Giving a signal: how protein phosphorylation helps Bacillus navigate through different life stages

Abstract

Protein phosphorylation is a universal mechanism regulating a wide range of cellular responses across all domains of life. The antagonistic activities of kinases and phosphatases can orchestrate the life cycle of an organism. The availability of bacterial genome sequences, particularly Bacillus species, followed by proteomics and functional studies have aided in the identification of putative protein kinases and protein phosphatases, and their downstream substrates. Several studies have established the role of phosphorylation in different physiological states of Bacillus species as they pass through various life stages such as sporulation, germination, and biofilm formation. The most common phosphorylation sites in Bacillus proteins are histidine, aspartate, tyrosine, serine, threonine, and arginine residues. Protein phosphorylation can alter protein activity, structural conformation, and protein–protein interactions, ultimately affecting the downstream pathways. In this review, we summarize the knowledge available in the field of Bacillus signaling, with a focus on the role of protein phosphorylation in its physiological processes.

This article reviews the literature on protein phosphorylation events in Bacillus species during different stages of their life cycle and pathogenesis.

Introduction

The Bacillus genus comprises ~300 annotated species, which are divided into different phylogenetic clusters based on 16S rRNA and evolutionary relationships (Ash et al. 1991, Parte 2018). Members of the Bacillus genus are rod-shaped, mostly aerobic, or facultative anaerobes, Gram-positive spore-forming bacteria. Two of the most studied clades in these clusters are the Bacillus subtilis and Bacillus cereus clades. The subtilis clade comprises ten species including B. subtilis, B. amyloliquefaciens, B. aerophilus, B. pumilus, B. atrophaeus, B. licheniformis, Bacillus sp. 586, Bacillus sp. 916, Bacillus sp. JS, and Bacillus sp. BT1B_CT2, and the pathogenic species such as B. anthracis, B. cereus, and B. thuringiensis are classified as part of the cereus clade (Rooney et al. 2009, Bhandari et al. 2013). Among these, B. subtilis, commonly found in soil environments and plant roots, is the most characterized species. It is used as a model organism to decipher physiological and regulatory mechanisms in all the Bacillus species (Vlamakis et al. 2013, Harwood et al. 2018, Ravikumar et al. 2018, Richts et al. 2019, Errington and Aart 2020). Subtilis clade members have a wide range of industrial and medical applications owing to their phenotypic and genotypic variations (Cui et al. 2018, Harwood et al. 2018, Jezewska-Frackowiak et al. 2018, Su et al. 2020).

The Bacillus life cycle is composed of three distinct phases: spore formation (the dormant stage), spore germination, and a replicating vegetative phase (Soule 1932, Sella et al. 2014). Bacillus spores, formed under nutrition-deprived conditions, are metabolically inactive entities that can remain viable for extended periods (Graham-Smith 1930, Khanna et al. 2020). These endospores are highly resistant to stress conditions encountered in nature and, therefore, can be found in a wide range of environments including water, air, and soil. Under favorable conditions, these spores germinate into the metabolically active vegetative form (Watabe 2013, Bressuire-Isoard et al. 2018, Christie and Setlow 2020). Pathogenic Bacillus species proliferate inside their vertebrate hosts (Nicholson et al. 2000, Nicholson 2002, Arora et al. 2017a). When pathogenic Bacilli infect their hosts, the spores germinate and secrete toxins such as enterotoxin and emetic toxin, as well as phospholipases and proteases that help the bacteria to survive in the host environment (Moayeri et al. 2015, Sharma et al. 2017, Ehling-Schulz et al. 2019, Enosi Tuipulotu et al. 2021). Another survival strategy adopted by the members of this genus is biofilm formation. A biofilm is a matrix of exopolysaccharides, proteins, and nucleic acids that acts as a shield and protects the bacterium from various antibiotics and the host’s immune response (Hobley et al. 2015, Hall and Mah 2017, Huang et al. 2020, Shemesh and Ostrov 2020). The transition of Bacillus from one phase to another depends on environmental and nutritional conditions, which are sensed by the bacterial regulatory proteins. The Bacillus regulatory network involves various sensory proteins present on the surface and their post-translationally modified forms, particularly phosphorylation. This review article focuses on the role of protein phosphorylation events during transitioning of Bacillus species between different stages as well as during infection in the human host. We tried to include the most relevant articles but may have missed some studies due to limitations of search engine criteria and article length.

Post-translational modifications (PTMs) play important roles in providing the required proteomic diversity in bacteria, despite smaller genome sizes. PTMs such as phosphorylation, glycosylation, acetylation, and methylation influence almost all aspects of cellular physiology (Arora et al. 2010, 2021, Higgins and Dworkin 2012, Sajid et al. 2015, Manuse et al. 2016, Mijakovic et al. 2016, Khan et al. 2018, Macek et al. 2019, Bonne Kohler et al. 2020, Singhal et al. 2020, Wang and Cole 2020). Among these PTMs, protein phosphorylation is one of the most widely documented and investigated modifications across the bacterial system (Fig. 1). There are dedicated protein kinases and phosphatases that are categorized based on the phosphate group transfer from ATP to specific amino acids, such as serine (Ser)/threonine (Thr) (Ser/Thr protein kinase-STK and Ser/Thr protein phosphatase-STP), tyrosine (Tyr) (bacterial protein Tyr kinase-BY kinase and protein Tyr phosphatase-PTP), histidine (His)/aspartate (Asp) (two-component system-TCS/His sensor kinase-phosphorelay), srginine (Arg) (Arg kinase and Arg phosphatase), cysteine (Cys) [phosphotransferase system (PTS) regulated protein (such as EIIA) phosphorylation of Cys residues using PEP as energy source], and atypical protein kinases that are unlike characterized kinases but still possess kinase activity. The major phosphorylation systems are shown in the Featured image (Hoch 2000, Stock et al. 2000, Pereira et al. 2011, Capra and Laub 2012, Grangeasse et al. 2012, Fuhrmann et al. 2013, Chao et al. 2014, Deutscher et al. 2014, Mijakovic et al. 2016, Elhawy et al. 2021, Huang et al. 2021, Zhang et al. 2021). Interestingly, many proteins are phosphorylated on multiple sites, as also identified in most phosphoproteome-based studies (Macek et al. 2007, Elsholz et al. 2012, Ravikumar et al. 2014, Birk et al. 2021). For example, 214 Ser/Thr/Tyr phosphosites were identified in 153 proteins (Birk et al. 2021) and 121 Arg phosphosites were identified in 87 proteins of Bacillus subtilis (Elsholz et al. 2012). It also indicates promiscuous activities of several kinases, with overlapping specificity, and the primary reason for the fallible connection between different studies.

Figure 1.

Network of protein phosphorylation in Bacillus. Protein phosphorylation in Bacillus regulates several aspects of their life cycle, including sporulation, germination, biofilm formation, protein synthesis, DNA regulation, cell architecture, stress responses, and virulence. The detailed regulatory processes are discussed in the text along with the unique aspects of cross-talk and cross-phosphorylation within the kinases of different classes.

TCSs are ubiquitous among bacterial systems, consisting of two proteins, a sensor His kinase (HK) and a response regulator (RR). When an external stimulus is sensed, the sensor HK is activated by autophosphorylation of a conserved His residue. This signal is then subsequently transferred to its cognate RR, which is usually a transcription factor, resulting in differential altered gene expression (Fabret et al. 1999, Bijlsma and Groisman 2003, Capra and Laub 2012, Groisman 2016). TCSs are known to be involved in various cellular pathways in Bacillus sp, such as stress response (Darmon et al. 2002, Dhiman et al. 2014, Diomande et al. 2014, Mike et al. 2014, Groisman 2016, Gupta et al. 2018), protease regulation (Perchat et al. 2011, Gupta et al. 2017), sporulation (Stephenson and Hoch 2002, Fujita and Losick 2003, White et al. 2006, Gopalani et al. 2016, Peng et al. 2017), metabolic pathways (Birkey et al. 1998, Tanaka et al. 2003, Repizo et al. 2006, Geng et al. 2007, Myers et al. 2016, Aggarwal et al. 2017, van den Esker et al. 2017), cell wall regulation (Howell et al. 2003, Dobihal et al. 2019, Wu et al. 2019), biofilm formation (Verhamme et al. 2007, Stubbendieck and Straight 2017, Zhou et al. 2018), antibiotic tolerance (Kesel et al. 2013, Dintner et al. 2014, Zhang et al. 2015, Koh et al. 2021), and pathogenesis (Duport et al. 2006, Vetter and Schlievert 2007, Brillard et al. 2008, Gohar et al. 2008, Stauff and Skaar 2009, van Schaik et al. 2009).

Our understanding of phosphorylation events at different residues such as Ser, Thr, Tyr, Arg, or Cys has evolved over the last three decades. For example, the kinases responsible for Tyr phosphorylation were named bacterial tyrosine (BY) kinases and were identified based on sequence homology with known Tyr kinases, such as the YwqD/YwqE Tyr kinase and phosphatase pair in B. subtilis (Mijakovic et al. 2003, 2005). Subsequently, more such pairs were characterized in B. subtilis with roles predicted in various cellular pathways based on substrate identification in phosphoproteome studies (Mijakovic and Deutscher 2015).

The importance of Ser/Thr phosphorylation was soon realized and with the advancement in genome sequencing and -omics technologies, various Ser/Thr kinases (STKs) and Ser/Thr phosphatases (STPs) were identified and characterized in B. subtilis and other microbes (Fischer et al. 1996, Moszer 1998, Obuchowski et al. 2000, Vijay et al. 2000, Iwanicki et al. 2002, Madec et al. 2002, Shakir et al. 2010, Lima et al. 2011, Pereira et al. 2011, Arora et al. 2012, Bidnenko et al. 2013, Borriss et al. 2018, Rajagopalan et al. 2018, Baros et al. 2020). Global phosphoproteome and in silico analysis further helped in the identification of substrates regulated by Ser/Thr phosphorylation (Levine et al. 2006, Macek et al. 2007, Miller et al. 2009, Ravikumar et al. 2014, Pan et al. 2015, Rosenberg et al. 2015, Arora et al. 2017b, Zhang et al. 2018, Shi et al. 2020, Birk et al. 2021). The Bacillus genus encodes two classes of STKs that are categorized based on sequence homology and conserved sequence motifs. These are eukaryotic-like Ser/Thr kinases (eSTKs) or Hanks-type kinases and atypical STKs (non-eSTKs), and two-component kinases such as Ser/Thr kinases with specific single substrates (Pereira et al. 2011, Arora et al. 2012, Shi et al. 2014a, Mijakovic et al. 2016, Nguyen et al. 2016, Stancik et al. 2018, Zhang et al. 2021).

The global phosphoproteome of Arg phosphorylation in B. subtilis identified >100 proteins with phosphorylated Arg sites across diverse classes of proteins involved in metabolism, cellular architecture, sporulation, and various stress-induced responses (Elsholz et al. 2012, Schmidt et al. 2014, Trentini et al. 2014, Singh et al. 2015, Fuhrmann et al. 2016, Mijakovic et al. 2016, Zhou et al. 2019). In the phosphoproteome study by Elsholz et al. (2012), of the 87 proteins identified to be phosphorylated on Arg residues, 17 were phosphorylated on multiple sites. These proteins were ClpC, RpoB, AroA, MtnK, OdhA, BdhA, ComfA, ComGA, ComK, RpoC, RpsM, GltA, Gmk, GroEL, GudB, MenB, and MtnA (Elsholz et al. 2012). Interestingly, genetic deletion of Arg kinase McsB in B. anthracis caused alteration in vegetative cells and spore morphology, defective growth at elevated temperatures, and reduced sporulation and germination efficiencies (Singh et al. 2015). These studies signify the physiological relevance of Arg phosphorylation in the Bacillus species.

In the following sections, we summarize the role of protein phosphorylation during different stages and pathways in the Bacillus (Table 1).

Table 1.

Phosphorylation targets in Bacillus sp. in various cellular pathways.

Kinase/phosphatase	Substrate/target	Experimental validation	References
Cell morphogenesis
Bsu PrkC	YvcK, YmfM (RodZ), WalR, CpgA, LtaS, YfnI, YvgJ, YqgS, FtsZ	I, II, IIIa, IIIb	Foulquier et al. (2014), Ravikumar et al. (2014), Libby et al. (2015), Absalon et al. (2008), Pompeo et al. (2018a)
Bsu PrpC	YvcK, YmfM (RodZ), WalR, CpgA, YfnI, FtsZ	I, IIIa, IIIb	Foulquier et al. (2014), Ravikumar et al. (2014), Libby et al. (2015), Absalon et al. (2008), Pompeo et al. (2018a)
Bsu WalRK	cwlO, lytE, iseA	I, IIIb	Salzberg et al. (2013), Dobihal et al. (2019)
Bas WalRK	ftsE, eag, kinB3	I	Dhiman et al. (2014)
DNA metabolism/regulation
Bsu PtkA	SSBs, YorK, SalA, MinD, RecA, DivIVA	I, II, IIIa, IIIb	Mijakovic et al. (2006), Macek et al. (2007), Jers et al. (2010), Shi et al. (2014b)
Bsu PtkB	MutL, RpoB, MinD, PolA	II	Shi et al. (2014b)
Bsu PtpZ	SSBs, MinD, PolA, RpoB, MutL	I, II, IIIb	Mijakovic et al. (2006), Shi et al. (2014b)
Bsu YabT	MinD, MutL, AbrB	I, II	Shi et al. (2014b), Kobir et al. (2014)
Bsu PrkD	SalA, AbrB	I, II	Shi et al. (2014b), Kobir et al. (2014)
Bsu PrkC	AbrB	I	Kobir et al. (2014)
Bsu STK	CodY, AbrB, RecA	IIIa	Macek et al. (2007), Soufi et al. (2010)
Bsu McsB	RecA, RpoB, Ssb, DivIVA	III​​​​​​​a	Elsholz et al. (2012), Schmidt et al. (2014)
Bsu YwlE	RecA, RpoB, Ssb, DivIVA	III​​​​​​​a	Elsholz et al. (2012), Schmidt et al. (2014)
Bsu DegSU	ComK	I	Dahl et al. (1992), Hamoen et al. (2003)
Metabolsim
Bsu HprK/P	Crh	I, IIIb	Landmann et al. (2012)
Bsu PrkC	YwjH, GlnA, Icd, AlsD	I	Pietack et al. (2010)
Bsu PtkA	Ugd, Asd, Ldh, Eno, FatR, SalA	I, II	Jers et al. (2010), Mijakovic et al. (2003), Petranovic et al. (2009), Derouiche et al. (2013), Shi et al. (2014b), Derouiche et al. (2015)
Bsu PtpZ	Ugd	I	Mijakovic et al. (2003)
Bsu STK	Pyk, Asd, Eno, GlnA, Icd, YwjH	III​​​​​​​a	Eymann et al. (2007), Macek et al. (2007)
Bsu McsB	Eno, GlnA	III​​​​​​​a	Schmidt et al. (2014)
Bsu YwlE	Eno, GlnA	III​​​​​​​a	Schmidt et al. (2014)
Bsu CitST	citM	I	Repizo et al. (2006)
Bsu GlnKL	glsA, glnT	I	Satomura et al. (2005)
Bsu YufLM	maeN, yflS	I	Tanaka et al. (2003)
Bsu PhoPR	ResDE	I	Birkey et al. (1998)
Bsu LytST	ysbA	I	van den Esker et al. (2017)
Bas PrkD	Pyk	I	Arora et al. (2012)
Bas PrkC	Eno	I	Virmani et al. (2019)
Bas PhoPR	phoA, pst	I	Aggarwal et al. (2017)
Protein synthesis
Bsu PrkC	Ef-Tu, Ef-G, CpgA	I, III​​​​​​​a, IIIb	Absalon et al. (2009), Ravikumar et al. (2014), Gaidenko et al. (2002), Shah et al. (2008), Dworkin and Shah (2010), Ravikumar et al. (2014), Absalon et al. (2009), Pompeo (2012)
Bsu PrpC	Ef-Tu, Ef-G, CpgA	I, III​​​​​​​a, IIIb	Gaidenko et al. (2002), Ravikumar et al. (2014), Absalon et al. (2009), Ravikumar et al. (2014)
Bsu STK	Ef-Tu, Ef-G	III​​​​​​​a	Levine et al. (2006), Eymann et al. (2007)
Bsu McsB	Ef-Tu, Ef-G	III​​​​​​​a	Schmidt et al. (2014)
Bsu YwlE	Ef-Tu, Ef-G	III​​​​​​​a	Schmidt et al. (2014)
Bsu WalRK	sasA	I	Libby et al. (2019)
Bas PrkC	Ef-Tu, Ef-G	I	Arora et al. (2017b), Arora (2013)
Bas PrpC	Ef-Tu	I	Arora et al. (2013)
Sporulation
Bsu YabT	RecA, RacA, SsbA, AbrB, YabA, Ef-Tu	I, II, IIIb	Bidnenko et al. (2013), Shi et al. (2014b), Derouiche et al. (2016), Kobir et al. (2014), Garcia Garcia et al. (2018), Pereira et al. (2015)
Bsu SpoIIE	RecA, RacA	I, II	Shi et al. (2014b)
Bsu PrkA	σK	I	Yan et al. (2015)
Bsu CotH	CotB, CotG	I	Nguyen et al. (2016)
Bsu SpoIIAB	SpoIIAA, σF	I	Clarkson et al. (2004), Duncan and Losick (1993)
Bsu SpoIIE	SpoIIAA	I	Duncan et al. (1995)
Bsu KinA-E	Spo0F, Spo0B, Spo0A	I	Burbulys et al. (1991), Jiang et al. (2000)
Bsu RapA	Spo0F	I	Perego et al. (1994)
Bsu Spo0E	Spo0A	I	Ohlsen et al. (1994)
Bsu CotH	CotB, CotG	I	Nguyen et al. (2016)
Bas BAS1213–1214	KinD	I	Gupta et al. (2018)
Bce LytSR	lrgAB, clhAB2	I	Chandramohan et al. (2009)
Bth LytSR	SpoIIP	I	Peng et al. (2017)
Germination
Bsu PrkC	Ef-G	I, III​​​​​​​a, IIIb	Gaidenko et al. (2002), Shah et al. (2008), Dworkin and Shah (2010), Ravikumar et al. (2014)
Bsu PrpC	Ef-G	I, III​​​​​​​a, IIIb	Gaidenko et al. (2002), Ravikumar et al. (2014)
Bsu McsB	Tig, SigA	I, III​​​​​​​a	Elsholz et al. (2012), Zhou et al. (2019)
Bsu YwlE	Tig, SigA	I, III​​​​​​​a	Elsholz et al. (2012), Zhou et al. (2019)
Bsu CotH	CotB, CotG	I	Nguyen et al. (2016)
Bas PrkC	Eno	I	Virmani et al. (2019)
Biofilm
Bsu KinC	Spo0A	I	Lopez et al. (2009), Shemesh et al. (2010)
Bsu KinD	Spo0A	I	Aguilar et al. (2010)
Bsu CssRS	TasA	I	Steinberg et al. (2020)
Bsu DegSU	YiT	–	Kobayashi and Ikemoto (2019)
Bsu PrkC	AhpA via AbrB, GroEL	III​​​​​​​a	Zwick et al. (2017), Ravikumar et al. (2014)
Bsu PrpC	GroEL	III	Ravikumar et al. (2014)
Bsu YabT	YabA	I	Garcia Garcia et al. (2018)
Bas PrkC	GroEL	I, IIIb	Arora et al. (2017b)
Stress response
Bsu McsB	CtsR, GroEL, ClpC, ClpP	I, III​​​​​​​a	Fuhrmann et al. (2009), Elsholz et al. (2012), Schmidt et al. (2014)
Bsu YwlE	CtsR, GroEL, ClpC, ClpP	III​​​​​​​a	Elsholz et al. (2012), Schmidt et al. (2014)
Bsu PtkA	DnaK	I, III​​​​​​​a	Shi et al. (2016)
Bsu PtkB	DnaK	I, III​​​​​​​a	Shi et al. (2016)
Bsu DesKR	desA	I	Cybulski et al. (2004)
Bsu LiaSR	liaI	I	Jordan et al. (2007), Kesel et al. (2013)
Bsu BceRS	bceA	I, IIIb	Dintner et al. (2014)
Bsu WalRK	cwlO, lytE	I	Takada et al. (2018)
Bsu CssRS	HtrA,HtrB	–	Westers et al. (2006), Noone et al. (2012)
Bsu RsbW	RsbV	I	Alper et al. (1996), Yang et al. (1996)
Bsu RsbP, RsbU	RsbV	I	Yang et al. (1996), Vijay et al. (2000)
Bsu RsbT	RsbS, RsbRA	I, IIIb	Yang et al. (1996), Chen et al. (2004), Kim et al. (2004b)
Bsu RsbX	RsbS, RsbRA	I	Yang et al. (1996), Chen et al. (2004)
Bas HssRS	–	–	Mike et al. (2014)
Bas HitRS	–	–	Mike et al. (2014), Pi et al. (2020)
Bas EdsRS	clsT	I	Laut et al. (2020)
Bas WalRK	–	–	Dhiman et al. (2015)
Bce CasKR	desA	I	Diomande et al. (2016)
Bce RsbK	RsbY	I	van Schaik et al. (2005), de Been et al. (2010)
Bth YvqEC	–	–	Zhang et al. (2015)
Bth YvcPQ	yvcS1S2, yvcR, kapD, BMB171_C4835	I	Zhang et al. (2016)
Virulence
Bas PTS	AtxA	IIIb	Tsvetanova et al. (2007), Raynor et al. (2018), Bier et al. (2020)
Bas STK	AtxA via CodY and AbrB	I	Strauch et al. (2005), van Schaik et al. (2009), Chateau et al. (2013)
Bas PrpN	CodY	I, IIIb	Gangwal et al. (2022)
Bas PrkC	CodY	I	Gangwal et al. (2022)
Bas HprK/P	HpR	I	Poncet et al. (2004), Choi et al. (2006)
Bas BrrAB	lef, pag, cya, atxA	I	Vetter and Schlievert (2007)
Bas BAS2109–2108	Proteases	I, IIIb	Gupta et al. (2017)
Bce ResDE	Enterotoxins (without PlcR)	I	Duport et al. (2006)
Bce YvfTU	Via PlcR	I	Brillard et al. (2008)

Protein kinases and phosphatases are represented by different colors. Pink: STK (Ser/Thr kinases); light blue: STP (Ser/Thr phosphatases); dark blue: BY-K (Bacillus Tyr kinases); orange: PTP (Bacillus Tyr phosphatases); maroon: Arg kinases; gold: Arg phosphatases; green: histidine (His)/aspartate (Asp) (two-component system-TCS/His sensor kinase-phosphorelay); red: atypical STKs and two-component like Ser/Thr kinases and phosphatases. Bacillus species are abbreviated as B. subtilis (Bsu), B. anthracis (Bas), B. cereus (Bce), and B. thuringiensis (Bth).

The experimental validation of proteins/targets is shown as:

I: in-vitro; II: interactomics-based study; III^a: phosphoproteome; and III^b: in-vivo.

Cellular architecture and growth

The Gram-positive bacterial cell wall is comprised mostly of multilayered peptidoglycan sheets and teichoic acids that serve as a protective barrier and help to maintain the bacterial shape and integrity. Bacterial growth and cellular integrity is a dynamic process that requires continuous hydrolysis of the cell wall to introduce breaks for the accumulation of newly formed peptidoglycan units into the cell wall (Vermassen et al. 2019). In B. subtilis, this controlled autolysis is carried out by endopeptidases such as CwlO and LytE, which are further regulated by the WalRK TCS (Yamaguchi et al. 2004, Salzberg et al. 2013, Dobihal et al. 2019, 2022). The WalRK regulon is mostly composed of genes encoding autolysins, autolysin inhibitors, ABC transporters, and genes involved in teichoic acid synthesis (Salzberg et al. 2013). Phosphorylation of the WalR RR at Asp53 by its cognate HK, WalK, blocks the promoter of autolysin inhibitor iseA, thereby causing an increase in the expression of these endopeptidases (Libby et al. 2015, Dobihal et al. 2019). Moreover, mutant walR bacilli have an L-shaped morphology and a complete loss of the bacterial cell wall, implicating the role of WalR in cell wall morphogenesis (Dominguez-Cuevas et al. 2012). In B. anthracis, though the functional relevance of the WalRK TCS has not been well-explored, its expression is induced in the presence of an inhibitor of cell wall synthesis, fosfomycin (Dhiman et al. 2015). Furthermore, WalR has been shown to bind with the promoter regions of eag (S-layer protein EA1), ftsE (ABC transporter), and kinB3 (HK) (Dhiman et al. 2014). Among these, EA1 is an important component of the S-layer surrounding the cellular envelope that imparts structural stability to the cell wall (Chateau et al. 2020, Fioravanti et al. 2022). Phosphorylation of WalR at Thr101 by PrkC, a membrane-localized eSTK with surface-exposed PASTA (peptidoglycan and Ser/Thr kinase-associated) domain, is important for the regulation of cell wall homeostasis during the stationary phase in B. subtilis (Libby et al. 2015). The two-site phosphorylation of WalR at Asp53 and Thr101 by WalK and PrkC, respectively, enhances WalR activity and blocks the expression of SasA, a protein involved in antibiotic tolerance (Libby et al. 2019) (Fig. 2).

Figure 2.

Role of protein kinases in Bacillus cell development and growth. The schematic diagram shows multiple proteins being regulated by phosphorylation during general cell development and growth with four specific processes illustrated: cell morphogenesis, protein synthesis, DNA regulation, and metabolism. Different classes of kinases are involved in each process that targets the substrates, which are involved in multiple pathways. Black solid arrows represent kinase-mediated substrate phosphorylation, red dotted arrows represent activation/upregulation, black lines with terminal bars represent inhibition or downregulation, and black dotted arrows represent regulatory links (either upregulation or downregulation).

Lipoteichoic acid (LTA) is another important component of the cell wall, i.e. threaded across peptidoglycan layers and is required for optimal bacterial growth and physiology. Synthesis of LTA in B. subtilis involves four enzymes: LtaS, YfnI, YvgJ, and YqgS, which are phosphorylated by PrkC (Pompeo et al. 2018a). CpgA (YloQ), a multidomain GTPase, is another target of the PrkC/PrpC pair that plays an important role in the maintenance of bacterial shape and peptidoglycan deposition in B. subtilis (Cladiere et al. 2006, Absalon et al. 2008, 2009). Though the studies on CpgA phosphorylation lack functional and genetic evidence, they suggest a possible link between peptidoglycan biogenesis and Ser/Thr phosphorylation (Fig. 2).

In the vegetative stage, division at the septal region results in the generation of two daughter cells termed vegetative cells. These vegetative cells display a specific rod-shaped morphology, which is mainly determined by various proteins associated with the cell wall and cytoskeleton (Jones et al. 2001, Errington and Wu 2017, Angeles and Scheffers 2021). MreB, an actin-like cytoskeletal protein is a key factor that determines the characteristic rod-shape of Bacilli, and the absence of mreB in B. subtilis alters the localization of penicillin-binding protein (PBP1), resulting in bulging cell phenotype (Formstone and Errington 2005, Carballido-Lopez 2006, Kawai et al. 2009). Interestingly, overexpression of YvcK, a protein with a similar localization pattern as MreB, was able to rescue the mreB mutant phenotype with proper PBP1 localization (Gorke et al. 2005, Foulquier et al. 2011). Ser/Thr phosphorylation-mediated regulation of YvcK (Thr304) by the PrkC/PrpC pair is shown to be critical for the rod-shaped morphology in mreB mutant cells of B. subtilis. A null mutant strain of mreB overexpressing the phosphoablative mutant YvcK (Thr304Ala) exhibits a bulging-type phenotype with mis-localized PBP1. However, mreB mutant bacteria overexpressing the phosphomimetic mutant YvcK (Thr304Glu) were able to restore rod-shaped morphology (Foulquier et al. 2014). Another cell shape-determining protein, RodZ (YmfM), is a target of the PrkC/PrpC pair in B. subtilis, although its role and functional implication is still unknown (Alyahya et al. 2009, Ravikumar et al. 2014, van Beilen et al. 2016) (Fig. 2).

Ser/Thr phosphorylation of the cell division protein FtsZ is important for efficient septum formation in prokaryotes (Garcia et al. 2016, Manuse et al. 2016, Hardt et al. 2017, Maurya et al. 2018, Rajpurohit et al. 2022). Interestingly, phosphoproteome analysis by Ravikumar et al. (2014) also identified FtsZ as a substrate of PrkC/PrpC in B. subtilis, and a study by our group reported septation defects and upregulated ftsZ levels in a prkC deletion mutant strain in B. anthracis (Dhasmana et al. 2021). These reports may provide a functional connection between PrkC and the regulation of cell division machinery in Bacillus genus via phosphorylation of FtsZ. In addition, a study on McsB Arg kinase (previously characterized as a Tyr kinase) in B. anthracis showed attenuated growth, elongated cell morphology (elongated cells), and defective septum formation in an mcsB null mutant strain, signifying the importance of Arg phosphorylation in bacterial growth and development (Mattoo et al. 2008, Singh et al. 2015). Furthermore, a new family of protein kinases belonging to the ubiquitous bacterial kinase family YdiB was shown to be important for normal bacterial growth (Karst et al. 2009, Nguyen et al. 2017).

Altogether, these studies highlight the widespread role of protein phosphorylation in the regulation of cell morphogenesis and growth in the Bacillus genus (Fig. 2).

Gene regulation

Protein phosphorylation is important in the regulation of various genetic processes including replication, transcription initiation, DNA condensation, and repair, via the phosphorylation of DNA/RNA binding proteins (Garcia-Garcia et al. 2016). Phosphoproteomics and interactomics studies have identified several proteins in B. subtilis that are phosphorylated at Ser/Thr/Tyr residues and are part of the gene regulatory network (Ravikumar et al. 2014, Shi et al. 2014b, Rosenberg et al. 2015). For instance, phosphorylation of a DNA single-stranded binding protein (Ssb) by the BY-kinase PtkA (formerly known as YwqD) and the YorK exonuclease specific for single-stranded DNA is important for DNA binding and exonuclease activities, respectively (Mijakovic et al. 2006, Jers et al. 2010) (Fig. 2). Inactivation of ptkA in B. subtilis causes the formation of multiple nucleoids the and accumulation of additional genetic material in the cells, indicating the role of Tyr phosphorylation in DNA replication and cell cycle regulatory pathways (Petranovic et al. 2007). Several other proteins involved in DNA and RNA metabolism that were identified as the target of BY-kinases (PtkA or PtkB) based on interactome analysis include RecA (recombinase), DivIVA (cell division protein), MinD (cell-division regulator), SalA (negative regulator of scoC expression, activator of PtkA kinase activity), PolA (DNA polymerase I), RpoB (DNA-directed RNA polymerase beta subunit), MutL (DNA mismatch repair protein), and TkmA/TkmB (protein tyrosine kinase activator) (Shi et al. 2014b). DivIVA is known to be regulated by protein phosphorylation in other bacterial species indicating that kinase-mediated regulation of cell division is conserved in diverse bacteria (Arora et al. 2014, Fleurie et al. 2014, Lee et al. 2014, Chaudhary et al. 2023).

The most studied Ser/Thr kinase and phosphatase pair involved in DNA regulation is the YabT kinase (Bidnenko et al. 2013) and the SpoIIE phosphatase (Duncan et al. 1995). These two enzymes are sporulation stage-specific and are critical to the development and maintenance of the spore genome (discussed in the sporulation section). Transcriptional regulators are another class of DNA-binding proteins that are targets of phosphorylation networks in the bacterial system (Dworkin 2015, Kalantari et al. 2015). The functional impact of Ser/Thr phosphorylation on the global transcriptional regulators CodY and AbrB has been studied using phosphorylation site mutants and DNA-binding experiments. Phosphorylation of AbrB at Ser86 hinders its DNA binding ability, leading to deregulation of target genes involved in the production of exoprotease, sporulation, and competence development in B. subtilis (Soufi et al. 2010, Kobir et al. 2014), while in both B. subtilis and B. anthracis, CodY phosphorylation was detected at a residue (Ser215) critical for its DNA binding activity (Joseph et al. 2005, Macek et al. 2007, Joon et al. 2017). Furthermore, our recent study in B. anthracis shows that CodY phosphorylation at Ser215 completely abolishes DNA binding with the promoter region of one of its target genes, atxA, leading to defects in toxin synthesis (Gangwal et al. 2022) (Fig. 2). Apart from these, protein phosphorylation also regulates various pathways involved in bacterial competence, the ability to uptake DNA from the environment, and its subsequent incorporation in the genome by specific protein channels and import machinery, leading to variations in the bacterial genotype and phenotype (O’Connell et al. 2022). In B. subtilis, the TCS RRs ComA and ComK are the most widely studied positive regulators of various early and late competence genes, respectively (Weinrauch et al. 1990, Dubnau et al. 1994, Maier 2020). ComK is shown to be phosphorylated by Arg kinase McsB and dephosphorylated by cognate phosphatase ywlE. Further, in the ywlE mutant of B. subtilis, ComK was found to be phosphorylated at six Arginine sites (R65, R157, R161, R165, R186, and R19). In the ywlE mutant, ComK-dependent gene expression was upregulated which suggests ComK activity is positively regulated by Arg phosphorylation (Elsholz et al. 2012). The phosphorylation status of another TCS RR, DegU, plays a critical role in the activation of competence genes and assists in the binding of ComK during competence (Dahl et al. 1992, Hamoen et al. 2003).

Cellular metabolism

Cellular metabolism plays a key role in bacterial growth and development via the modulation of important processes depending on the nutritional status and metabolic activity of the bacteria. In Bacillus, metabolic changes help to drive cellular machinery down to one of two paths, to produce either replicating vegetative cells or dormant spores. Glucose serves as a common energy source for heterotrophic bacteria and its absence triggers the uptake of other carbon sources, such as citrate, glutamine, pyruvate, or malate (Schilling et al. 2007). In B. subtilis, dedicated TCSs sense the presence of these additional carbon sources and thus increase the expression of membrane transporters facilitating their cellular intake. For example, CitST and GlnKL TCSs regulate the expression of Mg²⁺–citrate transporters and glutamine transporters, respectively (Satomura et al. 2005, Repizo et al. 2006). The presence of citrate or glutamine causes autophosphorylation of the respective HKs, CitS, or GlnK, followed by the activation of their respective RRs, which in turn upregulate the expression of transporters involved in the uptake of citrate or glutamine (Satomura et al. 2005, Repizo et al. 2006).

In B. subtilis, in the absence of glucose as a major carbon source, LytST TCS is required for the uptake of pyruvate via inducing the transcription of YsbA, a protein involved in the upregulation of pyruvate transporters (van den Esker et al. 2017). Similarly, in a minimal medium, the presence of malate as the sole carbon source activates the YufLM TCS. Phosphorylated YufM binds to the promoter region of maeN and increases the surface expression of malate transporters to facilitate cellular growth on malate (Tanaka et al. 2003). In B. anthracis, PhoPR is another functional TCS, which is activated upon phosphate starvation. PhoP regulates the expression of phosphate metabolism-associated genes, e.g. phoA and pst, which help to overcome the limiting phosphate availability (Aggarwal et al. 2017). A similar PhoPR TCS exists in B. subtilis, which is activated under phosphate starvation and stimulates the degradation of teichoic acid in the cell wall, releasing phosphate ions, and hence fulfilling the metabolic need for phosphate (Pragai et al. 2004, Myers et al. 2016). Furthermore, phosphorylated PhoP directly increases the expression of another important TCS called ResDE, which is important for aerobic as well as anaerobic respiration during phosphate starvation conditions in B. subtilis (Birkey et al. 1998) (Fig. 2).

In B. subtilis, HPrK/P-mediated phosphorylation of the carbon-flux-regulating histidine protein Crh (a paralog of HPr) acts as a regulatory switch in carbon metabolism (Landmann et al. 2012). Additionally, PrkC phosphorylates four major metabolic enzymes, namely Transaldolase (YwjH), Glutamine Synthetase (GlnA), Isocitrate Dehydrogenase (Icd), and α-Acetolactate Decarboxylase (AlsD) (Pietack et al. 2010). Bacillus anthracis dual-specificity protein kinase (DSPK) PrkD phosphorylates pyruvate kinase (Pyk), an enzyme that catalyzes the concluding step of glycolysis, resulting in the inhibition of its specific activity (Arora et al. 2012). Pyk was also detected in the phosphoproteome study of B. subtilis (Macek et al. 2007) (Fig. 2). Since pyk mutant in B. subtilis produce more carbon dioxide and have a reduced growth rate (Fry et al. 2000), the role of Pyk phosphorylation in the growth, metabolism, and pathogenesis of different Bacillus species needs to be studied further.

Several other reports on B. subtilis have identified the metabolic enzymes that are phosphorylated at Arg residues, indicating a possible role of the McsB/YwlE system in their regulation (Elsholz et al. 2012, Schmidt et al. 2014, Trentini et al. 2016, Zhou et al. 2019, Ogura 2020). A study by Ogura et al. (2004) showed glucose-mediated regulation of mcsB and ywlE expression in B. subtilis, suggesting that Arg phosphorylation plays a role in cellular growth in glucose-containing media. McsB and YwlE mediate reversible Arg phosphorylation of ClpCP protease and TsaD, a tRNA modification enzyme that regulates ylxR (a nucleoid-associated protein) expression through PylxS promoter. YlxR is known to regulate >400 genes, indicating the importance of glucose mediated induction of gene expression through McsB and YwlE (Ogura 2020). Interestingly, McsB has been shown to be involved in regulating protein turnover of its substrates, causing degradation of anomalous proteins through the ClpCP protease system, as discussed later in stress response section. B. subtilis McsB and its cognate Arg phosphatase YwlE were shown to reversibly phosphorylate and regulate Glutamate dehydrogenase GudB degradation in the cell (Stannek et al. 2014). The redox-sensitive modulator MgsR of SigB regulon, controls the expression of genes involved in oxidative or thiol-specific stress in B. subtilis. MgsR is phosphorylated by McsB, which mediates its degradation through Clp proteases (Lilge et al. 2020) (Fig. 2). Hajdusits et al. (2021) delineated that under stress conditions McsB forms a closed octamer-like compartment, which interconverts with monomers and other oligomers in a phosphorylation dependent manner. Interestingly, the active sites in the octamer are sequestered and only phosphorylate unfolded proteins that can enter the compartment, thus mediating their degradation. Contrarily, dimerized McsB can phosphorylate its substrates and regulate their activity (Hajdusits et al. 2021). Additional metabolic studies showed that PtkA-mediated phosphorylation of a Ugd family protein—UDP glucose dehydrogenase, is important for its catalytic activation in B. subtilis (Mijakovic et al. 2003, Petranovic et al. 2009) (Fig. 2). A similar PtkA-dependent phosphorylation mechanism activates aspartate semialdehyde dehydrogenase (Asd), converting aspartyl phosphate to aspartyl semialdehyde and inorganic phosphate (Jers et al. 2010). In different species of Bacillus, the role of UDP glucose dehydrogenase is implicated in the production of exopolysaccharides and cell wall organization, while Asd is involved in amino acid biosynthesis (Mijakovic et al. 2004, Jakobsen et al. 2009, Naerdal et al. 2011). In B. subtilis, PtkA has been shown to regulate the cellular localization of enzymes required for carbon metabolism, namely Ldh (Lactate dehydrogenase) and Eno (Enolase) (Jers et al. 2010). Phosphorylation of Eno by PrkC also affects its cellular localization and expression in B. anthracis (Virmani et al. 2019). Additionally, phosphorylation of Pgm by PrkC regulates its activity, indicating that PrkC regulates glycolysis at multiple steps (Virmani et al. 2023) (Fig. 2).

Protein phosphorylation can indirectly regulate metabolic enzymes through phosphorylation-mediated activation or inhibition of various transcriptional regulators. In B. subtilis, PtkA-mediated phosphorylation of a transcriptional regulator FatR, involved in the metabolism of polyunsaturated fatty acids, abrogates its DNA binding ability consequently leading to the derepression of the fatR-cyp102A3 operon (Derouiche et al. 2013). PtkA-mediated phosphorylation of another transcriptional regulator, SalA, represses scoC (an aprE repressor) and activates the expression of a B. subtilis exoprotease, AprE (Derouiche et al. 2015). AprE is an important metabolic enzyme, i.e. required by growing bacteria for the supply of nutrients via extracellular protein degradation. In different Bacillus species, AprE synthesis is regulated by various transcriptional regulators including CodY, AbrB, DegU, ScoC, Hpr, SinR, and SalA that are in turn regulated by protein phosphorylation events (Ogura et al. 2004, Derouiche et al. 2015, Barbieri et al. 2016, Liu et al. 2020, Zhou et al. 2021, Zolfaghari Emameh et al. 2022). These studies, therefore, suggest a functional correlation between metabolism and protein phosphorylation.

Protein synthesis

The optimal functioning of protein synthesis machinery is vital for cell survival and growth. Protein synthesis comprises of three basic steps: initiation, elongation, and termination. After initiation, there is a multistep elongation cycle involving three major elongation factors, EF-Tu, EF-Ts, and EF-G (Xu et al. 2021). These are highly conserved proteins that are essential for the survival of bacteria. EF-Tu is a GTPase that has diverse functional roles ranging from translation to pathogenesis and can interact with a variety of macromolecules including RNA, nucleotides, and other proteins (Krab and Parmeggiani 2002, Maracci and Rodnina 2016, Harvey et al. 2019). It alternates between active (GTP-bound) and inactive (GDP-bound) states, which is responsible for accurate selection of aminoacyl-tRNA and its binding to the ribosome (Bourne et al. 1991, Schmeing et al. 2009, Sajid et al. 2011a, Talavera et al. 2018). These states of EF-Tu are controlled by EF-Ts, the guanine nucleotide exchange factor. EF-G is also an essential GTPase that translocates the ribosomes along the translating mRNA (Rodnina et al. 1997, Agirrezabala and Frank 2009).

PrkC/PrpC-dependent reversible phosphorylation of the elongation factors EF-G and EF-Tu has been widely reported in the Bacillus genus (Gaidenko et al. 2002, Levine et al. 2006, Shah et al. 2008, Absalon et al. 2009, Shah and Dworkin 2010, Arora et al. 2013, 2017). Ribosome-associated proteins have also been shown to be the targets of Ser/Thr phosphorylation in B. subtilis (Absalon et al. 2009, Pompeo et al. 2012). For example, the ribosome associated GTPase CpgA is phosphorylated by PrkC in B. subtilis. Mutation of the phosphorylated residue Thr166 decreases the GTPase activity of CpgA as well as its affinity to 30S ribosomal subunits. The Bacillus strains expressing the CpgA-Thr166Ala variant show growth defects and exhibit a curly morphology, indicating the importance of ribosome-associated protein phosphorylation in maintaining B. subtilis growth and morphology (Pompeo et al. 2012) (Fig. 2).

Bacillus sp. also utilize secondary messengers known as alarmones or hyperphosphorylated (p)ppGpp nucleosides for protein synthesis during nutrient deprivation (Bange and Bedrunka 2020). These alarmones act as a cue for starving bacteria to shut down various essential cellular pathways, such as transcription and DNA replication, thereby helping them to conserve their energy resources (Potrykus and Cashel 2008, Steinchen and Bange 2016, Gourse et al. 2018). In B. subtilis, the cellular level of these alarmones is determined by dedicated (p)ppGpp synthetases such as SasA (small alarmone synthetase A) (Nanamiya et al. 2008), which are known to be regulated by Ser/Thr phosphorylation (Libby et al. 2019). The expression of sasA is regulated by the WalR transcription factor, a component of the WalRK TCS. Interestingly, WalR itself is regulated by PrkC/PrpC-mediated reversible phosphorylation, resulting in further activation of WalR activity and repression of sasA expression (Libby et al. 2019). Thus, the protein synthesis machinery in B. subtilis is subjected to regulation by phosphorylation, either directly by phosphorylation of translational factors or by (p)ppGpp-mediated inhibition (Fig. 2).

Sporulation

The process of spore formation and revival provides an excellent model for understanding the developmental processes in the bacterial system. During sporulation, the entire Bacillus metabolic pathways are reset with the help of various regulatory modifications such as protein phosphorylation to form a dormant spore (Errington 2003, Hoch 2017, Khanna et al. 2020). In B. subtilis, initiation of sporulation is triggered by adverse environmental conditions, such as, nutritional stress, oxygen tension, or redox changes. These signals are perceived by KinA-E HKs, which phosphorylate Spo0A, the master regulator of sporulation (Fig. 3) (LeDeaux and Grossman 1995, LeDeaux et al. 1995, Jiang et al. 1999, Fujita and Losick 2005, Aguilar et al. 2010). The activation of Spo0A is managed by a phosphorelay cascade emanating from Kin HKs (Jiang et al. 2000). For example, KinA-mediated phosphorelay involves Spo0F, Spo0B, and Spo0A (Burbulys et al. 1991). Activated Spo0A, in turn, represses the expression of AbrB, a transcription repressor of sporulation (Strauch et al. 1990). On the other hand, phosphatases RapA and Spo0E reset Spo0F and Spo0A to an unphosphorylated state (Ohlsen et al. 1994, Perego et al. 1994, Perego 2001). In B. anthracis, out of nine sensor HKs, five have been characterized and shown to initiate sporulation via this classic phosphorelay (Brunsing et al. 2005). Apart from these Kin HKs, two functional TCSs, BAS1213–1214 and BAS0540–0541, are characterized in B. anthracis with a possible role in sporulation (Gopalani et al. 2016, Gupta et al. 2018). The BAS1214 HK senses oxidative stress and phosphorylates its cognate RR, BAS1213. The phosphorylated RR thus increases its expression and that of sporulation kinase D, which causes a reduction in sporulation efficiency (Gupta et al. 2018). In the BAS0540–0541 TCS, the overexpression of the BAS0540 RR causes a reduction in spore counts, although the molecular mechanism is still unknown (Gopalani et al. 2016).

Figure 3.

Role of protein kinases during sporulation in Bacillus. The schematic diagram shows several pathways being regulated by phosphorylation during sporulation. Every pathway involves multiple proteins’ activity regulated through phosphorylation. Black solid arrows represent kinase-mediated substrate phosphorylation, red dotted arrows represent activation or upregulation, and black lines with terminal bars represent inhibition or downregulation.

The significance of TCSs is not limited to the initiation of sporulation, they are also involved in later stages of sporulation including forespore engulfment and mother cell lysis. In B. anthracis, LytSR TCS functions by dissipating proton motive force, acting as a signal for the induction of lrgAB and clhAB₂ transcription, which can affect sporulation efficiency and cell viability during the stationary growth phase (Chandramohan et al. 2009). In B. thuringiensis, the LytSR TCS is induced by the mother cell compartment sigma factor, SigE. Deletion of lytS-lytR causes defects in forespore engulfment by the mother cell. LytSR can induce the expression of a cell wall hydrolase, SpoIIP, however, the exact mechanism of this regulation is not known (Peng et al. 2017). Thus, multiple TCSs are important for controlling sporulation (Fig. 3).

The importance of Ser/Thr phosphorylation in context of sporulation in B. subtilis has been studied and reviewed extensively (Pompeo et al. 2016). Activation of prespore specific RNA polymerase (SigF) gene by Ser phosphorylation was one of the first discovered examples of regulatory Ser phosphorylation in B. subtilis (Min et al. 1993). This is achieved by the proteins present in the same operon: SpoIIAB (anti-SigF), SpoIIAA (anti-anti-SigF), and SpoIIE (serine phosphatase). SpoIIAB binds and inactivates SigF, while phosphorylation of SpoIIAA by SpoIIAB at a serine residue results in the release of SigF from the complex (Duncan and Losick 1993, Clarkson et al. 2004).

As cell-type-specific transcription factors are activated during different sporulating stages, the expression of specific kinases and phosphatases is also triggered during sporulation, indicating their requirement during this process (Kroos et al. 1999, Piggot and Hilbert 2004). For example, among the four characterized Hanks family Ser/Thr kinases in B. subtilis, the expression of YabT and PrkA peaks during sporulation initiation (Bidnenko et al. 2013, Yan et al. 2015). In B. subtilis, PrkA was shown to phosphorylate a 60-kDa protein in the crude extract at a Ser residue, but its functional relevance remained inconclusive due to lack of in-vivo data in the absence of this protein (Fischer et al. 1996). Later, the role of PrkA was linked to the sporulation process due to defective sporulation in prkA null mutant strain, possibly due to the downregulation of spore-specific transcription factor σK (Yan et al. 2015) (Fig. 3). Interestingly, another independent study in B. subtilis revealed sequence similarity of PrkA with ATP-dependent protease family proteins and PrkC-mediated regulation of PrkA protease activity was shown to be critical for the initiation of the sporulation process (Zhang et al. 2022). The expression of YabT kinase during sporulation initiation is required for the development and maturation of bacterial endospores in B. subtilis (Bidnenko et al. 2013). DNA binding proteins such as RecA, RacA, YabA, and single-stranded DNA binding proteins (Ssb) are the targets of YabT kinase (Bidnenko et al. 2013, Shi et al. 2014b, Derouiche et al. 2016, Garcia-Garcia et al. 2016, 2018) (Fig. 3). Among these, RecA (a DNA recombinase), and RacA (a chromosome-anchoring protein), are involved in maintaining the chromosomal integrity of the developing spore (Bidnenko et al. 2013, Shi et al. 2014b, Ramirez-Guadiana et al. 2016), while YabA is crucial for replication initiation during sporulation (Noirot-Gros et al. 2006, Garcia Garcia et al. 2018). Bacillus subtilis has two Ssb proteins, SsbA and SsbB, that are implicated in various pathways related to genome maintenance and natural competence (Lindner et al. 2004, Yadav et al. 2013, 2014, Paschalis et al. 2017). Mass spectrometry (MS) analysis revealed a novel phosphorylation site (Thr38) on the SsbA in B. subtilis (Derouiche et al. 2016), which was previously found to be phosphorylated on Tyr82 (Mijakovic et al. 2006) and Arg76 residues (Elsholz et al. 2012). This phosphorylation was primarily mediated by YabT, which enhanced the cooperative binding to single-stranded DNA (Derouiche et al. 2016). RecA is an SOS repair protein that plays an important role in bacterial DNA damage repair pathways (Nahrstedt et al. 2005, Million-Weaver et al. 2015, Torres et al. 2019). The deletion mutant of yabT and nonphosphorylatable mutant of RecA in B. subtilis exhibits same phenotype of increased sensitivity to DNA damage, indicating its role in DNA integrity maintenance (Bidnenko et al. 2013).

YabA negatively regulates replication initiation by decreasing cooperative binding of replication initiator DnaA to DNA (Noirot-Gros et al. 2006, Schenk et al. 2017). Phosphorylation of YabA by YabT kinase causes enhanced sporulation in B. subtilis, possibly correlating with decreased DNA replication and cell division (Garcia Garcia et al. 2018) (Fig. 3). YabT also phosphorylates the transition-phase transcriptional regulator AbrB, which is important for sporulation (Kobir et al. 2014). AbrB phosphorylation abrogates its DNA binding to the promoter of Spo0E, which is essential for maintaining the level of the active form of Spo0A-P (Molle et al. 2003, Shafikhani and Leighton 2004) (Fig. 3). YabT also phosphorylates EF-Tu, leading to decreased GTP hydrolysis and stabilization of interaction with the ribosome. This results in downregulation of protein synthesis by causing a halt in the protein elongation cycle in the cells undergoing a dormant state (Pereira et al. 2015). Furthermore, the functional implication of Ser/Thr phosphorylation in the context of sporulation has not been well-explored in the pathogenic Bacillus species. A recent report on B. anthracis demonstrated complete inhibition of the sporulation process in the absence of the STP PrpN (Gangwal et al. 2022). In a nutshell, these studies indicate the critical role of Ser/Thr phosphorylation in the sporulation pathway.

Bacillus spores have a multilayered structure consisting of exosporium (present in only a few species), a spore coat, cortex, and the core wall (Khanna et al. 2020). The spore coat is a thick sieve-like protein layer that protects the endospore. In B. subtilis, CotH, an atypical kinase present in the inner layer of the spore coat regulates the assembly of spore coat proteins (Naclerio et al. 1996, Nguyen et al. 2016, Scott and Newton 2016). CotH in B. subtilis and B. cereus acts as a kinase, phosphorylating two other spore coat proteins (CotB and CotG) on Ser residues, which is essential for efficient spore coat assembly (Nguyen et al. 2016, Freitas et al. 2020, Di Gregorio Barletta et al. 2022). Moreover, phosphorylation levels of CotB and CotG by CotH were shown to be sensitive to thermal variations, with higher efficiency at lower temperature (25°C) than at higher temperature (42°C) (Isticato et al. 2020, Di Gregorio Barletta et al. 2022). This makes CotH-mediated phosphorylation of CotB and CotG essential for proper spore coat morphogenesis (Fig. 3). Exosporium, an irregular-shaped layer consisting of hair-like projections is present in the outer layer of spore coat and is required for interaction with host cells and the surrounding environment (Bozue et al. 2007, Stewart 2015, Wang et al. 2016). In B. anthracis, ExsB, a CotG homolog present at the basal layer, is essential for stable attachment of exosporium to the spore coat. ExsB was found to be the highly phosphorylated exosporium protein, with at least 14 of its 19 Thr residues modified in its central region. This phosphorylation event is speculated to be controlled by a homolog of CotH, as occurs in the case of B. subtilis (McPherson et al. 2010, Freitas et al. 2020). Besides this, the deletion strain of McsB Arg kinase in B. anthracis showed defects in sporulation, in addition to cell growth and germination, confirming the role of Arg phosphorylation during the sporulation process (Singh et al. 2015) (Fig. 3).

Germination

The process of germination involves the progressive metabolic awakening of the dormant spore by reactivation of major biological processes including cell growth and protein synthesis machinery. This phenomenon is triggered by stimuli such as nutrient germinants that are sensed by surface receptors on the spore membrane and involve the temporal expression of about 30% of the bacterial genome as studied in the model organism B. subtilis (Keijser et al. 2007). Bacterial kinases are reported to be important in spore germination. The Ser/Thr kinase PrkC with surface-exposed PASTA domain, is a crucial germinant receptor, which mediates the muropeptide (small peptide fragments released from growing bacterial membranes)-dependent germination process in B. subtilis (Shah et al. 2008, Shah and Dworkin 2010, Squeglia et al. 2011) (Fig. 4). Furthermore, PrkC-mediated induction of the secretory peptidoglycan hydrolase YocH ensures the availability of muropeptides during the germination process by digestion of the surrounding bacterial peptidoglycan (Shah and Dworkin 2010). Mutant spores lacking prkC showed a muropeptide-dependent defective germination profile in B. subtilis and B. anthracis (Shah et al. 2008).

Figure 4.

Role of protein kinases during spore germination in Bacillus. The schematic diagram shows multiple proteins regulated by phosphorylation during the spore germination process. The phosphorylation of these proteins is shown by black solid arrows. Red dotted arrows represent activation/upregulation, and black lines with terminal bars represent inhibition/downregulation.

Following signal acquisition to exit dormancy, the protein machinery is activated to stimulate the growth of metabolically active vegetative cells (Sinai et al. 2015, Xing and Harper 2020). The translation factor EF-G is identified as a common substrate of PrkC/PrpC (Fig. 4), and its phosphorylation is important in the regulation of protein synthesis during germination in B. subtilis (Gaidenko et al. 2002, Shah et al. 2008, Shah and Dworkin 2010). As mentioned, the surface-exposed PASTA domain of PrkC can sense muropeptides, which activate PrkC and help in spore germination. Activated PrkC, in turn, transmits the signal inside the germinating spore and phosphorylates EF-G. This signaling module suggests the role of PrkC in exiting dormancy and facilitating spore germination through protein synthesis (Shah et al. 2008). Another translation factor, EF-Tu, was also identified as a common substrate of the PrkC/PrpC pair in B. subtilis and B. anthracis, though the functional relevance of this phosphorylation is still unknown (Absalon et al. 2009, Arora et al. 2013) (Fig. 4).

Arg phosphorylation also plays an important role during spore germination in B. subtilis (Zhou et al. 2019). A transposon-based genetic screen in B. subtilis showed that genetic disruption of the Arg phosphatase gene ywlE results in severe germination defects (Zhou et al. 2019). In this study, the significance of Arg phosphorylation in spore germination was also corroborated by an accelerated germination process in the absence of Arg kinase-McsB. Furthermore, Arg phosphoproteome of spores identified 18 proteins, including the translation factor Tig and housekeeping sigma factor SigA. The impact of Arg phosphorylation on these two proteins in the context of germination was assessed by using phosphomimetic and phosphoablative mutants and germination defects were observed in Tig (Arg45Asp) and SigA (Arg365Asp) mutants. The study showed arginine dephosphorylation of Tig and SigA as an important regulatory step for the re-establishment of bacterial transcriptional and translational machinery by enabling Tig association with ribosomes and SigA activation during the germination process (Zhou et al. 2019). Interestingly, the deletion of mcsB in B. anthracis leads to reduced germination efficiency, thus highlighting a strain-specific role of McsB in spore germination (Singh et al. 2015).

Apart from the protein synthesis pathway, the germination of bacterial spores involves the reprogramming of cellular metabolism. In fact, various studies have shown glycolytic enzymes as a target of Ser/Thr phosphorylation in B. subtilis and B. anthracis (Arora et al. 2012, Rosenberg et al. 2015, Virmani et al. 2019). In B. anthracis, phosphorylation of a glycolytic enzyme, enolase, by PrkC plays an important role in spore germination by modulation of enolase activity, expression, and cellular localization (Virmani et al. 2019) (Fig. 4).

In another class of atypical STKs, the Bacillus spore coat protein CotH was shown to be important during germination in B. subtilis (Naclerio et al. 1996). CotH-mediated Ser phosphorylation of two other spore coat proteins, CotB and CotG, is an essential requirement for spore germination (Nguyen et al. 2016). Although the mechanistic aspects of the observed germination defect have not been explored yet, it would be interesting to investigate the functional relevance of CotH in the Bacillus genus owing to the presence of its orthologs across different genera including spore-forming bacteria and even eukaryotic species (Nguyen et al. 2016) (Fig. 4).

Biofilm

Biofilm is a multicellular structure with complex cellular differentiation that helps bacteria to deal with external stresses such as nutrient deprivation, antibiotics, and low oxygen tension, allowing them to survive in adverse conditions (Hoiby et al. 2010, Kostakioti et al. 2013, Yin et al. 2019, Arnaouteli et al. 2021). As mentioned before, B. subtilis has five HKs (KinA–E) that autophosphorylate upon sensing environmental signals and regulate Spo0A phosphorylation (Burbulys et al. 1991). Apart from inducing sporulation, Spo0A is known to be vital in the formation of biofilm under low oxygen conditions, and deletion of spo0A restricts the cells to a monolayer pattern rather than a three-dimensional structure (Hamon and Lazazzera 2001, Mielich-Suss and Lopez 2015). Also, while KinA primarily controls sporulation, KinC-mediated activation of Spo0A is mainly linked to biofilm formation (Shemesh et al. 2010, Devi et al. 2015). With the aid of mathematical modeling, the activation of Spo0A by KinC was shown to be dependent on the bacterial growth phase and concentration of KinA (Chen et al. 2022). Another HK, KinD is reported to possess both kinase and phosphatase activities and is activated by lipoprotein Med (Aguilar et al. 2010, Banse et al. 2011). KinD-mediated phosphorelay fine-tunes Spo0A phosphorylation levels, thus becoming a switch to trigger either sporulation or biofilm formation (Aguilar et al. 2010). Small molecule inducers including l-malic acid released by tomato roots activate KinD, hence triggering the transcription of matrix-producing genes leading to bacterial biofilm formation on tomato roots (Chen et al. 2012). A combination of glycerol and manganese is also reported to initiate biofilm formation via specifically activating KinD-mediated signaling (Shemesh and Chai 2013). Also, in B. subtilis biofilm defects in spo0A mutants were rescued by mutations in abrB transcription factor, suggesting that Spo0A-mediated repression of abrB is essential for biofilm formation (Hamon and Lazazzera 2001).

In B. subtilis, the CssRS TCS stabilizes the biofilm by limiting the expression of the repressor tasA, thereby increasing the production of the extracellular matrix, which is crucial for successful biofilm formation (Steinberg et al. 2020). Also, the deletion of cssRS causes a sharp increase in the population of motile bacteria (Steinberg et al. 2020). Even after the biofilm is formed, another TCS, DegS-DegU induces the secretion of YIT toxin within the biofilm to destroy sensitive cells and attack any incoming competitor cells (Kobayashi and Ikemoto 2019). DegSU also controls the expression of extracellular proteases and enzymes that are crucial for biofilms (Kobayashi 2007). In rhizobacterium, B. amyloliquefaciens, the ResDE TCS is reported to sense oxygen deprivation. It triggers biofilm formation by increasing the expression of terminal oxidases (Zhou et al. 2018). Apart from histidine kinases, Ser/Thr kinases also regulate biofilm formation and maintenance (Fig. 5).

Figure 5.

Role of protein kinases in Bacillus biofilm formation. Biofilm formation in bacteria is a complex process and requires regulation at several steps. The figure is a schematic representation of phosphorylation-mediated regulation of biofilm formation in bacteria. As shown, different classes of kinases and their substrates are required for biofilm formation. Black solid arrows represent kinase-mediated substrate phosphorylation, red dotted arrows represent activation/upregulation, and black lines with terminal bars represent inhibition/downregulation.

The formation of biofilm, as already mentioned, depends on environmental cues to which the bacteria are exposed. These cues are sensed by surface proteins that mediate proper colonization. The Ser/Thr kinase PrkC, which possesses a surface-exposed sensor domain is crucial for biofilm formation. Interestingly, prkC deletion in B. anthracis also leads to the abrogation of biofilm formation. Mechanistic insights on the signaling pathway mediated by Ser/Thr phosphorylation driving biofilm formation in B. anthracis have emerged from the linkage of PrkC and one of its substrates, the GroEL chaperone (Arora et al. 2017b). GroEL has also been shown to be involved in biofilm formation in various other organisms like Mycobacteria and Streptococci (Ojha et al. 2005, Yin et al. 2019). MS studies revealed GroEL as one of the substrates of PrkC, and overexpression of native GroEL resulted in partial resumption of biofilm formation in a biofilm-defective prkC deletion strain (Fig. 5) (Arora et al. 2017b). Furthermore, phosphorylation of YabA by Ser/Thr kinase YabT negatively regulates biofilm formation by increasing the cellular level of Spo0A-P, the key regulator of genes involved in biofilm formation and sporulation (Garcia Garcia et al. 2018). Although the mechanism of tyrosine phosphorylation in biofilm formation is still unknown, null mutant strains of ptkA and ptpZ showed an altered biofilm phenotype in B. subtilis (Kiley and Stanley-Wall 2010). The biofilm formed in these conditions showed loss of “fruiting bodies” for sporulation and the absence of typical biofilm complex radial structures. The reason for this phenotype was attributed to the defective sporulation efficiency of the bacterial cells that were growing in the biofilm colony. Complete loss of biofilm formation was observed in the strains lacking both the BY kinases, ptkA and ptkB (Gerwig et al. 2014). Apart from this, flagellar motility of several Bacillus species that helps the bacteria to swim and slide across surfaces is often linked to biofilm initiation and development (Houry et al. 2010, Guttenplan and Kearns 2013, Liaqat et al. 2018, Li et al. 2022). Inhibition of motility promotes biofilm formation and in B. subtilis, flagellar motility and swarming properties of the bacteria are regulated by the phosphorylation status of the transcriptional regulator DegU by its cognate HK, DegS (Verhamme et al. 2007, Murray et al. 2009). Altogether, these studies indicate the central role of protein phosphorylation during biofilm formation in Bacillus (Fig. 5).

Stress response

Bacterial populations encounter various stressful conditions throughout their life cycle ranging from alterations in temperature, pH, osmolytes, nutrient deficiency, and exposure to antibiotics. To combat this, bacteria have evolved sophisticated stress responses that involve a wide range of cellular and morphological changes (Marles-Wright and Lewis 2007, Ultee et al. 2019, Cheng-Guang and Gualerzi 2020). This section presents current knowledge on the role of protein phosphorylation during stress conditions in Bacillus sp. including activation of SigB, the master regulator of general stress response (GSR) via a protein phosphorylation cascade involving Rsb (Regulator of sigma B) family proteins (Fig. 6).

Figure 6.

Ser/Thr phosphorylation in the activation of the SigB (σ^B) regulon. In unstressed conditions, the RsbT kinase forms a complex with other Rsb family proteins (RsbS antagonist and RsbR coantagonist with its paralogues). Upon environmental stress, the RsbT kinase phosphorylates RsbS and RsbR with its paralogues at Ser/Thr residues and is released from the complex. RsbT then interacts with the RsbU phosphatase resulting in its activation and dephosphorylation of a downstream antagonist, RsbV, which is further regulated by RsbW-mediated phosphorylation. Unphosphorylated RsbV dissociates the σ^B–RsbW kinase complex by binding to RsbW, thereby releasing σ^B, which in turn associates with RNA Polymerase and activates σ^B regulon gene expression. The RsbX phosphatase resets the entire stressosome machinery by dephosphorylation of RsbS and RsbR. Under energy-deficient conditions, a separate pathway is activated involving dissociation of another complex comprised of the RsbP phosphatase and a stress RR, RsbQ. The released RsbP results in the dephosphorylation of RsbV, which triggers downstream signaling pathways. During cold stress, bacteria respond by triggering another independent pathway involving RsbW. The complex of RsbW and σ^B modulates the expression of genes regulated by σ^B.

Protein Arg phosphorylation in the Bacillus is primarily linked to the maintenance of overall protein quality by regulated proteolysis of misfolded or aggregated proteins during stress conditions (Mijakovic et al. 2016). This is achieved by a regulatory system that involves McsB (Arg kinase)-mediated inhibition of CtsR (stress response transcriptional regulator) repressor activity, activation of the ClpCP proteolytic machinery, and phospho-Arg tagging of the misfolded proteins (Fuhrmann et al. 2009, Elsholz et al. 2011, Tao et al. 2012, Trentini et al. 2016). The kinase activity of McsB is further modulated by its repressor CtsR, binding of phospho-Arg polypeptides at its catalytic site, ClpC (stress response-related ATPase, AAA+ superfamily), McsA (Arg kinase activator protein), and finally by the activity of its cognate Arg phosphatase (YwlE) (Kirstein et al. 2005, 2007, Elsholz et al. 2011, 2012, Fuhrmann et al. 2013, Suskiewicz et al. 2019). Strains lacking mcsB and clpC in B. anthracis show growth defects at elevated temperature (43°C) (Singh et al. 2015). The importance of Arg phosphorylation in stress response pathways was demonstrated by the global phosphoproteome of the B. subtilis Arg phosphatase (ywlE) mutant strain. Phospho-Arg sites were detected in proteins involved in GSR (controlled by SigB), such as stress on the cell envelope by antibiotics and osmolarity changes, heat shock, cold shock, and oxidative stress (Elsholz et al. 2012). Given the widespread role of Arg phosphorylation in bacterial stress response, another Arg phosphoproteome study, focusing specifically on heat and oxidative stress, identified key bacterial stress RRs such as CtsR, GroEL, ClpC, and ClpP as targets of Arg phosphorylation in B. subtilis (Schmidt et al. 2014). Furthermore, the YwlE phosphatase is inactivated during oxidative stress conditions as a regulatory mechanism for the induction of stress-related genes and McsB-mediated Arg phosphorylation (Fuhrmann et al. 2016). These studies highlight the importance of protein Arg phosphorylation during stress conditions in Bacillus.

The documented role of Tyr phosphorylation in Bacillus stress pathways, on the other hand, is limited to only a few studies. The chaperone protein DnaK (Hsp70 family protein) is a widely studied heat shock protein, i.e. a member of chaperone machinery activated during stress conditions and is also reported to be a target of protein phosphorylation in other bacteria (Sherman and Goldberg 1993, Seeger et al. 1996, Peake et al. 1998, Mayer and Bukau 2005, Roncarati and Scarlato 2017, Rigo et al. 2020). DnaK was identified as a substrate in Ser/Thr/Tyr phosphoproteomes (Eymann et al. 2007). Later, it was identified that PtkA/PtpZ-mediated regulation of DnaK by phosphorylation at a Tyr residue was responsible for controlling its chaperone activity, which affects survival of B. subtilis under heat shock conditions. (Shi et al. 2016). Deletion mutants of two low molecular weight Tyr phosphatases, YwlE (also an Arg phosphatase) and YkfJ showed reduced bacterial resistance to ethanol stress in B. subtilis, suggesting their possible roles in generating resistance to stress (Musumeci et al. 2005).

Various environmental stresses encountered by bacteria activate specific TCSs. Two different TCS can activate the same set of downstream target genes. One such example is the Heme sensing TCS (HssRS) and the HssRS-interfacing TCS (HitRS) in B. anthracis (Mike et al. 2014, Pi et al. 2020). Alterations in cell envelope integrity activates HitRS, that in turn interacts with HssRS and coordinate heme and cell envelope stress response (Mike et al. 2014). Similarly, the HssRS and HitRS TCSs exist in B. thuringiensis, which indirectly control the growth in the presence of heme through the uncharacterized operon hrmXY (Schmidt et al. 2016).

In B. subtilis, adaptation to cold temperature is achieved by the DesKR TCS. DesK (HK) senses the ordered lipid pattern in the membrane and autophosphorylates, followed by DesR (RR) phosphorylation (Albanesi et al. 2004, Abriata et al. 2017). Active phospho-DesR then binds to the promoters of genes involved in the synthesis of the fatty acid desaturase DesA (Cybulski et al. 2004). DesK also responds to changes in pH; acidic pH breaks hydrogen bonds in the helix connecting the DesK transmembrane domain to the cytosolic domain, and thus abolishes the DesK-dependent synthesis of unsaturated acids, providing rigidity to the bacilli (Bortolotti et al. 2020). Apart from temperature and pH sensing, DesKR is highly expressed in low-pressure conditions in B. subtilis strain WN1106 (Fajardo-Cavazos et al. 2012). In B. cereus, adaptation to a cold environment occurs by higher expression of unsaturated fatty acid to maintain membrane fluidity at a lower temperature (Diomande et al. 2014). The CasKR TCS activates the expression of fatty acid desaturase DesA under cold conditions by ceasing repression of the desA promoter (Diomande et al. 2015, 2016).

In B. anthracis, a novel TCS named the Envelope Disruption System EdsRS is critical for managing the stress induced by targocil, an inhibitor that targets the cell envelope (Laut CL 2020). Compromised membrane integrity is detected by EdsS (HK), which results in its autophosphorylation and subsequent phosphorylation of EdsR (RR). Phosphorylated EdsR binds and activates the promoter of the BAS1661–BAS1663 operon encoding the cardiolipin synthase ClsT for the repair of the cell envelope in targocil-exposed vegetative cells. Exposure of Bacillus spores to targocil is reported to be highly toxic, as spore outgrowth requires rapid membrane generation (Laut CL 2020). In B. thuringiensis, the YvqEC and YvcPQ TCSs sense disturbances in the cell envelope and provide resistance against the cell wall targeting compounds vancomycin and bacitracin, respectively (Zhang et al. 2015, 2016). In B. subtilis, the LiaSR TCS manages the exogenous stress induced by the presence of peptide antibiotics targeting cell wall synthesis (bacitracin, nisin, vancomycin, and ramoplanin) (Mascher et al. 2004, Kesel et al. 2013). The LiaR-dependent promoter liaI is expressed during the transition from the exponential to stationary phase, suggesting an additional sensor signal for LiaS, other than antibiotics (Jordan et al. 2006, 2007). In B. subtilis, BceRS is a dedicated TCS that responds to the presence of bacitracin via its specific binding to BceB (permease) (Ohki et al. 2003, Dintner et al. 2014). BceS (HK) works in coordination with BceB (permease) and the level of BceS (HK) autophosphorylation is dependent on the BceA-bacitracin complex (Dintner et al. 2014).

WalRK plays an important role in cell wall homeostasis under physiological conditions in B. subtilis. Apart from this, WalRK is equally important for growth and cell proliferation under heat stress (Takada et al. 2018). WalK (HK) is activated upon heat stress and its activity is negatively modulated by two membrane proteins WalH and WalI (Takada et al. 2018). The presence of WalH and WalI is crucial to avoid overexpression of downstream endopeptidase genes, thus avoiding cell lysis. Even in B. anthracis, the expression of WalRK is induced either by temperature or in the presence of the bactericidal drug fosfomycin, an inhibitor of cell wall synthesis (Dhiman et al. 2015). In B. subtilis, the CssRS TCS is important in managing secretion stress, which happens when misfolded proteins accumulate outside the membrane and interfere with protein secretion. This inefficient protein secretion is detected by CssRS, which then increases the expression of the chaperonic proteases HtrA and HtrB (Westers et al. 2006, Noone et al. 2012).

Another bacterial GSR involves the activation of sigma factor, SigB (master regulator of GSR), which regulates the expression of over 100 genes in B. subtilis during stress conditions (Haldenwang and Losick 1979, Bernhardt et al. 1997, Hecker and Völker 1998). Activation of the SigB regulon is achieved by a signaling cascade that involves Ser/Thr phosphorylation of Rsb (regulator of sigma factor) family proteins comprising TCS such as Ser/Thr kinases (RsbT and RsbW); PP2C-type phosphatases (RsbP, RsbX, and RsbU); antagonists (RsbS and RsbV), and coantagonists (RsbRA, RsbRB, RsbRC, and RsbRD) (Price et al. 2001, Hecker et al. 2007, Rodriguez Ayala et al. 2020).

In normal conditions, Ser/Thr kinase RsbW phosphorylates the antagonist RsbV, causing its inactivation and also acts as a negative regulator of SigB that blocks its binding to the RNA polymerase core enzyme (Alper et al. 1996, Yang et al. 1996, Rodriguez Ayala et al. 2020). Environmental stress conditions (acid/ethanol/salt) trigger dephosphorylation of RsbV by RsbU. RsbU is activated by interacting with RsbT Ser/Thr kinase that acts as a mediator and conveys environmental stress signals to initiate the downstream signaling. In the absence of stress, RsbT is entrapped in a 25S multiprotein oligomeric complex consisting of RsbS (antagonist) and RsbRAs (coantagonist) and its paralogues such as YkoB (RsbRB), YojH (RsbRC), YqhA (RsbRD), and YtvA (Akbar et al. 2001, Chen et al. 2003, Kim et al. 2004a). Under stress conditions, RsbT phosphorylates RsbRA, which facilitates phosphorylation of RsbS. Then RsbT is released from the complex, thus activating RsbU (Chen et al. 2004, Kim et al. 2004b). Activated RsbU dephosphorylates RsbV, which then binds to the RsbW kinase (RsbV–RsbW), thereby dissociating the RsbW–SigB complex. The SigB released from the complex binds to RNA polymerase, thus activating the SigB regulon (Dufour and Haldenwang 1994). This signaling pathway is reset by RsbX STP that dephosphorylates RsbS and RsbRA, and serves as a fine-tuning mechanism resulting in the sequestration of RsbT in the stressosome complex (Yang et al. 1996, Price et al. 2001, Chen et al. 2004). Furthermore, the absence of the RsbX protein in the B. subtilis system results in uncontrollable activation of the SigB regulon (Voelker et al. 1997) (Fig. 6). Under energy stress conditions (glucose, oxygen, and phosphate starvation), RsbV dephosphorylation is mediated by a complex of RsbP phosphatase and RsbQ hydrolase, thus activating the SigB regulon (Vijay et al. 2000, Brody et al. 2001). In the case of cold shock, the signaling pathway works independently of RsbP, RsbU, and RsbV. The degree of stability of the RsbW–SigB complex regulates the silencing or transcription of stress-related genes (Brigulla et al. 2003) (Fig. 6).

In B. cereus, SigB activation is achieved by a protein complex comprising RsbK (sensor kinase), RsbW (kinase), RsbV (antagonist), and RsbY (phosphatase). Following stress conditions, the sensor kinase RsbK phosphorylates and activates RsbY, which in turn dephosphorylates the RsbV antagonist. Unphosphorylated RsbV sequesters RsbW (a negative regulator of SigB), thus activating the SigB regulon (van Schaik et al. 2005, de Been et al. 2010).

Virulence

While the majority of Bacillus species are nonpathogenic, a few of them belonging to the B. cereus group are known to cause disease in animals and humans (Ehling-Schulz et al. 2019). The pathogenicity of this group is attributed to two major factors-secreted toxins or capsule. These factors are paramount for the survival of the bacteria inside the host, and thus it is important to study the network that regulates their synthesis for the development of novel prophylactics (Moayeri et al. 2015). In B. anthracis, the most studied protein, which is involved in the synthesis of toxins and capsule is the master virulence regulator, AtxA (Anthrax toxin activator) (Fouet 2010, McCall et al. 2019). The B. anthracis null mutant of atxA is avirulent in the mouse model of infection (Dai et al. 1995). Furthermore, the cellular level of AtxA decides the fate of associated pathways and thereby modulates the pathogenic cycle. High levels of AtxA in the cell are conducive to toxin gene expression while inhibiting sporulation in B. anthracis (Dale et al. 2018). The global transcriptional regulators CodY and AbrB regulate the synthesis of AtxA (Strauch et al. 2005, van Schaik et al. 2009, Gangwal et al. 2022), and are themselves targeted by Ser/Thr phosphorylation machinery (Macek et al. 2007, Kobir et al. 2014). This regulation is achieved either by direct binding of the transcriptional regulator to the promoter region of the toxin-producing genes (Dale et al. 2012, Chateau et al. 2013) or by an unknown post-translational mechanism (van Schaik et al. 2009).

In the nonpathogenic B. subtilis, phosphorylation of AbrB at a Ser residue inhibits its DNA binding property (Kobir et al. 2014), whereas in B. anthracis, CodY phosphorylation at a Ser residue abrogates its DNA binding to the atxA promoter, leading to depletion of toxin proteins (Gangwal et al. 2022). In B. anthracis, PTS-mediated phosphorylation of AtxA at conserved His residues in the PRD domain (PTS-regulated domain) regulates its activity and affects toxin synthesis (Tsvetanova et al. 2007, Raynor et al. 2018). Interestingly, PTS proteins HPr and Enzyme I were also shown to be required for atxA expression and their deletion mutant B. anthracis strain showed defective toxin production and attenuated virulence in an animal model of anthrax (Bier et al. 2020). More recently, another STP—PrpN was shown to regulate AtxA synthesis via phosphorylation-mediated regulation of CodY DNA binding activity. CodY was confirmed as a target of the PrkC and PrpN and the null mutant strain of prpN demonstrated a significant reduction in anthrax toxins and AtxA. Functional implication of PrpN-mediated dephosphorylation on CodY activity was examined using phosphomimetic and phosphoablative CodY mutants. While phosphorylation of CodY at a Ser residue resulted in complete abolishment of its DNA-binding ability at the atxA promoter region, unphosphorylated CodY was reported as a positive regulator of anthrax toxins (Gangwal et al. 2022). This highlights the importance of protein Ser/Thr and His phosphorylation in maintaining the optimum AtxA level for toxin production (Fig. 7).

Figure 7.

Role of protein kinases in Bacillus virulence. The pathways associated with virulence are activated in pathogenic species such as B. anthracis. These pathways follow specific signals during the infection stage within the host, which activate a unique set of signaling events emanating usually from surface-bound kinases and phosphatases. The schematic shows these specific proteins and their interacting partners, which carry out the indicated functions. Black solid arrows represent kinase-mediated substrate phosphorylation, red dotted arrows represent activation/upregulation, and black lines with terminal bars represent inhibition/downregulation.

Toxin biosynthesis is often correlated with carbon sources in pathogenic bacteria (Poncet et al. 2009, Chiang et al. 2011). In Bacillus sp., this is mediated by a carbon catabolite protein (CcpA) and phosphotransferase system (PTS) component, HPr, via the classical carbon catabolite repression (CCR) mechanism (Khan and Banerjee-Bhatnagar 2002, Lorca et al. 2005, Singh et al. 2008). Hpr is phosphorylated by Hpr Kinase (Poncet et al. 2004, Choi et al. 2006) and PrkC (Macek et al. 2007, Pietack et al. 2010). While the functional relevance of phosphorylation by PrkC is still unknown, the phosphorylation by Hpr kinase plays an important role in modulating Hpr activity and binding to CcpA, resulting in differential expression of multiple genes in B. subtilis (Blencke et al. 2003, Deutscher et al. 2014) (Fig. 7).

Chain formation is another factor that contributes to the virulence of bacteria including B. anthracis by helping the pathogen to evade the host immune response (Moller et al. 2012, Rodriguez et al. 2012, Prashar et al. 2013, Jouvion et al. 2016). In B. anthracis, prkC deletion results in severe defects in chain formation as compared to wild type strain, indicating the role of PrkC in the regulation of chain length in B. anthracis (Dhasmana et al. 2021). Earlier studies have also shown that B. anthracis lacking the prkC/prpC pair showed impaired survival within macrophages and attenuated virulence in an animal model (Shakir et al. 2010). These studies provide a possible regulatory link between Ser/Thr phosphorylation and virulence via chain formation in B. anthracis.

When B. anthracis infects its host, it secretes lethal and edema toxins, which target smooth muscle cells and epithelial cells, exacerbating the infection. The BrrAB TCS regulates the expression of these toxins during infection. Deletion of any one of the components of BrrAB causes downregulation of atxA and a significant reduction in the toxin-producing ability of the pathogen, thus decreasing the virulence (Vetter and Schlievert 2007). In addition to exotoxins, secreted proteases facilitate the infection via degrading the host tissue and mobilizing the bacteria inside the host (Chung et al. 2006). A novel TCS encoded by BAS2109–2108 regulates the expression of eight intracellular and extracellular proteases (Gupta et al. 2017). Activated BAS2108 (HK) transfers the phosphate group to its cognate RR, BAS2109, which binds to the promoters of many proteases, such as metalloendopeptidase, bacillolysin, and several Ser proteases; few of these proteases are involved in pathogenesis. Additionally, the expression of BAS2109 (RR) increases in the presence of carbon dioxide or nutritional stress (Gupta et al. 2017) (Fig. 7).

The food-borne pathogen B. cereus has a TCS, ResDE, which controls the production of hemolysin BL and nonhemolytic enterotoxins under anaerobic conditions, thus controlling levels of virulence (Duport et al. 2006). ResE responds to the acidic environment and oxygen limitation in the gut and helps bacteria to secrete extracellular toxins to establish infection (Duport et al. 2006). YvfTU TCS controls the expression of a major pleiotropic virulence gene regulator, PlcR in B. cereus (Gohar et al. 2008, Brillard et al. 2008). Although plcR expression is decreased in yvfTU mutant bacilli, there are only minor differences reported in the expression of the plcR regulon (Brillard et al. 2008).

Cross-talk and cross-phosphorylation

A recent surge in the number of protein–protein interactome and phosphoproteome studies of bacterial systems including that of Bacillus have revealed multisite phosphorylation of several proteins by different kinases. These kinases exhibit promiscuous activity and can phosphorylate each other as well as have common substrates, thereby contributing to the biological complexity, adding another regulatory layer to the protein phosphorylation network in the bacterial system (Lin et al. 2009, Chao et al. 2010, Kobir et al. 2011, Baer et al. 2014, Shi et al. 2014a, b, Willett and Crosson 2017, Pinas et al. 2018, Shen et al. 2020). To differentiate these two regulatory networks, we have used the term “cross-talk” to define phosphorylation and dephosphorylation of a substrate at multiple amino acid residues by different kinases/phosphatases and “cross-phosphorylation” to define phosphorylation/dephosphorylation between different kinases/phosphatases (Fig. 8).

Figure 8.

Cross-talk and cross-phosphorylation in Bacillus. (A) Cross-talk network representing multisite phosphorylation of proteins by different classes of kinases and phosphatases in Bacillus. A protein–protein interaction network was created using Cytoscape 3.8.2 software. Interaction among different kinases/phosphatases and their substrates are represented with colored nodes and colored lines. STK/STP: Ser/Thr kinase and phosphatase, BY-K/PTP: Bacillus Tyr kinase and phosphatase, and Hpr K: Hpr kinase. Among the kinases depicted in the figure, only PrkC and PrpC have validated substrates in both B. anthracis and B. subtilis (labeled BA in parentheses). (B) The cross-phosphorylation network between kinases is shown by a protein–protein interaction network. Different classes of kinases and phosphatases are represented by colored nodes, and interaction among different kinases/phosphatases are determined by colored arrows and lines. Blue arrows represent validated in-vitro assays, pink lines indicate phosphoproteome validations, and orange lines denote yeast two-hybrid interactions between bait and prey. Arrows point to the target proteins that are phosphorylated.

In B. subtilis, an interaction network linking different classes of protein kinases/phosphatases was generated using yeast two-hybrid interactome studies and in-vitro kinase assays (Shi et al. 2014a, b). A direct interaction was predicted between Ser/Thr kinase-YabT and BY-kinase/PTP PtkB and cognate Phosphatase PtpZ. An indirect interaction was also observed via TkmA (the specific modulator for BY-kinase PtkA) between YabT and another BY-kinase, PtkB. The two BY-kinases, PtkA and PtkB show possible cross-interaction through the PtkA-modulator TkmA. Also, the Tyr phosphatase PtpZ interacts with TkmA and PtkA. RsbT, a two-component-like Ser/Thr kinase regulating the stressosome complex of B. subtilis interacts with BY-kinase/phosphatase PtkA/PtpZ and Ser/Thr kinase PrkD. DegS, an HK, was shown to be phosphorylated by Ser/Thr kinases including interacts with the PrkD and YabT/SpoIIE pair (Jers et al. 2011, Shi et al. 2014a, b).

Furthermore, cross phosphorylation among the kinases has been defined by categorizing the kinases into two broad classes. The first class consists of PrkD, PrkC, YabT, and PtkA kinases that can autophosphorylate and phosphorylate other kinases. Among these, PrkC, PrkD, and YabT show intermolecular phosphorylation, while YabT and PtkA can cross-phosphorylate each other. Also, PrkC can phosphorylate PtkA near its autophosphorylating site. The other class includes PtkA and two-component Ser/Thr kinases such as RsbW and SpoIIAB, which are phosphorylated by other Ser/Thr kinases. Additionally, HPr kinase/phosphorylase (PTS component) is phosphorylated by PrkD, PrkC, and PtkA, while RsbT interacts with PrkD, and YabT (Shi et al. 2014a). Another example of cross-phosphorylation involves phosphorylation of TCS proteins DegS (HK) and WalR (RR) by STKs. Phosphorylation of DegS by STKs (PrkD and YabT) at the Ser76 residue enhances the phosphotransfer to DegU (RR) and was found to be important in the regulation of competence development, complex colony formation, and swarming (Macek et al. 2007, Jers et al. 2011). Furthermore, PrkC-mediated phosphorylation of WalR (RR) at Thr101 regulates the expression of genes involved in cell wall metabolism (Libby et al. 2015). These studies suggest different kinase classes work in tandem in a network and illustrate the biological complexity of cross-phosphorylation in Bacillus genus (Fig. 8).

Interestingly, in a high-throughput experiment, multisite phosphorylation of substrates was predicted with different classes of kinases/phosphatases. For example, RecA is phosphorylated by YabT, PtkA and Arg kinase (Soufi et al. 2010, Elsholz et al. 2012, Bidnenko et al. 2013, Shi et al. 2014b). Phosphorylation of SalA by PtkA was also validated by an in-vitro kinase assay (Derouiche et al. 2015). Another phosphoproteome study reported phosphorylation of known substrates of Tyr kinase PtkA such as aspartate semialdehyde dehydrogenase (Asd), Enolase (Eno), and YjoA at Ser/Thr residues (Jers et al. 2010, Ravikumar et al. 2014). Out of these, Eno was identified as a substrate of the McsB/YwlE pair in an Arg phosphoproteome analysis (Schmidt et al. 2014). In B. anthracis Eno was also shown to be phosphorylated at Ser/Thr residues by PrkC (Virmani et al. 2019). This PrkC-mediated multisite phosphorylation of Eno imprints phenotypic memory in B. anthracis spores and is important for spore germination. The global transcriptional regulator AbrB is also phosphorylated at Ser and Tyr residues. Phosphorylation at the Ser residue is mediated by STKs (PrkC/PrkD/YabT), while Tyr phosphorylation was detected in the B. subtilis phosphoproteome (Kobir et al. 2014, Ravikumar et al. 2014).

Cross-talk among B. subtilis phosphorylation pathways involve Ssb (SsbA and SsbB) required for DNA replication that are phosphorylated by PtkA and McsB (Mijakovic et al. 2006, Elsholz et al. 2012). Interestingly, SsbA is also phosphorylated by Ser/Thr kinases (PrkD, PrkC, and most efficiently by YabT), which aids in the cooperative binding of SsbA tetramers to DNA (Derouiche et al. 2016). Another example of cross-talk involves phosphorylation of a PTS protein, Hpr, at multiple residues. Phosphorylation at Ser residues is mediated by either a bifunctional ATP dependent Hpr kinase/phosphorylase (Reizer 1989) or Ser/Thr kinase/phosphatase pair PrkC/PrpC (Singh et al. 2007, Pietack et al. 2010), while phosphorylation at His is catalyzed by another PTS protein, Enzyme I (Reizer 1989). Phosphorylation of Hpr at the Ser/Thr/Tyr residues was also observed in phosphoproteome studies of B. subtilis (Ravikumar et al. 2014, 2018) (Fig. 8).

Arg phosphoproteome analysis identified several substrates such as GroEL, DegU, and the elongation factors EF-Tu and EF-G in B. subtilis that were previously shown to be phosphorylated at other residues (described in above sections), indicating multisite phosphorylation and possible cross-talk among the different families of kinases (Elsholz et al. 2012, Schmidt et al. 2014, Trentini et al. 2014). EF-Tu and EF-G were also identified as targets of Tyr phosphorylation in the B. subtilis germinating spore phosphoproteome (Rosenberg et al. 2015).

Compared to B. subtilis, B. anthracis has lost some Tyr kinases during evolution. Consequently, two unique DSPKs, PrkD, and PrkG, were identified in B. anthracis, belonging to different classes defined on the basis of their target residues. PrkD belongs to “dual-specificity tyrosine phosphorylation-regulated kinase” (DYRK), which autophosphorylates on Ser, Thr, and Tyr but phosphorylates its substrates only on Ser and Thr. PrkG was identified as a bonafide DSPK that can autophosphorylate and mediate substrate phosphorylation on Ser/Thr/Tyr residues. Interestingly, the STP PrpC also exhibits dual specificity (Arora et al. 2012). It can dephosphorylate both PrkD and PrkG, in addition to PrkC (Obuchowski et al. 2000, Shakir et al. 2010).

Conclusions and future directions

In this review, we assessed the role of protein phosphorylation in different life stages of Bacillus species (Table 1). Bacillus sp. have the ability to metamorphosize into different forms such as spores, vegetative cells, or biofilms, which is vital for their survival in the environment or in the host. The shift in metabolic needs between these stages is a complex process (Fouet and Mock 2006, Tan and Ramamurthi 2014, Christie and Setlow 2020, Setlow and Christie 2020). Besides basic control of gene expression, PTMs such as phosphorylation, provide an additional layer of regulation. Over the years, the importance of His/Asp and Ser/Thr/Tyr protein phosphorylation has been unraveled in different cellular pathways. More recently, phosphorylation of other residues including Lys, Arg, and Cys have also been discovered. Genetic regulation extends beyond the identification of direct signaling pathways, as many of these phosphorylation events are controlled by more than one phosphorylation scheme, as discussed in the cross-talk section.

Most of the early research on the role of protein phosphorylation was performed using the model organism B. subtilis (Cozzone 1988, Perego et al. 1989, Mukai et al. 1990). Over the years, it has been established that protein phosphorylation schemes are ubiquitous throughout the Bacillus species, though some members of the Cereus clade have lost some Tyr kinases and gained dual-specificity protein kinases that can perform the function of both Ser/Thr and Tyr protein phosphorylation (Arora et al. 2012).

Of the different phosphorylation systems, early discoveries emphasized the role of His/Asp-based TCSs in sensing environmental stress and regulating cellular behavior, while recent advances in this field indicate that Ser/Thr/Tyr/Arg protein phosphorylation may play an even larger role. Though many phosphorylated proteins have been recognized in recent literature, our understanding of upstream sensing stimuli is still limited. Among the Ser/Thr protein kinases, the role of muropeptides as sensory signals to PrkC is well-established (Dworkin and Shah 2010, Squeglia et al. 2011, Pompeo et al. 2018b). Recent studies on the PrkC homolog PknB in Mycobacterium tuberculosis indicate that lipid moieties can also be sensory signals (Kaur et al. 2019). Further work is needed to understand if in addition to muropeptides, lipid II, can also act as sensing signals to PrkC. These studies show that protein kinase signaling in Bacillus species is highly evolved and regulated by specific sensory signals.

Recent advancement in the MS techniques have facilitated increase in phosphoproteome studies. They range from nonspecific simple gel-based studies to more specific and complex MS studies. Improved phosphopeptide enrichment methods coupled with novel MS-based systemic approaches provides a better blueprint of the global phosphoproteome (Liu et al. 2021, Paulo and Schweppe 2021, Pina et al. 2021, Xiao et al. 2023). An in-depth description of these techniques is beyond the scope of this review but has been reported elsewhere (Engholm-Keller and Larsen 2013, Ed Dudley 2014, Yague et al. 2020). Importantly, phosphorylation at specific residues in a protein differs in lability, stoichiometry, and abundance. For instance, compared to phosphorylation at other residues, phospho-Asp/His and Arg are relatively unstable (Gonzalez-Sanchez et al. 2013, Huang et al. 2021). For this reason, enrichment and phosphoproteome studies on Asp/His/Arg phosphorylation are challenging and extensive research is needed in this field.

Unlike protein kinases, there is limited literature on the role of cognate protein phosphatases in cellular processes (Kennelly 2002, Sajid et al. 2015). Also, many regulatory mechanisms of phosphatases have been reported. For example, in M. tuberculosis, in a feedback loop system, PstP (homolog of B. anthracis PrpC) is phosphorylated by its cognate kinases PknA and PknB, causing its activation, though a similar mode of regulation has not been reported in Bacillus (Sajid et al. 2011b). The phosphatases of stressosome complexes are also shown to be regulated by a similar mechanism (Misra et al. 2019). The existing literature also suggests that similar to eukaryotes, a cross-talk and cross-phosphorylation network is also present in the bacterial systems and might provide another layer of regulation for phosphorylation pathways, thus enabling the efficient regulation of principal processes such as growth, DNA repair, cellular metabolism, and stress response.

In conclusion, phosphorylation is the key regulator of every aspect of Bacillus life, yet information about what triggers these events and how they maintain control over different pathways and cross-talk with each other is somewhat limited. Having several conserved processes, the Bacillus species are one of the best prokaryotic model systems utilized to study various aspects of bacterial physiology (Getz et al. 2019). Continued studies in this field can help to unravel more bacterial signaling systems and networks.

Conflict of interest

None declared