent progress in understanding the Bacillus subtilis and Mycobacterium tuberculosis ECF factor regulons 1 FAMILIES OF BACTERIAL SIGMA FACTORS Bacterial factors belong to two large and apparently unrelated protein families the 70 and the 54 families Gross et al 1998 1992 Helmann 1994 Helmann and Chamberlin 1988 Within the 70 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 factors a group of closely related but non essential paralogs group 2 and the more divergent alternative factors group 3 To this classification we can now add the ECF factors group 4 and the newly emerging TxeR family group 5 The nomenclature of factors and their genes has generated considerable confusion In general most factors in E coli and other Gram negative bacteria are given the designation of RNA polymerase subunits rpo Examples include the primary encoded by the rpoD gene and the heat shock factor encoded by the rpoH gene The factors themselves are often identified by a superscript to reflect their molecular mass in kDa RpoD is 70 RpoH is 32 For many of those factors identified genetically the is still identified by the original gene name examples include FecI in E coli and AlgU in P aeruginosa also known now as E In B subtilis and most other Gram positive organisms an alternative scheme has been adopted in which each alternative factor is given a letter designation and the corresponding genes are given sig designations By convention the primary factor is A and is encoded by the sigA gene and the alternative factors are identified by other letters In some cases other nomenclature is still in place for example in some species group 2 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 factors then there are letters in the alphabet 2 1 Group 1 The primary factors The Group 1 factors include E coli 70 and its orthologs Lonetto et al 1992 These factors are essential proteins responsible for most transcription in rapidly growing bacterial cells and are thus often referred to as the primary factors As a group the primary 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 factors recognize promoters of similar sequence TTGaca near 35 and TAtaaT near 10 where uppercase refers to more highly conserved bases 2 2 Group 2 Non essential proteins highly similar to primary factors 4 In some species there are factors that are closely related to the primary but dispensible for growth These group 2 factors include the E coli 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 Buttner and Lewis 1992 Like the group 1 factors the group 2 proteins contain all four of the conserved sequence regions characteristic of primary factors Lonetto et al 1992 Moreover the regions of factor that determine promoter selectivity are often nearly identical between the group 1 and 2 factors Thus it is likely that the group 1 and 2 proteins have extensive overlap in promoter recognition The most extensively studied group 2 is the E coli RpoS S stationary phase factor Hengge Aronis 1999 2000 Many promoters transcribed by the 70 containing holoenzyme are also recognized by S and only a few truly 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 This was dramatically illustrated when SELEX methods were used to determine the optimal binding sequence for the S holoenzyme the resulting consensus was identical to that already documented for 70 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 factors and the key to selectivity is the differential tolerance of non consensus bases For example 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 70 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 factors The functions of these 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 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 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 2 3Group 3 Secondary factors In 1992 Lonetto et al assigned the remaining known alternative factors to group 3 These proteins could all be clearly recognized as 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 factor e g E coli 70 or B subtilis A the fraction associated with group 3 factors can be greatly increased under conditions of stress or during developmental processes Hecker and Volker 1998 Price 2000 By reprogramming RNAP these factors function as global regulators allowing the coordinate activation of numerous unlinked operons As a class the group 3 factors are regulated in diverse ways some at the level of synthesis others by proteolysis and others by the reversible interaction with an anti factor Haldenwang 1995 Helmann 1999 Hughes and Mathee 1998 Kroos et al 1999 The group 3 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 factors are 5 associated with conserved promoter sequences For example the promoter selectivity of the flagellar 28 sub family is conserved between diverse bacteria Helmann 1991 and the B subtilis 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 factor allowing transcription initiation from coincident start points Helmann and Moran 2002 2 4Group 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 factors Lonetto et al 1994 Mark Buttner identified the gene for the alternative factor E in S coelicolor and noted that it had only distant similarity to known factors At about the same time Mike Lonetto in Carol Gross lab noted that S coelicolor E the recently identified E coli E and several other known regulatory proteins formed a distinct sub family within the 70 family of regulators Prior to the seminal paper of Lonetto et al 1994 many of the ECF 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 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 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 factor In keeping with the classification scheme introduced by Lonetto et al 1992 I propose that the ECF factors be referred to as group 4 Note that previously Wosten 1998 assigned these factors as sub group 3 2 of the group 3 factors However ECF factors are significantly more divergent in sequence and in many organisms they equal or exceed in numbers the group 3 factors Therefore it seems fitting that they define their own group with the 70 family This view is further supported by the assignment of ECF factors as a unique group within the conserved orthologous groups COG database Tatusov et al 2000 As a class the ECF 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 factor is cotranscribed with a transmembrane anti 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 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 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 factors S coelicolor R controls a cytoplasmic stress response see below 6 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 factors Table 1 2 in E coli 7 in B subtilis 10 in Mycobacterium tuberculosis and 50 in Streptomyces coelicolor 2 5Group 5 The TxeR sub family The discovery of the ECF sub family of factors taught us that the biochemical identification of one or two regulators as 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 factor controlling toxin gene expression in Clostridium difficile This regulatory protein functions biochemically as a factor despite the fact that the sequence of the protein bears little discernable resemblance to other members of the 70 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 70 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 PtoxA 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 factors appear to have similar promoter recognition properties Recent biochemical studies have confirmed