A proteomic view of cell physiology of the industrial workhorse Bacillus licheniformis

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A proteomic view of cell physiology of the industrial workhorse Bacillus licheniformis Birgit Voigt a,∗ , Rebecca Schroeter b , Thomas Schweder b , Britta Jürgen b , Dirk Albrecht a , Jan Maarten van Dijl c , Karl-Heinz Maurer d , Michael Hecker a a

Institute of Microbiology, Ernst-Moritz-Arndt-University, F.-L.-Jahn-Str. 15, Greifswald, Germany Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst-Moritz-Arndt-University, F.-Hausdorff-Str. 3, Greifswald, Germany c Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, HPC EB80, PO Box 30001, Groningen, The Netherlands d AB Enzymes GmbH, Feldbergstr. 78, Darmstadt, Germany b

a r t i c l e

i n f o

Article history: Received 27 March 2014 Received in revised form 26 May 2014 Accepted 3 June 2014 Available online xxx Keywords: Bacillus licheniformis Starvation Stress Marker proteins Marker genes

a b s t r a c t Bacillus licheniformis is known for its high protein secretion capacity and is being applied extensively as a host for the industrial production of enzymes such as proteases and amylases. In its natural environment as well as in fermentation processes the bacterium is often facing adverse conditions such as oxidative or osmotic stress or starvation for nutrients. During the last years detailed proteome and transcriptome analyses have been performed to study the adaptation of B. licheniformis cells to various stresses (heat, ethanol, oxidative or salt stress) and starvation conditions (glucose, nitrogen or phosphate starvation). A common feature of the response to all tested conditions is the downregulation of many genes encoding house-keeping proteins and, consequently, a reduced synthesis of the corresponding proteins. Induction of the general stress response (␴B regulon) is only observed in cells subjected to heat, ethanol or salt stress. This paper summarizes our current knowledge on general and specific stress and starvation responses of this important industrial bacterium. The importance of selected marker genes and proteins for the monitoring and optimization of B. licheniformis based fermentation processes is discussed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Bacillus licheniformis, a Gram-positive bacterium closely related to the Bacillus paradigm Bacillus subtilis, is commonly found in soil and other natural habitats. Like most members of the genus Bacillus, B. licheniformis is known for the efficient secretion of numerous hydrolytic enzymes directly into the environment. This enables the cells to degrade and grow on a variety of different substrates. Since in natural environments cells often encounter unfavorable conditions, such as low or high temperatures, oxidative stress or changes in osmotic pressure provoked by changes in the concentration of salts and other compounds, cells have evolved complex regulatory networks to react to such conditions. When grown in an abundance

∗ Corresponding author. Tel.: +49 3834 864237; fax: +49 3834 864202. E-mail addresses: [email protected] (B. Voigt), [email protected] (R. Schroeter), [email protected] (T. Schweder), [email protected] (B. Jürgen), [email protected] (D. Albrecht), [email protected] (J.M.v. Dijl), [email protected] (K.-H. Maurer), [email protected] (M. Hecker).

of nutrients, most bacteria first use their preferred nutrient source, which allows for the highest growth rate. However, in natural environments abundance of nutrients is scarce. Bacteria have therefore developed strategies to deal with such nutrient starvation conditions (Antelmann et al., 2000; Stülke and Hillen, 2000; Tam le et al., 2007). In this respect it is noteworthy that Bacillus species, such as B. subtilis and B. licheniformis, have evolved highly successful mechanisms to overcome environmental stresses and insults, which is probably the reason why they are highly robust both in nature and industrial fermentations (van Dijl and Hecker, 2013; Westers et al., 2004; Wiegand et al., 2013b). The capability of secreting large amounts of proteins and the fact that B. licheniformis is regarded as safe, have made this bacterium attractive for biotechnological applications. Accordingly, B. licheniformis has been used for decades as a host for industrial fermentation processes, for example to produce antibiotics and enzymes like proteases and amylases (Schallmey et al., 2004). In such large scale fermentation processes B. licheniformis is exposed to different stress situations which could hamper its productivity. It has been demonstrated that bacteria are able to recognize and react to substrate and pH gradients in industrial bioreactors in a time

http://dx.doi.org/10.1016/j.jbiotec.2014.06.004 0168-1656/© 2014 Elsevier B.V. All rights reserved.

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range of several seconds by inducing specific and general stress genes (Schweder et al., 1999). It is assumed that the metabolic burden caused by these stress responses may provoke negative effects on the production process (Schweder, 2011). The forced production of large amounts of heterologous proteins can lead to misfolding and degradation of target proteins which consequently negatively influence the yield of active enzymes (Buescher et al., 2012; Gasser et al., 2008; Nicolas et al., 2012; Sarvas et al., 2004; Wiegand et al., 2013b). The study of the responses of the host cells to different stress and starvation conditions is therefore mandatory to evaluate fermentation processes, and to develop more efficient protein production processes with microbial hosts. The sequencing of the genome of B. licheniformis was a prerequisite for the proteome analysis based on mass spectrometric protein identification, and it furthermore enabled the development of genomic DNA arrays for transcriptome studies (Rey et al., 2004; Veith et al., 2004). Master gels of B. licheniformis cells growing in defined or complex media have been established (Voigt et al., 2004). Such master gels included up to 300 identified protein spots. Using these proteome data, physiological studies shedding light on the cell‘s responses to nutrient starvation conditions (glucose, nitrogen or phosphate starvation) and to different stress conditions (heat, ethanol, salt or oxidative stress) were performed (Hoi le et al., 2006; Schroeter et al., 2011, 2013; Voigt et al., 2006, 2007, 2009, 2013). To gain more comprehensive insights into the cell physiology, proteomic analyses were combined with analyses of the transcriptome. Thereby, we explored the early responses to physical stress and nutrient starvation at two levels: first at the mRNA level by genomic DNA microarrays and secondly at the level of newly synthesized proteins. In this study we mainly used two-dimensional (2D) gel-based proteomics although liquid chromatography combined with tandem mass spectrometry (LC–MS/MS) would allow the identification of larger numbers of proteins and could potentially allow a higher coverage of the proteome. However, to study newly synthesized proteins the 2D gel-based analysis of [35 S]methionine labeled proteins is still the best method available. In this review, we present a proteomic overview of the cell physiology of B. licheniformis and, where relevant, we compare the behavior of this industrial workhorse to that of B. subtilis. Importantly, we use in silico-generated “fusion gels” that combine the 2D gel images of proteins collected during different stress or starvation conditions. In doing so, we have been able to visualize and identify general and specific marker proteins for different stress and starvation conditions. This library of marker proteins can now be applied as a facile tool to monitor and manage industrial fermentation processes.

as specific stress and starvation proteins which are synthesized only in response to a defined stress or starvation stimulus. These stress/starvation-specific proteins directly deal with a single stimulus. Starvation-specific proteins may help to take up the limiting substrate with high affinity, to search for new, alternate substrates or to replace the limiting substrate by other substrates. Stressspecific proteins may neutralize the factor provoking the stress, may help to adapt to the stress or repair damage caused by the stress. General stress proteins, on the other hand, induced by a different set of stress/starvation stimuli, are not directed against a specific stimulus, but may provide a general protection to the nongrowing cell, no matter which stimulus induced the non-growing state. A critical element in the general stress/starvation response is the sigma factor B (␴B ) regulon. The ␴B -dependent genes are induced under many different conditions and encode numerous general stress proteins that confer multiple, non-specific and preventive resistances to cells (Hecker et al., 2007).

2. General stress and starvation responses

3.2. Specific responses to glucose starvation

In bacterial cells two main groups of stress-responsive proteins can be distinguished. The first group is formed by house-keeping proteins performing house-keeping functions during growth. Proteins involved in transcription and translation, in central carbon metabolic pathways and in amino acid and nucleotide biosynthesis belong into this group. These proteins are no longer synthesized when cells enter the non-growing state induced by stress or starvation. In fact, many of these proteins will be degraded when growth is arrested by unfavorable conditions as has been shown for B. subtilis and Staphylococcus aureus (Gerth et al., 2008; Michalik et al., 2009). The second group comprises proteins upregulated under stress and starvation conditions. Proteins of this second group are often synthesized not at all, or only in low amounts in exponentially growing cells. Among the proteins belonging to the second group are general stress and starvation proteins, which are synthesized in response to different environmental stimuli, as well

When B. licheniformis cells are growing exponentially on glucose, most glycolytic enzymes accumulate to higher levels than observed for cells growing on non-glycolytic carbon sources (Voigt et al., 2004). On the other hand, most TCA cycle enzymes and the ATP synthase are strongly repressed during glucose excess when glutamate is added to the medium. This suggests that, during growth on high amounts of glucose, ATP synthesis via substrate phosphorylation is almost sufficient for growth. Part of the glycolytic intermediates is secreted by an overflow metabolism. Thanh et al. (2010) found that mainly acetate and small amounts of acetoin and 2,3-butanediol were released into the medium by B. licheniformis cells growing on excess glucose. The observed glucose-dependent accumulation of enzymes for acetate synthesis is consistent with these findings (Voigt et al., 2004). When glucose becomes limiting, most glycolytic enzymes and proteins involved in overflow metabolism are downregulated

3. Response of B. licheniformis cells to nutrient starvation 3.1. General and specific starvation responses The first group of proteins regulated in response to starvation comprises house-keeping proteins, the synthesis of which is diminished in response to starvation. In starved cells many of the house-keeping proteins are still present and may be active as indicated by the fact that they were visible on stained protein gels for at least one hour after transition phase (Voigt et al., 2007). Proteins whose synthesis is upregulated in response to starvation can be sub-classified into general starvation proteins, which are in fact upregulated in response to different starvation conditions (see Section 3.5), and specific starvation proteins which are upregulated only in response to a single condition. In contrast to B. subtilis, the ␴B regulon was not induced during glucose or phosphate starvation of B. licheniformis (Hoi le et al., 2006; Voigt et al., 2007). This is probably due to the fact that the rsbQP operon, responsible for the ␴B induction upon nutrient limitation (Völker et al., 1994), is missing from the genome of B. licheniformis. A further general starvation response is sporulation. However, in B. licheniformis a significant induction of sporulation was only detected in phosphate-starved cells. The cytoplasmic serine protease IspA, which could be involved in the regulation of sporulation under phosphate starvation conditions (Chen et al., 2003) was displaying the highest level of upregulation in response to phosphate starvation. This and other specific starvation responses of B. licheniformis are summarized and discussed in the following sections.

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(Voigt et al., 2007). In contrast, synthesis of TCA cycle enzymes and of some proteins of the electron transport chain is enhanced. Furthermore, gluconeogenesis is induced, as indicated by an elevated synthesis of the phosphoenolpyruvate carboxykinase (PckA). Induction of the glyceraldehyde-3-phosphate dehydrogenase (GapB), catalyzing an irreversible step during gluconeogenesis has been observed at the transcriptional level (Voigt et al., 2007). Regulation of glycolysis, TCA cycle and gluconeogenesis in B. licheniformis is probably similar to B. subtilis since most of the involved regulators, such as CcpA, CggR, CcpC and CcpN, are encoded by the B. licheniformis genome (Blencke et al., 2003; Jourlin-Castelli et al., 2000; Ludwig et al., 2001; Moreno et al., 2001; Rey et al., 2004; Servant et al., 2005; Veith et al., 2004). After exhaustion of glucose the overflow metabolites released into the medium during exponential growth are rapidly consumed (Thanh et al., 2010). This can also be inferred from the upregulation of proteins involved in the usage of acetate and acetoin (Voigt et al., 2007). Involvement of the acoABCL operon in acetoin usage has been demonstrated by Thanh et al. (2010). An acoB mutant is no longer able to use acetoin and, consequently, the acetoin concentration in the medium remains high. In B. subtilis the acoABCL operon is ␴L -dependent and its transcription is induced by AcoR (Ali et al., 2001). AcoR and SigL expression are negatively controlled by CcpA (Ali et al., 2001; Choi and Saier, 2005). A ␴L -promoter has been found upstream of the B. licheniformis acoA gene (Veith et al., 2004) and the acoR gene is induced during glucose starvation (Voigt et al., 2007) pointing to a similar regulation of the acoABCL operon in this organism. B. licheniformis possesses genes for the glyoxylate cycle (aceBA operon) (Veith et al., 2004). The glyoxylate cycle enables the usage of C-2 substrates, like the overflow metabolites acetate, acetoin and 2,3-butanediol, as sole carbon sources. During glucose starvation the glyoxylate cycle is induced, as indicated by the elevated expression of the isocitrate lyase and the malate synthase genes (Voigt et al., 2007). At the proteome level only the isocitrate lyase has been found at elevated levels. Interestingly, a B. subtilis strain transformed with the glyoxylate cycle genes from B. licheniformis is able to use C-2 compounds, such as acetate and acetoin, as carbon sources demonstrating that the glyoxylate cycle is sufficient for the usage of such compounds (Kabisch et al., 2013). Kabisch et al. (2013) could demonstrate that for the function of the glyoxylate cycle in B. subtilis it is necessary to concurrently transfer a small hypothetical gene encoded in front of the aceBA operon. This gene encodes a small protein containing conserved cysteines in two CXXC motifs (Pfaffenhäuser, 2012). A number of intracellular proteins involved in the metabolism of alternative carbon sources are synthesized at elevated levels in glucose-starved B. licheniformis cells (e.g. alpha-glucosidase, MalL and 6-phospho-beta-glucosidase, LicH) (Voigt et al., 2007). Additional genes encoding such proteins were found to be induced, but the corresponding proteins were not identified in the 2D gels of intracellular proteins. Also, some transport-related proteins, very likely required for the uptake of alternative carbon sources, are upregulated, including some ABC transporter components and a fructose specific PTS enzyme (Voigt et al., 2007). In B. subtilis genes involved in the utilization of alternative carbon sources are often regulated by carbon catabolite repression, i.e. they are repressed by binding of CcpA to cre boxes in the promoter regions of these genes as long as glucose is present (Blencke et al., 2003; Galinier et al., 1999; Moreno et al., 2001). A similar mode of regulation in B. licheniformis is indicated by the detection of potential cre boxes in the promoter regions of many genes encoding enzymes for alternative carbon source usage (Voigt et al., 2007). Catabolite repression of an amylase gene of B. licheniformis has been also demonstrated (Laoide et al., 1989). Although numerous extracellular carbohydrate-hydrolyzing enzymes are encoded in the B. licheniformis genome, only few such genes are actually

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upregulated and only few of the corresponding enzymes were secreted by glucose-starved cells (Voigt et al., 2006, 2007). The enhanced synthesis of proteins involved in catabolism of amino acids and lipids after exhaustion of glucose suggests that B. licheniformis is able to use non-carbohydrate organic substances as carbon source. Specifically, a significant upregulation of several cytoplasmic and secreted peptidases and proteases has been observed upon glucose starvation (Voigt et al., 2006, 2007). Repression of an extracellular protease of B. licheniformis by glucose, suggesting a carbon catabolite repression mechanism, has been described by Laishley and Bernlohr (1968). Furthermore, proteins involved in the degradation of various amino acids, such as arginine, proline, glutamine and threonine are upregulated in glucose-starved B. licheniformis cells. According to Laishley and Bernlohr (1968) arginine degradation in B. licheniformis is under carbon catabolite repression since it was repressed by glucose. Arginine degradation is also under CcpA control in B. subtilis (Belitsky et al., 2004). The synthesis of several enzymes for fatty acid degradation, generating C-2 and C-3 compounds, is also enhanced in glucose-starved B. licheniformis cells (Voigt et al., 2007). This induction of lipid degradation has also been reported for glucose-starved B. subtilis cells (Koburger et al., 2005). In B. licheniformis the C-2 compounds can be used via the glyoxylate pathway while the C-3 compounds, such as propionyl-CoA, can be fed into the TCA cycle via the methylcitrate pathway. In this pathway, propionyl-CoA is condensed with oxaloacetate to 2-methylcitrate. The 2-methylcitrate is converted via several reactions to succinate. Synthesis of enzymes involved in this pathway was shown to be induced in B. licheniformis as well as in B. subtilis (Koburger et al., 2005; Voigt et al., 2007). 3.3. Specific responses to nitrogen starvation It has been demonstrated that B. licheniformis can grow on different nitrogen sources thereby reaching different growth rates (Bernlohr et al., 1988; Golden and Bernlohr, 1985). As for B. subtilis, glutamine and ammonium are preferred nitrogen sources for B. licheniformis (Fisher, 1999). The global nitrogen metabolism regulator in B. subtilis is TnrA (Wray et al., 1996). In B. licheniformis transcription of the tnrA gene is strongly induced during nitrogen starvation suggesting a similar role of the corresponding protein under these conditions (Voigt et al., 2007). Synthesis of some other proteins with regulatory functions was found to be altered during nitrogen starvation, e.g. synthesis of the regulator CodY is downregulated. In B. subtilis CodY controls expression of several nitrogen metabolism genes depending on the energy status of the cells and the availability of branched chain amino acids (Shivers and Sonenshein, 2004). In contrast, synthesis of YvyD, a protein with similarity to sigma-54 modulating factors from Gram-negative bacteria, was upregulated. Nitrogen starvation provokes induction of genes encoding ammonium (nrgA, BLi01175) and nitrate transporters (nasA), as well as genes encoding glutamine ABC transporter components (Voigt et al., 2007). Furthermore, upregulated synthesis of the glutamine synthetase and the glutamate synthase has been observed in nitrogen-starved cells (Voigt et al., 2007). Both enzymes are responsible for ammonium assimilation. Induction of these enzymes during nitrogen starvation has been also observed in other bacteria (Amon et al., 2008; Tam le et al., 2007). The glutamine synthetase is encoded in an operon together with its own transcriptional repressor GlnR, which revealed a similar upregulation under nitrogen starvation conditions in B. licheniformis (Voigt et al., 2007). Supposedly, the B. licheniformis glnRA operon is repressed by GlnR as long as ammonium is present in the medium as is the case in B. subtilis (Brown and Sonenshein, 1996).

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Synthesis of most enzymes catalyzing amino acid anabolic reactions is downregulated in nitrogen-starved cells (Voigt et al., 2007). Repression at the transcription level has been observed mainly for genes involved in arginine, isoleucine and leucine biosynthesis. On the other hand, genes for histidine and tryptophan biosynthesis were shown to be transcribed at an elevated level. Consistent with these findings, the synthesis of the corresponding proteins, such as HisB and HisH and TrpA, B and D, is elevated (Voigt et al., 2007). Furthermore, different intracellular peptidases and proteases are detectable at elevated levels. A weak increase in the level of the intracellular ATP-dependent protease ClpP and the ATPase subunit ClpC suggests the occurrence of a protein stress probably induced by truncated polypeptides. The induction of proteases is very likely aimed at the mobilization of alternative nitrogen sources, for example from proteins that are no longer needed in starved cells. Consistent with this view, also an elevated secretion of extracellular proteases and peptidases has been observed (Voigt et al., 2006, 2007). At the same time, several peptide and amino acid transporters are upregulated presumably for increased uptake of peptides and amino acids from the growth medium. Induction of other processes aimed at the mobilization of alternative nitrogen sources has furthermore been observed at the transcriptional level (Voigt et al., 2007). For example, the nasA-F operon, encoding the assimilatory nitrate and nitrite reductase and a nitrate transporter are upregulated under these conditions. 3.4. Specific responses to phosphate starvation The main response of phosphate-starved B. subtilis cells is the induction of the PhoPR regulon, (Allenby et al., 2005; Antelmann et al., 2000; Hulett, 2002; Pragai and Harwood, 2002). The PhoPR regulon is controlled by the sensor kinase PhoR and the transcriptional regulator PhoP. Expression of PhoPR regulon members in B. subtilis serves to overcome the effects of phosphate limitation. Both genes encoding the two-component system PhoPR are present in the B. licheniformis genome and are induced under phosphate starvation conditions (Hoi le et al., 2006). The transcriptional activator PhoP was also observed to be transiently upregulated in the intracellular proteome of phosphate-starved B. licheniformis cells. To overcome the phosphate limitation, genes encoding components of the high affinity phosphate transporter are strongly induced in phosphate-starved B. licheniformis cells (Hoi le et al., 2006; Voigt et al., 2006). It is interesting to note that the membrane bound lipoprotein PstS accumulates in the exoproteome of phosphate-starved cells. In B. subtilis, several PhoPR-controlled proteins are secreted in order to mobilize alternative phosphate sources (Allenby et al., 2005; Antelmann et al., 2000). A similar response was reported for phosphate-starved B. licheniformis cells. For example, the phosphatases PhoB and PhoD were found to be secreted into the growth medium at a higher level when phosphate becomes limiting (Hoi le et al., 2006; Voigt et al., 2006). However, although there is a relatively strong induction of the phosphatases at the transcriptional level under phosphate starvation conditions, the corresponding proteins represent only a minor fraction of the exoproteome. Notably, studies on the secretion of alkaline phosphatases by B. licheniformis cells grown in phosphate-limited media were initiated more than 30 years ago (Chesbro and Lampen, 1968). Subsequently, the characteristics and localization of the phosphatases were studied (Hulett et al., 1976; Hulett et al., 1986). Phosphateconcentration dependent expression of a B. licheniformis alkaline phosphatase in B. subtilis was shown by Lee et al. (1991). Kumar et al. (1983) presented first evidence for the dependence of the expression of an alkaline phosphatase of B. licheniformis on the

PhoP-PhoR two-component system since a mutant defective in the corresponding chromosomal region was shown to be deficient in the secretion of this phosphatase. Further enzymes needed for the acquisition of phosphate from alternative sources, such as a glycerophosphoryl diester phosphodiesterase and proteins involved in the degradation of nucleic acids, are secreted in high amounts upon phosphate starvation (Hoi le et al., 2006; Voigt et al., 2006). Induction of nucleic acid degrading enzymes during phosphate starvation was described for other organisms too (Ishige et al., 2003; Znamenskaya et al., 1995). The dominant protein in the extracellular phosphate starvation proteome of B. licheniformis is phytase (Hoi le et al., 2006; Voigt et al., 2006). A phytase is also encoded by the B. subtilis genome, but this protein was not detected in the exoproteome of phosphate-starved cells (Antelmann et al., 2000). Phytate is the main storage form of phosphate in many plants, and the strong secretion of phytase upon phosphate starvation indicates that phytate serves as a major alternative phosphate source for B. licheniformis. At the transcriptional level the tua operon encoding proteins that allow an economic use of phosphate by replacing the phosphate-containing cell wall teichoic acids by phosphate-free teichuronic acids is induced in phosphate-starved B. licheniformis cells (Hoi le et al., 2006). At the same time mRNA levels of the tag operons for the synthesis of teichoic acids are decreased. The tag as well as the tua genes belong to the PhoPR regulon in B. subtilis (Liu and Hulett, 1998). To define the putative PhoPR regulon and to identify potential new regulon members in B. licheniformis a bioinformatics analysis for a genome-wide prediction of PhoP binding sites has been performed using the conserved PhoP-binding sequence of B. subtilis (Hoi le et al., 2006). Potential PhoP-binding sites were detected in the promoter regions of a number of genes induced during phosphate starvation in B. licheniformis, such as phoD, phoB, phy, yfkN, yhcR, pstS, pstBA and ispA. Potential new members of the PhoPR regulon in B. licheniformis are the genes alsD and alsS for which PhoP-binding sites were predicted and which were found to be induced under phosphate starvation conditions both by transcriptome and proteome analyses. Intriguingly, the induction of part of the putative PhoPR regulon was also observed in B. licheniformis cells during an upshift of the external pH (Hornbaek et al., 2004). It has been suggested that this induction could be due to precipitation of the phosphate in the medium at high pHex values. 3.5. General and specific marker proteins for starvation To identify general and specific proteins upregulated during glucose, nitrogen or phosphate starvation, a 2D PAGE approach was used (Hoi le et al., 2006). Protein spot quantification was performed with the Delta 2D software and expression ratios were calculated based on the relative spot volume. Fusion gels were generated in silico by combining the gel images of [35 S]-labeled proteins collected during the respective starvation responses using the “fusion gel” function of the Delta 2D software. Subsequently, proteins were color-coded according to their expression profile and specific and general stress proteins could be visualized (Fig. 1). Among the proteins synthesized at elevated levels under all three conditions are ClpC and ClpP, which suggests that all three conditions result in a protein folding stress. However, the Clp proteins do not accumulate under these conditions as evidenced by the finding that their amounts in stained gels did not increase significantly. The Clp proteins belong to the CtsR regulon of B. licheniformis (Nielsen et al., 2010). In addition, a few other proteins, like DhaS, AlsS, YlbP, YqhS and BLi03994, are upregulated in cells grown under all three starvation conditions. Notably, proteins specifically induced under

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Fig. 1. Changes in the cytoplasmic proteome of glucose-, nitrogen- and phosphate-starved B. licheniformis cells. Proteins were labelled with l-[35 S]-methionine during the exponential growth phase (OD 0.4) and 60 min after entry into stationary growth phase. Proteins were separated in a pH gradient 4–7. A fusion image containing the proteome images of the starvation conditions and the exponential growth phase proteome image was created using the “union fusion” method of the Delta 2D software (Decodon GmbH, Greifswald). Color coding of the protein spots was done in such a way that all proteins belonging to a spot subset, i.e. all proteins induced in response to one starvation condition or combinations of starvation conditions received a defined, subset-specific color (see color scheme). Only proteins induced more than 2.0 fold compared to the exponential growth phase were included in the color coding. Quantification, gel fusion and color coding was done with the Delta 2D software (Decodon GmbH Greifswald). (G: glucose starvation, N: nitrogen starvation, P: phosphate starvation). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

particular starvation conditions were also identified. For example, the induction of AcoA, B and L, AcuA, KduI, BglH and RocD is specific for glucose-starved cells, induction of GlnA, BLi01909, and TrpA, D and E is specific for nitrogen-starved cells, and induction

of PhoP, PstBA and S, YlaK and YdhD is specific for phosphatestarved cells. These specific proteins can therefore be used as marker proteins for the respective starvation responses in future studies.

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4. Response of B. licheniformis cells to environmental stress 4.1. General and specific stress response Similar to the nutrient starvation studies two groups of proteins that are regulated in response to physical environmental stress could be determined. The first group includes again the house-keeping proteins. All tested stress conditions initiate a downregulation of house-keeping proteins, as do all investigated starvation conditions. This includes for example proteins involved in translation, such as aminoacyl-tRNA synthetases and translation elongation factors. Furthermore, synthesis of many enzymes employed in central carbon metabolism, amino acid and nucleotide biosynthesis is also diminished. The second group of proteins exhibits upregulation during stress. This group includes the general stress proteins that are upregulated under most tested stress conditions (see Section 4.5) and specific proteins for each stress. Among the general stress proteins are the ␴B -dependent proteins. Differently from starvation, the tested stress stimuli (except oxidative stress) lead to an induction of the ␴B regulon. The specific stress responses of B. licheniformis are summarized in the following paragraphs. 4.2. Specific response to oxidative stress Oxidative stress has been provoked by incubating growing B. licheniformis cells with hydrogen peroxide (Schroeter et al., 2011). Many of the upregulated proteins/genes are known to have functions in the oxidative stress response from studies with other organisms (Mostertz et al., 2004). Proteins belonging to a putative PerR regulon are among the ones showing the highest synthesis rates early after exposure to hydrogen peroxide (Schroeter et al., 2011). Synthesis of the catalase KatA was found to be strongly enhanced. MrgA, a DNA-binding stress protein, is also upregulated at a high rate, but this upregulation is only transient. This protein can be observed in two forms, a low molecular mass monomer and a high molecular mass multimer. The multimeric form shows a higher upregulation than the monomer suggesting a fast and stable assembly. Furthermore, the two subunits of the alkylhydroperoxide reductase (AhpC and F) are synthesized at an elevated level upon hydrogen peroxide stress. Putative new members of the B. licheniformis PerR regulon are BLi04114 (similar to Fur-family transcriptional regulators) and BLi04115 (putative ferrochelatase), which show a massive induction after peroxide stress (BLi04114 only found in the transcriptome) (Schroeter et al., 2011). Both genes are located directly downstream of the katA gene and NorthernBlots indicated co-transcription of the three genes upon oxidative stress (Schroeter et al., 2011). The proteins Tpx (thiol peroxidase, involved in detoxification), TrxA (thioredoxin), TrxB (thioredoxin reductase, together with TrxA involved in thiol homeostasis) and YugJ (similar to NADH-dependent butanol dehydrogenase), show a transient moderate upregulation and are members of a putative B. licheniformis Spx regulon (Schroeter et al., 2011). Among the upregulated proteins is also the subunit B of the excinuclease ABC (UvrB). In B. subtilis the excinuclease is a member of the SOS regulon, which responds to DNA damage (Au et al., 2005). Further members of the putative B. licheniformis SOS regulon were found to be upregulated at the RNA level (Schroeter et al., 2011). The Clp machinery might be involved in the degradation of proteins damaged by the oxidative stress as evidenced by a weak induction of ClpC and ClpP in stressed cells. Four other proteases were found to be induced at the transcriptional level and might contribute to the degradation of damaged proteins. An increase of free amino acids, revealed by analysis of the intracellular metabolome, supports the idea that protein degradation occurs in hydrogen peroxidestressed cells (Schroeter et al., 2011). Among the other proteins

upregulated during this stress are SufB and SufC, belonging to a recently described iron/sulphur cluster biogenesis system (Albrecht et al., 2010; Schroeter et al., 2011). The SUF system is induced in oxidatively stressed Escherichia coli cells and replaces the components of the housekeeping iron/sulphur cluster biogenesis system (ISC system) during oxidative stress (Albrecht et al., 2010; Lee et al., 2004; Zheng et al., 2001). Surprisingly, induction of the genes encoding the two enzymes of the glyoxylate cycle (see Section 3.2) was observed in B. licheniformis cells stressed with hydrogen peroxide. Most likely this induction is provoked by oxidative damage of the isocitrate dehydrogenase (Murakami et al., 2006). 4.3. Specific response to salt stress The response of B. licheniformis to salt stress has been analyzed after an osmotic shock with 6% NaCl (Schroeter et al., 2013). The initial response of B. subtilis to an increase in osmotic potential in the environment is the import of potassium ions. However, expression of the potassium transport systems KtrAB and KtrCD is neither in B. subtilis nor in B. licheniformis influenced by environmental conditions (Schroeter et al., 2013; Yakimov et al., 1995). Since a high intracellular potassium concentration is unfavorable, the cells accumulate compatible solutes for adaptation to osmotic stress as a second response (Whatmore et al., 1990). Compatible solutes can be acquired from the environment via specific Opu-transporters. Indeed, the expression of the respective genes is induced after an increase of osmotic pressure in B. subtilis (Hahne et al., 2010; Steil et al., 2003). Although the genes encoding the Opu transporters in B. licheniformis were significantly induced upon osmotic stress, the corresponding proteins did not accumulate with the exception of OpuE (Schroeter et al., 2013). Compatible solutes can also be synthesized de novo or by transformation of precursor molecules. Proline is the major compatible solute produced by B. subtilis (Kohlstedt et al., 2014; Whatmore et al., 1990). B. licheniformis possesses two pathways for proline synthesis from glutamate. The anabolic pathway, used during physiological growth conditions, with the proteins ProA1, ProB1, ProG and ProI, and the osmostress-mediated pathway with the proteins ProHB2A2 (Veith et al., 2004). The transcriptome data revealed an induction of the osmostress-specific pathway also reflected by a significantly increased accumulation of ProB2 and ProH later during the stress (Schroeter et al., 2013). Also, the arginase RocF and the pyrroline carboxylate dehydrogenase RocA were shown to accumulate. Surprisingly, the synthesis of GltA, a subunit of the glutamate synthase converting 2-oxoglutarate to glutamate, the precursor of proline synthesis, is downregulated in salt-stressed B. licheniformis cells as well as in salt-stressed B. subtilis cells (Höper et al., 2006). Höper et al. suggested that in B. subtilis GltA can be replaced upon salt stress by the ␴B -dependent protein YerD, a putative second glutamate synthase, (Hahne et al., 2010; Höper et al., 2006). However, in the genome of B. licheniformis there is no homologue of yerD and so far no second glutamate synthase has been annotated (Veith et al., 2004). Enzymes involved in cell wall metabolism are also among the upregulated proteins in salt-stressed B. licheniformis (Schroeter et al., 2013). For example, higher synthesis rates were measured for the UTP-glucose-1-phosphate uridylyltransferase (GtaB) and penicillin-binding proteins (PbpC, PbpX). In addition, some proteins involved in peptidoglycan synthesis and cell shape-determining proteins showed increased accumulation in stressed cells. Upregulation of penicillin binding proteins was observed for salt shocked B. subtilis cells too (Hahne et al., 2010). Besides the typical salt-mediated changes, a higher synthesis rate of proteins belonging to an oxidative stress response has been observed (Schroeter et al., 2013). These changes are also known

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from B. subtilis and B. cereus indicating that salt stress induces a secondary oxidative stress (Browne and Dowds, 2001; den Besten et al., 2009; Höper et al., 2006). Interestingly, the afore-mentioned PerR-regulated DNA-binding stress protein MrgA which occurs in cells subjected to oxidative stress in a monomeric as well as a high molecular weight multimeric form (Schroeter et al., 2011) was found only as the monomeric form in salt-stressed cells even though its synthesis is highly induced. The proteins SufB, C, D and U were also synthesized at an elevated level. The induction of these proteins indicates an enhanced need for the formation or repair of Fe–S clusters. Furthermore, several pentose phosphate pathway enzymes accumulated to higher amounts after two hours of salt stress. This accumulation could be related to the secondary oxidative stress, as many enzymes involved in ROS-detoxification rely on NADPH (Holmgren, 1989), which is produced through the pentose phosphate pathway. Lastly, proteins involved in chemotaxis (CheV, W, Y, Yfms) and motility (MotB, SwrC) accumulate in B. licheniformis cells after 120 min salt stress (Schroeter et al., 2013). The flagellin protein (Hag) is also present at higher concentrations in cells after salt stress. In B. subtilis chemotaxis and motility are negatively regulated by the addition of NaCl as indicated by a downregulation of the related genes and proteins (Höper et al., 2006; Steil et al., 2003). 4.4. Specific responses to heat and ethanol stress The response of B. subtilis to heat stress has been extensively studied (Helmann et al., 2001; Schumann, 2003). Different regulons like the HcrA regulon, the CtsR regulon and the CssSR regulon are specifically upregulated during heat stress. Furthermore, the ␴B -dependent general stress regulon is induced in heat-stressed B. subtilis cells. The response of bacteria to ethanol stress is less well characterized. Ethanol targets the cell envelope and induces the ␴M regulon (Seydlova et al., 2012; Thackray and Moir, 2003). Furthermore, it has been reported that ethanol stress also induces the ␴B regulon in B. subtilis (Boylan et al., 1993). The response of growing B. licheniformis cells to heat and ethanol stress displays some similarities to that of B. subtilis (Voigt et al., 2013). Proteins belonging to the HrcA regulon like DnaK, GroL and GroS are among the strongest upregulated proteins under both stress conditions. The resemblance of the HcrA regulon of B. licheniformis to that of B. subtilis was confirmed by Nielsen et al. (2010) using an hcrA mutant. The respective proteins have important chaperone functions assisting in the refolding of proteins denatured by heat or ethanol treatment. When refolding fails, the denatured proteins can be degraded by the Clp protease complex consisting of one of the ATPase subunits (e.g. ClpC, ClpE) and the proteolytic ClpP subunit (Gerth et al., 2004; Kock et al., 2004). Elevated synthesis of the ClpC, ClpE and ClpP proteins was observed in heatand ethanol-stressed B. licheniformis cells with ClpE being the protein with the highest synthesis rate after heat stress (Voigt et al., 2013). Both heat and ethanol stress increase the synthesis of the putative serine protease HtrB that is known to be regulated by the two-component system CssRS in B. subtilis (Darmon et al., 2002; Voigt et al., 2013). In heat-stressed B. licheniformis cells, proteins related to oxidative stress like MrgA, OhrA, SufD, SufS and TrxB are synthesized in higher amounts than in control cells (Voigt et al., 2013). None of these proteins is upregulated after ethanol stress but, among the ␴B -dependent proteins induced after ethanol treatment are proteins known to confer resistance to oxidative stress (e.g. Dps, OhrB, SodA, YcdF). Ethanol stress is known to target the cell envelope (Takahashi et al., 2001). In B. subtilis the extracytoplasmic sigma factor ␴M and genes from its regulon are upregulated (Thackray and Moir, 2003).

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In B. licheniformis only two putative ␴M -dependent proteins are synthesized at an elevated level (Voigt et al., 2013). 4.5. General and specific marker proteins for stress Color-coded gel images of all stress conditions have been created to distinguish proteins with elevated synthesis under all stress conditions from proteins specific for each individual stress (Fig. 2). A common response to physical stress is the increased synthesis of proteins belonging to the CtsR regulon, e.g. ClpC and ClpP. In contrast, the ATPase ClpE, which can also form a complex with the protease subunit ClpP, is upregulated only during heat and ethanol stress and ClpE is therefore a specific marker for the latter stresses. Contrary to nutrient starvation conditions, induction of the general stress response (␴B regulon) can be observed under all stress conditions except oxidative stress. The strongest induction of the ␴B regulon is observed in ethanol-stressed cells. Among the proteins induced by more than one stress are proteins involved in handling oxidative stress. Proteins known to be involved in response to oxidative stress and that are upregulated in hydrogen peroxidestressed B. licheniformis cells are also upregulated in heat- and especially in salt-stressed cells. Notably, the color-coded gel imaging reveals that some KatA spots, the high molecular mass MrgA spots and BLi04115 are specific marker proteins for hydrogen peroxide provoked oxidative stress. Specific marker proteins for salt stress are for example PbpX, OppD, YkrY and NrdF. Lastly, proteins like YodC, YutC, YbcF and YqiW are specifically upregulated upon heat stress, and YwaD and YwlF upon ethanol stress. 5. Responses of B. licheniformis to high-level protein secretion When B. subtilis is engineered to secrete proteins at high levels it mounts a so-called secretion stress response that has been related to the accumulation of misfolded proteins at the membrane-cell wall interface (Darmon et al., 2006; Hyyryläinen et al., 2001; Noone et al., 2012; Westers et al., 2006). This stress response is controlled by the CssRS two-component regulatory system and its main feature is the CssRS-dependent upregulation of the membraneassociated and extracellular proteases HtrA and HtrB (Antelmann et al., 2003; Darmon et al., 2002; Krishnappa et al., 2013, 2014). In addition, the CssRS system antagonizes the overproduction of membrane proteins in B. subtilis (Zweers et al., 2009). In B. licheniformis, the secretion stress response has thus far not been studied in great detail. However, Wiegand et al. (2013a) investigated fermentation stage-dependent adaptations of this bacterium to production of the protease subtilisin by whole transcriptome RNA sequencing. Intriguingly, this study suggested the absence of a CssRS-mediated stress response during stages of high-level subtilisin production as evidenced by the downregulation of the htrA and htrB genes. Also, the transcription of the auto-regulated cssR and cssS genes was reduced under these conditions. This implies that no misfolded subtilisin accumulated in the cell wall during the fermentation process, suggesting that the post-translocational subtilisin folding is either very effective or that the overproduced subtilisin contributes to the efficient degradation of any misfolded subtilisin molecules and/or other secreted proteins. The latter would be in line with the observation that extracellular proteases of B. subtilis are responsible for substantial degradation of this bacterium’s own membrane- and cell wall-associated proteins (Krishnappa et al., 2013, 2014). Furthermore, the study by Wiegand et al. (2013b). showed that most genes encoding components of the major secretion (Sec) pathway, through which most Bacillus proteins are secreted (Tjalsma et al., 2000, 2004), were substantially downregulated in the late fermentation stages when subtilisin was

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Fig. 2. Changes in the proteome of B. licheniformis cells subjected to oxidative, heat, ethanol and salt stress.

produced at the highest levels. It is presently unknown to what extent this down-regulation of sec gene expression impacts on the levels of active Sec translocons in the membrane, and how this influences the secretion of subtilisin. Answering these questions will require a quantitative analysis of the amounts of different Sec proteins in the membrane and a kinetic analysis of subtilisin secretion rates at different stages during fermentation. In contrast to the sec genes, genes for the so-called TatAyCy twin-arginine translocation (Tat) system, which is generally dedicated to the transport of folded proteins (Goosens et al., 2013), were upregulated. The biological significance of this observation has not yet been clarified, but several different scenarios are conceivable. First, the stress imposed by high-level subtilisin production could lead to a possibly increased requirement for particular Tat-dependently exported proteins. Second, Wiegand et al. (2013b) speculated that part of the overproduced subtilisin might be secreted via TatAyCy. Although

subtilisin is normally secreted through the Sec pathway of B. subtilis, this protein can be secreted via Tat when provided with an appropriate twin-arginine signal peptide (Kolkman et al., 2008). Furthermore, an overflow mechanism from the Sec into the Tat secretion pathway has been suggested for a secreted lipase that was heavily overproduced in B. subtilis (Kouwen et al., 2009). It is thus conceivable that the elevated expression of the tatAyCy genes upon high-level subtilisin production is indicative for an overflow secretion mechanism. However, alternative explanations for the observed upregulation of the tatAyCy genes cannot be ruled out until a mutagenesis study has been performed in which these tat genes and genes for Tat-dependently exported proteins have been deleted and the kinetics of subtilisin secretion in the resulting mutant strains have been investigated. Furthermore, it would be informative to measure the secretion rates of other reporter proteins in the subtilisin-overproducing strain. Altogether, the findings

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of Wiegand et al. (2013a) warrant further investigations on the responses of B. licheniformis to the overproduction of secreted proteins as this may lead to the identification and resolution of important secretion bottlenecks. 6. Outlook To monitor and optimize industrial fermentation processes, a comprehensive understanding of the physiology of the respective bacterial host organisms is mandatory. Knowing the physiological details will facilitate the detection of particular bottlenecks in the respective process. In turn, this will pinpoint potential targets for strain or process development with the ultimate objective to improve productivity (Schweder, 2011; Wiegand et al., 2013b). One problem represents for example the large scale environment of industrial fermentation processes, which can cause gradients due to the feeding and mixing strategies in fed-batch fermentations (Enfors et al., 2001; George et al., 1998). Established industrial bacterial host cells respond quickly to local concentrations of nutrients (Bylund et al., 1998; Schweder et al., 1999). Thus, the size of nutrient or pH gradient zones and the specific response of the production cells in these zones could influence the performance of a fermentation process (Schweder, 2011; Schweder and Hecker, 2004; Schweder et al., 1999). In this regard, protein signatures for stress and starvation stimuli uncover relevant marker genes or proteins for growing and non-growing cells that will point to critical conditions in the fermentation process. Such markers are therefore highly valuable tools for bioprocess monitoring and control. As reviewed in the present paper, a comprehensive library of marker genes and proteins has been established by analyzing gene expression and protein synthesis of B. licheniformis cells subjected to various starvation and stress conditions. Since most of the applied conditions simulate potentially critical conditions during fermentation, the identified makers can now be used to monitor and manage fermentation processes and to develop facile tools for on-line process control including, for example, electrical biochips (Jürgen et al., 2005; Pioch et al., 2008; Schweder, 2011). This opens up completely new avenues for achieving maximal yields in industrial fermentations with the B. licheniformis cell factory. References Albrecht, A.G., Netz, D.J., Miethke, M., Pierik, A.J., Burghaus, O., Peuckert, F., Lill, R., Marahiel, M.A., 2010. SufU is an essential iron-sulfur cluster scaffold protein in Bacillus subtilis. J. Bacteriol. 192, 1643–1651. Ali, N.O., Bignon, J., Rapoport, G., Debarbouille, M., 2001. Regulation of the acetoin catabolic pathway is controlled by sigma L in Bacillus subtilis. J. Bacteriol. 183, 2497–2504. Allenby, N.E., O’Connor, N., Pragai, Z., Ward, A.C., Wipat, A., Harwood, C.R., 2005. Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis. J. Bacteriol. 187, 8063–8080. Amon, J., Brau, T., Grimrath, A., Hanssler, E., Hasselt, K., Holler, M., Jessberger, N., Ott, L., Szokol, J., Titgemeyer, F., Burkovski, A., 2008. Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on the OmpR-type regulator GlnR. J. Bacteriol. 190, 7108–7116. Antelmann, H., Darmon, E., Noone, D., Veening, J.W., Westers, H., Bron, S., Kuipers, O.P., Devine, K.M., Hecker, M., van Dijl, J.M., 2003. The extracellular proteome of Bacillus subtilis under secretion stress conditions. Mol. Microbiol. 49, 143–156. Antelmann, H., Scharf, C., Hecker, M., 2000. Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J. Bacteriol. 182, 4478–4490. Au, N., Kuester-Schoeck, E., Mandava, V., Bothwell, L.E., Canny, S.P., Chachu, K., Colavito, S.A., Fuller, S.N., Groban, E.S., Hensley, L.A., O’Brien, T.C., Shah, A., Tierney, J.T., Tomm, L.L., O’Gara, T.M., Goranov, A.I., Grossman, A.D., Lovett, C.M., 2005. Genetic composition of the Bacillus subtilis SOS system. J. Bacteriol. 187, 7655–7666. Belitsky, B.R., Kim, H.J., Sonenshein, A.L., 2004. CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression. J. Bacteriol. 186, 3392–3398. Bernlohr, R.W., Saha, A.L., Young, C.C., Toth, B.R., Golden, K.J., 1988. Nutrientstimulated methylation of a membrane protein in Bacillus licheniformis. J. Bacteriol. 170, 4113–4118.

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