starvation responses in Bacillus subtilis and Staphylococcus aureus

Research in Microbiology 160 (2009) 245e258 www.elsevier.com/locate/resmic

Physiological proteomics and stress/starvation responses in Bacillus subtilis and Staphylococcus aureus Michael Hecker*, Alexander Reder, Stephan Fuchs, Martin Pagels, Susanne Engelmann Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Jahnstrasse 15A, 17487 Greifswald, Germany Received 20 February 2009; accepted 23 March 2009 Available online 3 May 2009

Abstract Gel-based proteomics is a useful approach for visualizing the responses of bacteria to stress and starvation stimuli. In order to face stress/ starvation, bacteria have developed very complicated gene expression networks. A proteomic view of stress/starvation responses, however, is only a starting point which should promote follow-up studies aimed at the comprehensive description of single regulons, their signal transduction pathways on the one hand, and their adaptive functions on the other, and finally their integration into complex gene expression networks. This ‘‘road map of physiological proteomics’’ will be demonstrated for the general stress regulon controlled by sB in Bacillus subtilis and the oxygen starvation response with Rex as a master regulator in Staphylococcus aureus. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Stress/starvation responses; Regulatory network; Stimulons; Modulons; sB modulon; Rex modulon; Bacillus subtilis; Staphylococcus aureus

1. Introduction: proteomics and stress/starvation responses As a result of the longstanding interaction between bacteria and a continuously changing set of stress and starvation stimuli in natural ecosystems, a very complex adaptational network has been developed. Deeper insights into these stress/ starvation gene expression networks are crucial for understanding microbial physiology in general. Starvation for essential nutrients such as carbon, energy, nitrogen, oxygen or phosphorous sources as well as, oxidative, osmotic, heat, cold or acid stress are growth-restricting stimuli in nature. These environmental stimuli induce a more or less large set of genes whose proteins are responsible for adaptation to the growthrestricting conditions.

* Corresponding author. E-mail addresses: [email protected] (M. Hecker), alexander. [email protected] (A. Reder), [email protected] (S. Fuchs), [email protected] (M. Pagels), susanne.engelmann@ uni-greifswald.de (S. Engelmann). 0923-2508/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2009.03.008

More than 20 years ago, we used the panorama view of proteomics to visualize the responses of Bacillus subtilis to stress and starvation for the first time [20,22,69]. We found a dramatic response of B. subtilis to a various set of stress stimuli visualized by 2D gel-based proteomics. The same pattern of almost 50 proteins was found to be strongly induced after imposition of heat, salt and other stress stimuli [66]. Later it turned out that the expression of these proteins is controlled by the alternative sigma factor, SigB (sB) (reviewed by [18,54]). By a combination of these gel-based proteomics data with studies on the molecular genetics of sigB, the sBdependent general stress response of B. subtilis was discovered and characterized (reviewed by Price [54]) and is one of the most obvious and comprehensive responses of the cell to a different set of growth-restricting stimuli. Even if gel-based proteomics only covers a part of the proteins expressed, this technique is still a valuable toolbox to address physiological issues such as stress and starvation responses [19]. Most of the main cellular functions of growing and non-growing cells can be monitored by a gel-based protein expression profiling [42]. This statement is not only valid for metabolic pathways e most of them are covered by gel-based

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proteomics e but also for the monitoring of rapid stress or starvation responses. One of the most convincing advantages of gel-based proteomics compared to the more sensitive gelfree approaches is its amenability for visualizing the protein synthesis pattern in response to environmental stimuli, because proteins newly synthesized after stress/starvation can be directly visualized and separated from the proteins accumulated before stress/starvation using the 35S-methionine labelling approach [5]. The dramatic changes in the gene expression pattern in response to stress/starvation stimuli are preferably analyzed using DNA arrays in a more comprehensive way than by proteomics. Whereas changes in the gene expression pattern occurring within only a very few minutes can be detected by DNA array studies, much more time is required to visualize changes on the proteome level, because protein accumulation needs some time to be detected by staining procedures. Using radioactive labeled amino acids, however, these changes can be visualized in a 3-min time scale because only newly synthesized and thereby radioactive labeled proteins can be visualized by autoradiography. A combination of transcriptomics and proteomics offers a new quality in analyzing the extremely complex gene expression networks because the majority of genes/proteins induced or repressed by the stimuli can be visualized. These adaptational networks consist of a large set of specific and more general stress and starvation boxes which are tightly connected. Proteomics is an excellent tool to put proteins with known or still unknown functions into the single

stress/starvation boxes within the network and to study their adaptive functions. The basic steps for exploring the modules of the network are: (i) The definition of stimulons e that means the entire set of proteins/genes induced or repressed by the stimulus; (ii) The dissection of stimulons into single regulons based on the study of mutants in global regulators; (iii) The study of the interaction between the single regulons creating overlapping areas within the adaptational network (e. g. by genes controlled by more than one global regulator); (iv) The definition of modulons, a network of regulons controlled by a master regulator that directly controls the main regulon and secondary regulators that are, in turn, responsible for the regulation of downstream located subregulons; (v) The sequential regulation of gene expression in a timelapse scale (e.g. gene expression programs during sporulation of B. subtilis, see [23] for review). The first step in analyzing stress/starvation adaptation is to look for all proteins induced or repressed by the stimulus, because all newly induced proteins will together accomplish the stress/starvation adaptation. The dual channel imaging technique used in our laboratory has been proven to be particularly suited to search for proteins induced or repressed by stress/starvation stimuli [5]. This method allows a rapid assignment of proteins to stimulons or regulons simply by

Fig. 1. Dual-channel image for simultaneous display of protein synthesis (red image) versus protein amount (green image) of B. subtilis in response to heat shock (48  C). Cytoplasmic proteins were labeled with L-[35S]methionine after exposure to heat shock and separated by 2D gel electrophoresis. Proteins were stained using silver nitrate for detecting protein amounts, and protein synthesis was determined from autoradiograms. The resulting 2D gel images were analyzed using DECODON Delta2D software. The proteins of the heat shock stimulon appear in red, since they display strongly induced protein synthesis.

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a comparative analysis of protein amount (protein staining) versus protein synthesis (autoradiography) in a single gel. Two digitized gel images have to be superimposed in alternative additive dual-color channels. The first channel showing accumulated proteins visualized by sensitive staining techniques is false-colored green. The second image (autoradiograph) showing the proteins labeled during a 5-min pulse with 35 S-L-methionine is false-colored red. When the two images are combined, accumulated and simultaneously synthesized proteins in growing cells are colored yellow. After the imposition of a stress/starvation stimulus, however, proteins not previously accumulated in the cell but newly induced are colored red. Identifying these red-colored proteins provides a simple technique for characterization of all proteins induced by a single stimulus and thus definition of the stimulon structure. In B. subtilis, heat stress induces more than 100 redlabeled proteins, some of still unknown function (Fig. 1). The allocation of unknown proteins to well defined stimulons is a simple procedure for a first, still preliminary prediction of their function: The unknown members of the heat stimulon will somehow be involved in adaptation to heat stress. In most cases, stimulons consist of more than one regulon. Hence, the next step in exploring the network is the dissection of stimulons into regulons. The procedure is to look for

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proteins that are no longer induced in a mutant in the global regulator that controls the regulon. Following this approach, two main regulon groups were found: (i) proteins induced by only one stress/starvation stimulus (or a group of functionally related stimuli such as paraquat or hydrogen peroxide for oxidative stress) with a specific adaptive function against that stimulus only, and (ii) general stress or starvation proteins induced by a different set of stress stimuli or starvation stimuli or both. Color coding technology was used to define such stress/starvation-specific and general stress proteins in B. subtilis and Staphylococcus aureus. For this visualization method, induced marker proteins for various stress and starvation stimuli were color-coded according to their induction profile (details see legends of Figs. 2 and 3, [14,61]). In this review article, two regulons were selected to show that proteomics is a good starting point for analyzing the structure of regulons: the sB regulon in B. subtilis and the Rex regulon in S. aureus. It will be shown that the proteomic view of regulons, however, might be just the beginning of detailed follow-up studies aimed at the comprehensive description of regulon structure and the understanding of molecular mechanisms of signal reception/transduction necessary for activation of regulons. Furthermore, the use of environmental stimuli requires new protective proteins whose function in adaptation

Fig. 2. Fused proteome maps of B. subtilis exposed to heat, salt, hydrogen peroxide and paraquat stress (A) or starvation for ammonium, tryptophan, glucose and phosphate (B). The protein synthesis patterns (autoradiograms) of B. subtilis exposed to different stress (A) or starvation (B) conditions were combined to generate a fused stress or starvation proteome map, respectively. Induced marker proteins were color-coded according to their expression profile. Accordingly, proteins can be classified as specifically or generally induced stress or starvation proteins using the respective color codes.

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Fig. 3. Dual-channel image of wild type (green image) versus sigB mutant (red image) of B. subtilis. Cytoplasmic proteins of the wild type and sigB mutant were labeled with L-[35S]methionine separated by 2D gel electrophoresis. The resulting protein synthesis patterns (autoradiograms) were analyzed using DECODON Delta2D software. Several enzymes appearing in red were synthesized only in the sigB mutant (indicated by white circles).

networks needs to be analyzed. Finally, both regulons form superregulons, implying that both control subregulons which are downstream-integrated into complex adaptational gene expression networks. 2. The sB-modulon in the regulatory network of B. subtilis 2.1. Introduction To survive in their natural ecosystem, it was necessary to develop sensitive signal reception and transduction pathways for rapid and economic expression of an adaptive response finely tuned to fulfill the demands of the environmental stress stimulus without wasting the limited resources. Consequently, this thin line between losing the struggle to survive and being able to overcome starvation and physical stress conditions forced tight interconnection of cellular responses and developmental programs to form sophisticated regulatory networks that ensure flexibility and resilience in the face of environmental changes [6,46]. In this regulatory network, alternative sigma factor sB and its general stress regulon play a crucial role. For instance, the sB-dependent general stress regulon is tightly integrated into the heat stress stimulon that, in addition to the sB regulon, consists of a few specific heat stress regulons such as the HrcA-regulon and the CtsR-regulon controlling GroE/DnaK-machinery and Clp proteins respectively, as well as CssS/R-and the SigI-regulons, to mention only the most essential gene groups within this large stimulon. The sB-dependent general stress regulon of B. subtilis was chosen as one example to illustrate the tight interaction of

different regulons with gene expression networks of starved or stressed cells, on the one hand, and hierarchical regulation, on the other, with sB as a master regulator in a sB-controlled general stress modulon. In B. subtilis, the sB-mediated general stress response with its more than 150 known regulon members is one of the most essential and noticeable components of the adaptational gene expression network upon severe stress in exponentially growing cells as well as starving non-growing cells. The complex structure of this regulon was discovered almost 20 years ago by a proteome-based approach [24,66] showing the same set of proteins strongly induced by various stress/starvation stimuli (Fig. 3). A proteomic and molecular genetic view of this regulon marked the beginning of follow-up studies in the early nineties, ending up in the discovery of a highly sophisticated gene expression network in non-growing cells (reviewed by Price [54]). 2.2. Regulation of sB Characteristic features of the general stress response include the activation of sB caused by a large set of diverse stimuli and comprehensive stress resistance to the cell conferred by induced general stress proteins [16,22,29,54,67]. Remarkably, this resistance also covers such stimuli as alkali stress, that do not activate sB but could be considered prophylactic adaptation [16]. Both activation and inactivation of the alternative sigma factor sB are well-studied examples of tight regulation by a complex signal transduction cascade, including autoregulation and inhibitory feedback loops. Regulation of sB activity is subject to a partner switching

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mechanism in which interactions among the partners are driven by reversible serine and threonine phosphorylations. The central partner switching module consists of alternative sigma factor sB, its anti-sigma factor RsbW and the RsbW antagonist RsbV. Under optimal growth conditions, sB is captured in an inactive state by interaction with RsbW that sufficiently phosphorylates and inactivates its own antagonist RsbV and prevents sB from binding to the core RNA polymerase [22,54]. At least three main signalling pathways are able to trigger the activation of sB [9,27,65]: First, the stressosome, a multicomponent protein complex, [10,11,34] integrates environmental stress stimuli like salt, ethanol [7,66], acid [37], Mn2þ [17], sodium nitroprusside (SNP) [45] and bluelight stress [15], exposure to high and low temperature [4,21,70], as well as cell wall stress caused by addition of antibiotics such as bacitracin and vancomycin [44]. It is assumed that the stressosome oligomer has evolved to provide the potential for cooperativity in the signal integration process, enabling the response to be adjusted depending on the magnitude of the chemophysical impact [43]. Furthermore, the energy stress module is capable of sensing starvation of glucose, phosphate and oxygen [65,66], as well as treatment with azide, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mycophenolic acid that cause a drop in the cellular ATP level [72]. The third sB-activating pathway becomes active during continuous growth close to the minimal and maximal growth temperatures of B. subtilis [9,27]. Whereas the first two cascades act through environmental stress phosphatase RsbU [71] and energy stress phosphatase RsbP [64] upon the phosphorylation state of RsbV, activation of the third pathway is RsbV-independent and persistent, in contrast to the characteristic transient activation usually observed upon imposition of stress. Although the level of sB activation is finely tuned to match the strength of incoming stress signals, [43] this system deviates fundamentally from the widespread one- and two-component systems that usually convert a single signal into a single outcome, transcriptional regulation of small signal- or stress-specific regulons [46]. 2.3. Integration of sB into an adaptational network e the sB modulon Activation of sB is a good example of cooperativity and integration of multiple inputs for inducing a single orchestrated output: activation of the master regulator of global and a broad general stress response. In addition to induction of the general stress response, each of the above mentioned stimuli also results in enhanced transcription of stress- or starvationspecific genes, together representing the respective stimulon. Individual regulons within a modulon, whether specific or general, usually do not exist separately but exhibit overlapping properties with each other. Identification of sB-dependent genes that differ in their induction kinetics and/or amplitude from the typical sB-mediated induction pattern, or which show induction by stresses other than sB-activating stimuli, represent genes which are subject to complex control mechanisms.

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Based on global transcriptome and proteome studies, it was already possible to link many members of the general stress regulon to other known cellular responses and regulatory units. The simplest scenario of double control is sB-dependency combined with a second constitutively active sA promoter, as is the case for ctc, gtaB and sigB itself [3,8,32,33,59,62]. The genes opuE and opuD encoding proline and glycineebetaine uptake systems are induced from a sBand a second sA-dependent promoter that is, in addition, activated by osmotic stress [60,68]. sB-dependent regulation of clpP and the clpC operon is also subjected to control by sA and repressor CtsR, linking the general stress response to the CtsR regulon of the heat shock stimulon [12,38,39]. Amino acid starvation, which does not activate sB, also induces the general stress genes yvyD and ytxHIJ from a sH-dependent promoter [13,63]. Diamide stress, which also does not activate sB, induces the general stress genes trxA, sodA, gabD and yraA through activation of the global regulator Spx, that is most likely acting on sA-dependent promoters [48,49]. Finally, genes csbB and ydjB are jointly controlled by ECF sigma factors sW and sx [30,31] as well as sB [1,52]. These well known examples were chosen to emphasize the parallel network that is run by other global regulators whose activity is detached from the primary activity of sB or the respective sB promoters. Furthermore, data on the general stress regulon suggests additional scenarios of double or multiple controls that are more difficult to discover and characterize. These mechanisms are assumed to be directly and/or temporally coupled to the presence of a sB promoter and/or the activity of sB. The first example refers to genes like yabJ, yhaR and yqhZ. These candidate sB regulon members are preceded by ‘‘promoters‘‘ that are conserved in every position known to be crucial for recognition by sB, but remain uninducible by the known sB-activating stress stimuli heat, ethanol and salt ([53], J. Bandow and M. Hecker, unpublished data). The most obvious interpretation of this phenomenon would be the presence of operator elements within these promoter regions which are blocked by repressor molecules or need to be bound by a second activating factor. Nevertheless, this kind of sophisticated control is hard to detect because these promoters represent an interface where perception of two signals has to occur at the same time to achieve induction of the respective gene. One signal alone is not sufficient for promoter activation and will be erased in the signalling cascade. This scenario is very likely to occur, due to a large number of genes that are preceded by a well conserved but inactive sB-type promoter (unpublished data). Although this mode of double control still needs to be substantiated by experimental data, at least one well known example fulfills the criteria mentioned above. The clpC operon is induced from the sB- and the sA-dependent promoters in response to heat shock due to activation of sB and derepression by CtsR inactivation [38,39]. In contrast to heat shock conditions, the clpC operon is just slightly induced in glucose-starved cells [40] and ectopic expression of sB in a rsbW-minus background does not induce the clpC operon

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because the CtsR repressor prevents transcription at the sBdependent promoter even if sB is active under these circumstances [38,39]. Another observation was that each of the three independently performed transcriptional profiling studies [25,53,55] assigned about 120e190 genes to the sB regulon, but just a relatively small number (app. 25%) of these almost 300 general stress genes were identified by all three studies. Based on stringent genetic characterization, the affiliation of a gene with the regulon required the presence of a fairly conserved and responsive sB promoter. This resulted in the definition of a core sB regulon of about 150 genes and the identification of apparently sB-regulated genes that were not preceded by a sB-type promoter, for instance ykzC, ykyB or yutK [53]. Due to this observation and the fact that more than 20 of the general stress genes encode putative regulatory proteins, it was assumed that some gene expression cascades must be triggered by sB-dependent induction of global regulators that activate their corresponding regulons in a second step [25,53,55]. This assumption was confirmed by the recent characterization of the global regulator MgsR (YqgZ) (modulator of the general stress response) that established a sB-triggered global gene regulation cascade [56]. The MgsR protein represents a paralogue of the prototype of a new family of redox-sensitive transcriptional regulators classified as anti-a factor, the global regulator of the diamide stress response, Spx, originally discovered by Nakano and colleagues [48e50,56]. The novel regulatory function of Spx and MgsR is exerted by interaction with the C-terminal domains of the RNA polymerase a-subunits [49,51] rather than directly contacting the cis-active operator regions ([48,56], unpublished data). The expression of MgsR upon ethanol stress is exclusively dependent on sB leading to hierarchically and temporally ordered expression of a subregulon within the framework of the general stress response [56]. Some new insights could be deduced from this finding that are important for a more comprehensive understanding not only of sB ‘‘regulon’’ structure and function, but also of the architecture of the whole regulatory network: (i) Regulation by MgsR is an example of a signal processing and integration step in the regulatory cascade that is achieved by oxidation of the redox-sensitive CXXC-motif in MgsR which renders the protein active (unpublished data); this activation step leads to stress‘‘specific’’ fine tuning of target gene expression embedded in the ‘‘general’’ stress response; (ii) MgsR affects genes like gsiB, yhxD, ydbD and ydaD that are solely expressed by primary sB-dependent promoters; and (iii) genes that cannot be associated with sB-type promoters like yhxC, yhfQ and yjcF representing a direct connection to other regulatory systems due to sB-independent regulatory potential of MgsR ([56], unpublished data); furthermore, (iv) identification of a negatively regulated group of genes provides a first explanation for gene repression observed after imposition of stress in analogy to the repressor function of Spx [49,51,56]; finally, (v) the nitric oxide responsive regulator NsrR [58] is induced in a sBand MgsR-dependent manner [56]. NsrR is a transcriptional

repressor of the ResDE regulon and directly affects genes like nasD, hmp and fnr [47], thus potentially representing another level of sequential gene expression and network integration. Alongside the identification of MgsR, there are a few more promising candidates among the general stress proteins for observing additional sequential gene expression patterns. These may include known global regulators such as Spx or potential regulatory proteins like YdgC similar to the TetR/ AcrR family, YabT similar to serineethreonine protein kinases, YfkJ similar to proteinetyrosine phosphatases, and YdaK which is similar to the ActA response regulator of Myxococcus xanthus [25,53,55]. In this sense, sB is a master regulator which offers the opportunity for expression of secondary regulatory proteins and thereby connects many other regulons in the network, forming a higher-order sB ‘‘modulon’’. Perhaps it is a common feature of the general stress response that secondary stress-specific systems guarantee the necessary fine tuning and enable regulatory cross-talk under multiple stress conditions. Due to its central role in stress physiology, the level of complexity and the amount of data available on the sB response in B. subtilis, it should be applied to a system’s biology approach. It has been pointed out during the last decade that transcriptomics using DNA microarrays is a comprehensive and powerful tool for defining the structure of stimulons, modulons and regulons, and that proteomic approaches are indispensable for the visualization of both post-transcriptional regulation and post-translational modification, targeting and degradation processes [19]. Only the combination of data gained from all these experimental methods will enable us to model this important adaptational response and to modularize the complex network into functional smaller subnetworks. To define subsystems and to separate their function from other modules will make them more amenable to direct analysis and help us gain a holistic view on the underlying mechanisms. Finally, this methodology will enable us to visualize dynamic properties of the genetic network and to discover interconnections and components that have yet to be discovered (Fig. 4). 2.4. Function of sB-dependent general stress proteins One of the main functions of sB-dependent general stress proteins seems to be the development of multiple, nonspecific, and preventive stress resistance in cells no longer able to grow: multiple because the response includes resistance to many different stress stimuli, nonspecific because the growthinhibiting stimulus allows cells to cope with stresses to which they have not yet been exposed by triggering cross-protection against different stress conditions, and preventive because the still-active and vigorous cell from the growth phase to the non-growth phase transition might be equipped with protective proteins in the event of future stress (see [22,54] for review). Most of the 150 sB-dependent general stress genes code for proteins with still unknown functions. Nevertheless, their

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Fig. 4. Graphic representation of the SigB modulon by a combination of known modes of regulation of SigB-dependent general stress genes. The core SigB regulon (green) comprised of genes solely transcribed by SigB. General stress genes that are additionally expressed by a second constitutively active SigA promoter (brown). A subgroup controlled by SigB, SigA and the global repressor CtsR (black) or the two sigma factors A and B and the global regulator Spx (red). SigBdependent genes controlled by other alternative sigma factors like SigH (orange) or SigW and/or SigX (pale green). Genes were preceded by a well conserved SigB-type promoter, but failed to be induced by SigB-activating stimuli. These genes were very likely subject to thus far uncharacterized regulatory mechanisms and still need to be linked to the SigB modulon and the regulatory network (cyan). SigB-dependent expression of the global regulator MgsR (blue) and the jointly regulated group of general stress genes (light blue). SigB- and MgsR-dependent expression of nitric oxide responsive regulator NsrR may reflect a link to NsrR target genes and the ResDE regulon (dark red). MgsR regulated genes that could not be associated with SigB-type promoters, representing a direct connection to other regulatory systems due to the SigB-independent regulatory potential of MgsR (purple).

membership in the sB regulon facilitates an initial prediction of their function in the development of nonspecific, multiple, and preventive stress resistance. A comprehensive stresssensitivity screening program of 94 mutants in unknown but sB-dependent genes obtained from a collection of a EuropeaneJapanese consortium revealed a surprisingly high proportion of mutants impaired in single or multiple stress resistance, particularly in ethanol, cold shock resistance/ survival, or both. These data show the pivotal role of the sB response in stress adaptation. Even if these studies enable us to assign individual stress proteins to specific facets of multiple stress resistance, detailed physiological follow-up studies are required to understand the specific functions of the individual proteins within the single kind of stress protection. A first detailed study was performed on YerD, which seems to be involved in osmotic stress resistance [28,29]. McsB, the third gene of the clpC operon, which is at least under partial sB control, encodes for a protein kinase whose function in heat induction of clpC was analyzed. ctc in turn encodes a ribosome-associated protein that can bind to the 5S rRNA and might be required for accurate translation under stress conditions. In the future, the list of known sB-dependent proteins that are subject to detailed study will be extended, thus offering great potential for discovering new and interesting proteins with important functions in different facets of stress physiology and adaptation.

3. The anaerobic gene expression network of S. aureus e the Rex modulon 3.1. Introduction e a first proteomic view of the aerobic/anaerobic shift Shifting S. aureus cells from aerobic to anaerobic conditions results in remarkable reprogramming of the gene expression pattern leading to a switch in energy metabolism from respiration to fermentation. The first approach to this issue was again a proteomic view, this time of the wild type compared to a hemB mutant under aerobic conditions. This mutant was no longer able to form active respiration chains and thereby it is dependent on fermentation processes to gain energy for living. The proteome of this aerobically grown hemB mutant revealed a typical anaerobic signature with strong induction of pyruvate formate lyase and related fermentation enzymes, on the one hand, and repression of the pyruvate dehydrogenase complex and the TCA cycle on the other [36]. A more detailed proteomic and transcriptomic view of wild type cells shifting from aerobic to anaerobic conditions was presented by Fuchs et al. [14]. A dual channel imaging approach was used to compare the protein synthesis pattern before and after the shift by using a 35S-L-methionine pulse labelling approach. The proteomic view visualized reprogramming of the metabolism as expected, showing strong

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Fig. 5. Synthesis patterns of selected proteins representing different branches of the cellular metabolism of S. aureus under anaerobic conditions. Cytoplasmic proteins were labeled with L-[35S]methionine at different time points after shift to anaerobic conditions and separated by 2D gel electrophoresis. Protein synthesis was determined from autoradiograms. The resulting 2D gel images were analyzed using DECODON Delta2D software (t0 samples are in green, anaerobic samples and the 600 aerobic control are in red). Proteins newly synthesized after anaerobic shift appear in red. In contrast, proteins whose synthesis was repressed appear in green. Enzymes shown belong to glycolysis (A), fermentation (B), the PDH and TCA cycle (C), and miscellaneous (D). Bar graphs indicate relative synthesis rates (logarithm to the base 2) of individual proteins at different time points (individual scale for each protein). PfkA, 6-phosphofructokinase; TpiA, triosephosphate isomerase; GapA1, glyceraldehyde 3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase; PdhB/PdhC/PdhD, pyruvate dehydrogenase complex E1/E2/E3 component; AcnA, aconitate hydratase; SucA, 2-oxoglutarate dehydrogenase E1 component; SucC, succinyl-CoA synthase, beta subunit; Mqo1, malate:quinone oxidoreductase; Ldh1, L-lactate dehydrogenase; PflB, formate acetyltransferase; Adh1, alcohol dehydrogenase, zinc-containing (SACOL0660); Hmp, flavohemoprotein.

activation of glycolysis and fermentation, among others strong induction of pyruvate formate lyase, alcohol dehydrogenases and lactate dehydrogenase 1 on one hand, and repression of the TCA cycle on the other (Fig. 5). This proteomic approach also uncovered cellular reactions never seen before, such as strong induction of a member of the Clp machine, ClpL, but the function of this chaperone under anaerobic conditions is still a matter of debate. In addition to the proteomic view of basic carbon core metabolism, first insights into the regulation of this complex gene expression network were possible. Strong induction of SrrA, a response regulator of a two-component system, and strong downregulation of RsbW, the anti-sigma factor of sB, are examples of regulators that respond to oxygen shift. The proteomic approach was complemented by a metabolomic study revealing the main fermentation products in the extracellular space.

Using a combined metabolomic, proteomic, and transcriptomic approach (the latter revealed genes coding for proteins with still unknown function, but probably involved in fermentation, such as a nitrite/formate secretion protein (SACOL0301), a lactate proton symporter (SACOL2363) and a nitrite transport system (SACOL2386)), a metabolic and gene expression network of cells grown under anaerobic conditions was presented (Fig. 6). A more complex color coding approach that compares fermentation and nitrate respiration is shown in Fig. 7. The pattern of proteins whose synthesis was affected during adaptation to fermentation or nitrate respiration shares a clear overlap, including prominent fermentation enzymes such as pyruvate formate lyase, lactate dehydrogenase (Ldh1) and alcohol dehydrogenases, as well as the response regulator SrrA. Nevertheless, we found several protein spots whose synthesis was changed exclusively under

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Fig. 6. Scheme of the physiological changes of S. aureus under anaerobic conditions. The scheme summarizes major changes during adaptation to anaerobic conditions based on proteomic and transcriptomic data (Fuchs et al., 2007). Changes in fundamental cellular processes and metabolic pathways are shown as solid (induction) or dotted (repression) lines. Arrows indicate changes at the transcriptional level in the respiratory chain, transporters and virulence factors (up ¼ induction; down ¼ repression). GapR, gap transcriptional regulator; ClpL, ATP-dependent Clp protease, putative (SACOL2563); SrrA, DNA binding response regulator; SrrB, sensor histidine kinase; NirR, transcriptional regulator; CCR, c-catabolite repression.

Fig. 7. Fused proteome maps of S. aureus under anaerobic conditions in the presence or absence of nitrate. Protein synthesis patterns (autoradiograms) based on fermenting or nitrate respiring cells were combined to generate a fused proteome map. Significantly changed proteins were color-coded according to their expression profiles (see Fuchs et al., in prep.). Green or blue spots were only changed under fermentative or nitrate respiratory conditions, respectively. In contrast, orange spots represent a set of proteins whose expression was changed under both conditions.

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fermentation or nitrate respiration. For instance, synthesis of several proteins involved in DNA and RNA metabolism were repressed after oxygen limitation only if nitrate was absent (Fuchs et al., in prep.). 3.2. The Rex modulon structure and signal transduction pathways For the proteomic view of anaerobiosis of S. aureus, we followed the ‘‘road map’’ of physiological proteomics: after comprehensive analysis of the anaerobic stimulon (see Fig. 5), we analyzed regulons and global regulators involved in control of this anaerobic gene expression network. It turned out that the Rex protein is the main regulator that represses many genes of the anaerobic protein network in the presence of oxygen. A first proteomic view of a rex mutant compared to the wild type under aerobic conditions is presented in Fig. 8. In the presence of oxygen, only a small subset of proteins is clearly derepressed, including lactate dehydrogenase Ldh1 and response regulator SrrA (see Fig. 8). Under anaerobic conditions, however, derepression of anaerobic proteins was more pronounced (Fig. 9) indicating that some genes still need an anaerobic signal for full expression even if the Rex repressor has been removed. A Rex consensus sequence could be deduced from a set of verified Rex binding sites located at typical repressor motives in front of anaerobically induced genes. Transcription data confirmed the proteomic results just mentioned: only a subgroup became clearly

active under aerobic conditions in the rex mutant (Pagels et al., in prep.). The first question raised concerns the mechanism of Rex derepression under anaerobic conditions. The earliest cellular symbol for a shift from aerobic to anaerobic conditions might be a remarkable drop in the intracellular NAD/NADH ratio. This simple ‘‘signal transduction system’’ really works in the most plausible way: while NAD enhances the repressor’s activity, NADH competes with NAD and inactivates Rex that is no longer able to bind to its target region and to repress the genes involved. The second question is: Why do all Rexregulated genes not behave in the same way? Obviously, there is fine adjustment in expression of many anaerobically induced genes that need, in addition to inactivation of Rex, a second regulatory protein that activates their transcription under anaerobic conditions. This regulatory protein is required to activate a class of genes which are characterized by promoters probably too weak to be already active after removal of Rex. A second group of genes reaches maximal transcription in the rex mutant even under aerobic conditions. This group comprises what is necessary for anaerobic lactate fermentation, i.e. ddh (D-lactate dehydrogenase) and ldh1 (L-lactate dehydrogenase). These genes appear to be tightly controlled by Rex and are characterized by one or even two high-affinity Rex binding sites located at the transcription start site or at the promoter region. Obviously, the Rex signal transduction system is the only mechanism that controls and prevents expression of these genes under aerobic conditions. It turned

Fig. 8. Dual-channel image of a rex mutant (red) versus wild type (green) of S. aureus under aerobic conditions. Cytoplasmic proteins were labeled with 35 L-[ S]methionine at OD500 0.5 and separated by 2D gel electrophoresis. Protein synthesis was determined from autoradiograms. The resulting 2D gel images were analyzed using DECODON Delta2D software. Proteins derepressed in the rex mutant appear in red. Ldh1, L-lactate dehydrogenase; SrrA, DNA binding response regulator; PflB, formate acetyltransferase; AdhE, alcohol dehydrogenase, iron-containing (SACOL0135); Adh1, alcohol dehydrogenase, zinc-containing (SACOL0660).

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Fig. 9. Global and detailed view of protein synthesis in the rex mutant (red) and wild type (green) of S. aureus under anaerobic conditions. Cytoplasmic proteins were labeled with L-[35S]methionine 30 min after anaerobic shift and separated by 2D gel electrophoresis. Protein synthesis was determined from autoradiograms. The resulting 2D gel images were analyzed using DECODON Delta2D software. Proteins derepressed in the rex mutant appear in red. The detailed view of selected members of the Rex regulon (B and C) includes comparisons between the rex mutant (6) and wild type (wt) under aerobic ([þO2]) and anaerobic conditions ([O2]). The color code of the proteins is displayed in the heading. Proteins could be divided into two classes based on their expression profiles: proteins whose expression was exclusively controlled by Rex (Class I, B), and proteins subject to additional regulation (Class II, C). Ldh1, L-lactate dehydrogenase; SrrA, DNA binding response regulator; PflB, formate acetyltransferase; AdhE, alcohol dehydrogenase, iron-containing (SACOL0135); Adh1, alcohol dehydrogenase, zinc-containing (SACOL0660).

out that products of these genes could be required for the cell even under aerobic conditions. For instance, induction of the lactate dehydrogenase Ldh1 might be indispensable to life even when oxygen is available but the respiratory chain has been damaged by nitric oxide [26,57]. Other gene products which are not only under Rex control probably provide more specific responses to the lack of oxygen. For instance, oxygen-sensitive pyruvate formate lyase introducing mixed acid fermentation is subjected to further regulation which allows expression only under conditions in which oxygen cannot be used as terminal electron acceptor. Several additional gene regulators that need activation by an anaerobic stimulus are known, such as ArcR for the arginine deiminase pathway, the response regulator NreC for nitrate/ nitrite respiration, NirR for nitrite reductase and the response regulator SrrA for some fermentation pathways. It is interesting to note that NirR and SrrAB themselves are under negative Rex control, implying stepwise activation of the genes under double control. It has been suggested that the anaerobic signal that activates the response regulator SrrA

might be generated by a high reduction state of menaquinones (Kohler et al., 2008). All in all, this regulatory network can be described as a signal transduction/gene expression system with Rex as the main regulator (Fig. 10). The deactivation of Rex induced by a NADH increase in the absence of oxygen, however, is e at least for most of the genes e necessary but not yet sufficient for gene expression. Finally, at least in the case of the arginine deiminase pathway, a link to carbon control exists because this pathway is likely repressed by the CcpA protein in the presence of glucose, the main global regulator of glucose control [41]. Fig. 10 summarizes current data available thus far on the regulatory network and the central role of Rex in the Rex modulon. 3.3. The genes involved and their function The Rex regulon defined by electrophoretic mobility shift assays, northern analysis and proteomics covers gene products involved in anaerobic metabolism, transport processes and regulation. On the metabolic side, Rex-controlled genes code

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Fig. 10. Scheme of the Rex superregulon in S. aureus. This scheme summarizes the regulatory cascade controlled by the Rex repressor. Aerobic respiration by the respiratory chain heavily influences the intracellular NAD/NADH ratio. A drop in that ratio leads to inactivation of Rex and consequently to derepression of ldh1 and ddh, resulting in maximal transcription of these genes. Lactate fermentation is a one-step pathway leading to NAD regeneration, probably as the immediate response. However, the Rex superregulon also contains several subregulons comprising mixed acid and ethanol fermentation or nitrite/nitrate respiration. Rex controls expression of these genes directly and/or indirectly via other regulators. This complex network enables incorporation of several signals into the regulatory cascade, leading to finely adjusted adaptation. Colors of enzymes and transporters are adapted to regulators controlling their expression. MQ, menaquinone; HE, hemin cofactor; ox, oxidized form, red, reduced form; SrrA, DNA binding response regulator; SrrB, sensor histidine kinase; ArcR, transcriptional regulator, Crp/ Fnr family (SACOL2653); Rex, redox-sensing transcriptional regulator (SACOL2035); NreABC, control of nitrate reduction (SACOL2389eSACOL2391); PflB, formate acetyltransferase; Adh1, alcohol dehydrogenase, zinc-containing (SACOL0660); AdhE, alcohol dehydrogenase, iron-containing (SACOL0135); NirC, formate/nitrite transporter family protein (SACOL0301) NirB/NirD, nitrite reductase large/small subunit; NarG/NarH/NarI, respiratory nitrate reductase alpha/ beta/gamma subunit; ArcA, arginine deiminase; Ldh1, L-lactate dehydrogenase; Ddh, D-lactate dehydrogenase; LctP2, L-lactate permease (SACOL2363); Ald1, alanine dehydrogenase.

for L- and D-lactate dehydrogenase, pyruvate formate lyase and alcohol dehydrogenases SACOL0660 and SACOL0135, all involved in NAD regeneration under anaerobic conditions. Also, the alanine dehydrogenase Ald1, which is strongly induced after oxygen limitation, is under Rex control. Its function under anaerobic conditions is still unclear. Rex binding could also be confirmed for the upstream regions of genes coding for the nitrate/nitrite respiration system and two proteins very similar to lactate and nitrite/formate transporters (SACOL0301 and SACOL2363). On the regulatory side, we found genes coding for SrrA and NirR as part of the Rex regulon which also have an impact on gene expression and are probably involved in fine tuning and adjusting gene expression. 4. Outlook A panoramic view of proteomics offers the opportunity of visualizing cellular events never before witnessed. This

proteomic view, however, should not end up on endless lists with proteins, but should promote new ideas and hypotheses stimulated by the view of cells in a new and wider context. This route, from proteomic data to a new hypothesis, was on our minds when we started to analyze the response of B. subtilis cells to stress and starvation more than 20 years ago. The visual inspection of the protein induction pattern shown by gel-based proteomics revealed a new, comprehensive and probably crucial stress response which, among other approaches, initiated systematic studies on the structure, regulation and function of a complicated gene expression network with sB as the main regulator. These proteomics efforts were substantially complemented by studies on the molecular genetics of sigB and its signal transduction pathways [18,54], ending up in a comprehensive regulation network described in this review article. This route from proteomics to cell physiology has been extended in our group to related physiological issues focusing on protein stress and

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the CtsR regulon [35], the thiol-stress modulon [2] and the anaerobic gene expression network in S. aureus chosen for this review article (Pagels et al., in prep.). This systematic application and integration of all ‘‘-omics’’ technologies to physiological responses will not only provide new information on crucial chapters of microbial physiology, but will also pave the way to a more quantitative description and modelling of the stress/starvation responses via a systems biology perspective, an attractive challenge for future research. Acknowledgements We thank all colleagues, PhD students and cooperating partners who were involved in this work. We are very grateful to Richard A. Proctor for providing the rex mutant. This work was supported by grants from the BMBF (031U107A/-207A; 031U213B and within the framework of the SYSMO Program by grant 0313978A), the DFG (1887/7-4; GK212/3-00; SFB/ TRR34), the EU (Bacell Health Program LSHG-CT-2004503468; Staphdynamics) and the Land MV. References [1] Akbar, S., Price, C.W. (1996) Isolation and characterization of csbB, a gene controlled by Bacillus subtilis general stress transcription factor sigma B. Gene 177, 123e128. [2] Antelmann, H., Hecker, M., Zuber, P. (2008) Proteomic signatures uncover thiol-specific electrophile resistance mechanisms in Bacillus subtilis. Expert Rev. Proteomics 5, 77e90. [3] Benson, A.K., Haldenwang, W.G. (1992) Characterization of a regulatory network that controls sigma B expression in Bacillus subtilis. J. Bacteriol. 174, 749e757. [4] Benson, A.K., Haldenwang, W.G. (1993) The sigma B-dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock. J. Bacteriol. 175, 1929e1935. [5] Bernhardt, J., Bu¨ttner, K., Scharf, C., Hecker, M. (1999) Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis. Electrophoresis 20, 2225e2240. [6] Birkey, S.M., Liu, W., Zhang, X., Duggan, M.F., Hulett, F.M. (1998) Pho signal transduction network reveals direct transcriptional regulation of one two-component system by another two-component regulator: Bacillus subtilis PhoP directly regulates production of ResD. Mol. Microbiol. 30, 943e953. [7] Boylan, S.A., Redfield, A.R., Brody, M.S., Price, C.W. (1993) Stressinduced activation of the sigma B transcription factor of Bacillus subtilis. J. Bacteriol. 175, 7931e7937. [8] Boylan, S.A., Thomas, M.D., Price, C.W. (1991) Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor sigma B of Bacillus subtilis. J. Bacteriol. 173, 7856e7866. [9] Brigulla, M., Hoffmann, T., Krisp, A., Vo¨lker, A., Bremer, E., Vo¨lker, U. (2003) Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation. J. Bacteriol. 185, 4305e4314. [10] Chen, C.C., Lewis, R.J., Harris, R., Yudkin, M.D., Delumeau, O. (2003) A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis. Mol. Microbiol. 49, 1657e1669. [11] Delumeau, O., Chen, C.C., Murray, J.W., Yudkin, M.D., Lewis, R.J. (2006) High-molecular-weight complexes of RsbR and paralogues in the environmental signaling pathway of Bacillus subtilis. J. Bacteriol. 188, 7885e7892.

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