University of Groningen Cell envelope related processes in Bacillus subtilis van den Esker, Mariëlle Henriëtte

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Cell envelope related processes in Bacillus subtilis

van den Esker, Mariëlle Henriëtte

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Publication date: 2018

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van den Esker, M. H. (2018). Cell envelope related processes in Bacillus subtilis: Cell death, transport and cold shock. Rijksuniversiteit Groningen.

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521545-L-bw-vdEsker 521545-L-bw-vdEsker 521545-L-bw-vdEsker 521545-L-bw-vdEsker Processed on: 25-7-2018 Processed on: 25-7-2018 Processed on: 25-7-2018

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General introduction

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1. Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has been isolated in the 19th_{century from} hay, already suggesting its plant-associated niche 1_{. The presence of genes involved} in colonizing plant roots and consuming plant material further indicates that the rhizosphere is the natural habitat of B. subtilis1,2_{. Some}_{B. subtilis strains are even able to} enter the plant, making it an endophyte 3,4_._{B. subtilis is well adjusted to surviving in this} environment, as it is able to swim and swarm, and form complex biofilms 5,6_{. Furthermore,}

B. subtilis can utilize a wide variety of carbon sources, from simple carbohydrates like

glucose and pyruvate, to more complex nutrients including plant-derived rhamnose and arabinose 7,8_{. In harsh conditions,}_{B. subtilis forms spores: small, dormant cells encased by} a thick, tough wall that is resistant to many adverse conditions. When the environment becomes more favorable, spores germinate and grow out into vegetative cells again. In this way, B. subtilis is able to survive extreme circumstances.

B. subtilis encounters many other species in the soil, such as uni- and multicellular

eukaryotes, fungi and other bacteria. Interaction with these other species and formation of multicellular biofilms are the rule rather than the exception 9_{. Apart from direct contact,} excretion of secondary metabolites allow organisms to send and receive signals and in that way, communicate with the outside world 10_{. Interactions can affect both species involved,} and may be beneficial, neutral or detrimental to one or both species. One example includes the interaction between bacteria and fungi (BFI) in which both species can exchange nutrients. Moreover, BFIs can be beneficial to bacteria because they receive physical protection from the fungi. Sometimes, they even use the fungal hyphae for transport to areas that they would not be able to reach by themselves 11_.

The diverse phenotypes of B. subtilis, the ability of natural competence which allows

natural transformation, its fast growth when cultivated, and the GRAS (Generally Recognized As Safe) status made this bacterium a popular candidate for laboratory studies, and it is now the most widely used Gram-positive organism among researchers. It is also a popular bacterium for industrial applications, since it is able to excrete large amounts of proteins. B. subtilis belongs to the large phylum of Firmicutes, characterized by

a low C+G content. The genome of B. subtilis was first sequenced in 1997, and re-sequenced

in 2009 1,12_{. It has a genome of 4.2 Mbp, and around 4,100 genes encode for proteins of} which 261 are essential when B. subtilis is grown in LB medium at 37°C 13,14_{. Most essential} genes encode for proteins involved in information processing, cell envelope synthesis, shape maintenance, division and energetics 13_{. Many different strains of}_{B. subtilis have} been described, but strain 168, a derivative of a strain isolated in Marburg, is most often

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employed as model organism. Its high transformability led to a fast spread around the world, and many vectors and (mutant) strains are now available in a genetic stock center (BGSC) making it easy for scientists to access material for research and cloning 15,16_.

2. The cell envelope of

Bacillus subtilis

Bacteria are able to live in various conditions, which can sometimes be hostile and unpredictable. In order to protect them, they have evolved a complex cell envelope that acts as a barrier to the outside world. This cell envelope fulfills many functions, and is indispensable for division, growth, morphogenesis and communication with the environment. The cell envelope can fall in two main categories, positive or Gram-negative, depending on their ability to retain a crystal violet dye (Fig. 1). The Gram-negative cell wall consists of a cytoplasmic membrane, surrounded by a distinct periplasmic space containing a thin peptidoglycan layer. On the outside, an outer membrane is present, with lipopolysaccharides attached to it. Gram-positive bacteria lack a defined periplasmic space and outer membrane, although microscopy observations suggested the presence of a thin periplasm-like layer directly outside the cell membrane 17,18_{. The cytoplasmic}

Figure 1. Schematic overview of the Gram-positive and Gram-negative cell envelope. In contrast

to Gram-negative organisms, Gram-positive bacteria do not show a distinct periplasm and lack an outer membrane. LTA: lipoteichoic acid; LPS: lipopolysaccharide; WTA: wall teichoic acid. Proteins are not shown. Figure adapted from Swoboda et al. (2010) 136_.

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membrane is surrounded only by a thick PG layer (30-100 nm) with other molecules, e.g. teichoic acids, attached to it 19_.

This PG layer is essential for morphology; when the cell wall of B. subtilis is removed,

the bacterium loses its shape 20_{. Due to the presence of the thick cell wall,}_{B. subtilis can} withstand fluctuating osmotic pressure. The semipermeable cell wall enables entrance and removal of compounds, whereas the cell membrane is much more selective, and harbours specific transport proteins for the uptake and excretion of compounds. These two layers of the envelope form an interface that provides structure, flexibility and strength, while also forming a selective barrier to the environment 21_.

2.1. The cytoplasmic membrane

The cytoplasmic membrane of B. subtilis consists of a phospholipid bilayer of 5-10 nm

thick with many proteins and other molecules embedded in it. At the surface of the membrane, essential metabolic processes take place, like the electron transport chain that generates energy. Moreover, the membrane acts as a selective barrier, allowing the uptake of useful components and the excretion of waste compounds. Few small, uncharged molecules can pass the membrane via diffusion, but most solutes are transported via membrane transport proteins 22_{. Specific types of proteins are present for every process,} such as nutrient uptake, ion transport or protein excretion. Next to transport proteins, proteins are present in the membrane that provide structural support to the cell, and that can detect environmental signals. Widespread sensor proteins are two component regulatory systems, that consist of a sensor histidine kinase located in the membrane, and a response regulator in the cytoplasm. When the histidine kinase detects a change in the environment, it phosphorylates the cognate response regulator that subsequently induces or represses the transcription of target genes 23_{. Thus, the cytoplasmic membrane} is the primary and most selective barrier of the bacterial cell, but it is not able to provide enough rigidity and protection to the outside environment on its own.

2.2. The cell wall

The cell wall of B. subtilis is required to withstand turgor pressure and maintain

the rod shape of the cell. The cell wall is mainly composed of peptidoglycan and teichoic acids. Peptidoglycan (PG) consists of a matrix of two alternating sugars, i.e. N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM) with 4-5 amino acids attached to NAM. Different NAM molecules are cross-linked via a peptide interbridge that couples the amino acids together. In that way, a strong mesh-like layer is formed that acts as an exoskeleton 24_{. The initial steps of PG synthesis take place at the cytoplasmic}

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surface. Subsequently, the precursor is transported across the membrane and is added to the growing PG chain by a trans-peptidation reaction. The final steps of glycan strand polymerization and cross-linking are catalyzed by a class of enzymes called penicillin-binding proteins (PBPs) 25_{. The PG formed acts as scaffold for anchoring other polymers} to the cell wall.

The most abundant polymers present in the B. subtilis cell wall are anionic

carbohydrate strands called teichoic acids, which can make up to 60% of the total cell wall mass 26,27_{. Teichoic acids (TAs) are either linked to the cell membrane (lipoteichoic acids;} LTAs), or to the PG of the cell wall (wall teichoic acids; WTAs) from where they extend beyond the PG layer (Fig. 1). TAs consist of a linkage unit and a phosphodiester-linked polyol repeat unit. This repeat unit varies between species, for example in Staphylococcus aureus and B. subtilis W23 it consists of a ribitol 5-phosphate, whereas the unit in B. subtilis

168 comprises a glycerol 3-phosphate (GroP) 28_{. These repeated units are phosphate} rich, thereby creating an anionic charge among the membrane. This negative charge has several functions including the regulation of autolysin activity and ion homeostasis (e.g. magnesium binding) 29_{. The charge of TAs can be altered by D-alanylation, which adds a} positively charged residue to the carbohydrate strand. The occurrence of this modification is affected by e.g. growth media, pH and temperature 30_{. When phosphate concentrations} in the medium are low, the cell switches to the production of teichuronic acids, which are basically the same as WTAs, but contain uronic acid without phosphor 31_.

WTAs are, like PG, synthesized at the wall-membrane interface. The initial steps of assembly occur at the cytoplasmic surface (Fig. 2). WTA-synthesizing proteins are encoded by tag genes (teichoic acid glycerol): tagO encodes a protein that catalyzes the first

assembly step of the linking unit of the teichoic acid, which is finalized by TagAB. The glycerol-phosphate chain is attached to this unit by TagDF. Subsequently, the chain can be modified by TagE with α-glucose, and transported through the membrane by TagGH. The exact mechanism of this flipping has not yet been elucidated. The final step is the coupling of the polymer to the C6-MurNAC unit of the PG by TagTUV 28_.

Although it was thought for a long time that WTAs are essential, a recent study revealed that it is possible to delete tagO 32,33_{. This results in the absence of WTAs in the} cell wall, and aberrant morphology as cells become rounded and cripple 33,34_{. Deletion of} other tag-genes is not possible, unless tagO is deleted. It is thought that this is connected to

the accumulation of toxic intermediates in the cell 33_{. Hence, all}_{tag genes, apart from tagO,} are indispensable in B. subtilis if the upstream process is not interfered with. LTAs are also

not essential, but their deletion leads to malformed morphology as well, e.g. misplaced septa in the cell wall 34_{. WTAs and LTAs cannot be deleted together, demonstrating the}

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redundancy of both polymers 34_{. The abundance of TAs already show their importance} in cellular functioning, but the molecular mechanisms behind their roles are not well understood 28_.

3. Functions of the cell envelope

The cell envelope is an intricate system that is essential for maintaining a proper physiological homeostasis within the cell, thereby allowing growth and division. Furthermore, the envelope plays an important role in adaptation to the environment by sensing changes and responding accordingly, e.g. by induction of specific signaling pathways resulting in adjusted gene expression. Each component of the cell wall is needed for proper functioning, although not all functions are well understood. Especially the exact role of WTAs remains enigmatic, but the amount of WTAs present in the cell wall suggest that they are required for almost every function it fulfills. Here, some particular functions of the B. subtilis cell envelope are discussed, as well as its connection with the

intracellular environment.

Figure 2. Wall teichoic acid structure and synthesis in B. subtilis168_{. Picture is adapted from Sewell}

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3.1. Nutrient uptake

One of the main prerequisites for growth is the availability of nutrients. Although media used in the laboratory are generally rich to achieve fast growth, B. subtilis encounters

frequent periods of scarcity in its natural habitat, resulting in low growth rates 35,36_. Besides, the cell constantly has to adapt to varying available nutrient sources 37,38_. Therefore, many cellular activities are focused on nutrient acquisition, like motility, chemotaxis and the secretion of exoproteases. After reaching the metabolic compound, uptake should take place in order to catabolize it. Much research has been performed to elucidate the exact mechanisms by which hydrophilic nutrients are transported through the hydrophobic cell membrane 39_{. Summarized, specific uptake proteins are expressed} that act either passively or actively 40_{. Passive uptake requires no energy, and can for} instance be achieved by (facilitated) diffusion, or via transport channels. Active processes do demand energy, e.g. ATP, to allocate nutrients against the concentration gradient. Well-known nutrient transporters include ABC-transporters, antiporters and symporters. Hence, in order to optimize nutrient translocation, many specialized transport proteins can be synthesized by the cell depending on the nutrient source 39_.

Transport proteins are usually produced after the cell senses the presence of a certain metabolite. A system that is often used for nutrient sensing, are two-component regulatory systems discussed above 41–43_{. These systems induce expression of transporters} only when a metabolic compound is present. Furthermore, carbon catabolite control maximizes metabolic efficiency when multiple carbon sources are present. The global regulator CcpA coordinates the carbon flow to and from crucial metabolites (e.g. pyruvate) by transcriptional control, and ensures utilization of the available carbon sources in a preferred order 44_{. In}_{B. subtilis, glucose and malate are favored carbon sources} 45,46_{. Consequently, when these carbon sources are present, transcription of genes involved} in uptake and catabolism of other resources is inhibited. CcpA regulates the expression of around 300 genes via catabolite responsive elements (cre), which are specific sequences

present in the operators or open reading frames of the affected genes 47,48_{. Among these 300} genes are transport proteins required for carbon uptake 47_{. Transcription can be induced} or repressed, depending on the exact position of the cre site. In this way, B. subtilis has

efficiently optimized its metabolism, thereby increasing its chances for survival.

3.2. Regulation of autolysin activity and cell death

Although most proteins in the cell envelope are located in the cytoplasmic membrane, several types of proteins are present in the cell wall as well. One major class of enzymes are murein hydrolases, or autolysins, located in the PG layer. These enzymes catalyze the

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hydrolysis of PG bonds, thereby enabling incorporation of new PG strands. Autolysins are thus essential for proper growth and expansion of the cell wall, but also for cell division and separation. Murein hydrolases are divided in several classes, depending on which exact bond they break in the NAM and NAG molecules 49_{. The activity of autolysins is} potentially lethal, and must therefore be tightly regulated. Studies, mainly performed in

S. aureus, have indicated that the charge of TAs are important, as the activity of autolysins

can be influenced even by slight pH changes 29,50,51_{. Ion binding by the anionic TA network,} together with D-alanylation, is believed to create regional changes in the pH, thereby regulating autolysin activity and localization 52–54_.

It was long believed that bacteria try to avoid overactivity of autolysins and subsequent lysis as much as possible, but it recently became clear that they can commit suicide under certain conditions. Examples includes mother cell lysis during sporulation and lysis during the formation of biofilms 55_{. Biofilms are communities of bacteria} encapsulated in an extracellular matrix produced by the bacteria, which provides protection from external hazards. The composition of the matrix differs between species, but components often include polysaccharides, proteins and extracellular DNA (eDNA) 56_{. In}_{S. aureus, eDNA is provided by a small part of the population that lyses using an} elaborate genetic program. This program, encoded by the Cid/Lrg network, has been compared to the apoptotic Bax/Bcl-2 network in eukaryotes 57_{. Later, it became clear} that the Cid/Lrg network not only regulates cell death during biofilm formation, but also under certain conditions in liquid cultures 58,59_.

The Cid/Lrg network consists of the cid and lrg operons that play an important

role in controlling murein hydrolase activity 55,57_{. The secondary structures of CidA and} LrgA exhibit a high degree of similarity to holins that regulate host cell lysis in phages by forming pores in the membrane of the host cell 60_{. Therefore, it was presumed that} these proteins are analogous to each other and that CidA and LrgA act as bacterial holin/ antiholin-like proteins that control cell death in S. aureus 61_{. Experiments have indeed} shown that LrgA –the antiholin-like protein- decreases murein hydrolase activity and increases penicillin tolerance, whereas CidA –the holin-like protein- increases murein hydrolase activity 58,62_{. LrgA is regulated by the two-component regulatory system LytSR} that senses the membrane potential and induces lrgA transcription when the membrane

potential dissipates, thereby trying to rescue the cell 63,64_{. The}_{cidABC operon is controlled} by CidR, a LysR-type transcriptional regulator that also regulates acetoin and acetate formation via cidC and alsSD. Overflow metabolism, and specifically the balance between

acetate and acetoin formation in S. aureus determines the cells fate, revealing a link

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Interestingly, it was found that 46% of all bacterial species for which genome sequences are available, possess homologues of the Cid/Lrg regulatory network 57_{. Thus,} it seems that the Cid/Lrg network is widely conserved in bacteria, but only a few studies have been performed to gain more insight in the function of the Cid/Lrg network in other species 67–69_{. In}_{B. subtilis, homologues of the cid and lrg operon, lytSR and cidR have been} identified as well. However, the role of this network in B. subtilis is so far elusive.

3.3. Cold adaptation

Under normal physiological temperatures, the cell membrane is a fluid structure and the temperature is optimal for enzymatic activity. For the mesophile B. subtilis, the optimum

temperature lies between 30°C and 37°C, but it is able to survive in temperatures ranging from 11°C to 55 °C 70_{. When the temperature suddenly changes, a heat- or cold shock is} provoked leading to adaption of the cell by optimizing its transcription and translation machinery 71,72_{. This happens primarily by induction of heat- or cold shock proteins (HSPs} and CSPs). These proteins enable proper transcription, translation and folding of the proteins, e.g. by acting as chaperones 70,73_{. Initially, the cold shock response (CSR) causes} a complete blockage in growth, but after acclimation to the new conditions, growth continues at lower rates. The CSR initiates the production of cold-induced proteins, enabling proper transcription and translation. Moreover, during the the CSR, the membrane composition is adjusted to increase its fluidity 74_.

Figure 3. A model of the Cid/Lrg regulatory network in S. aureus. The holin-like protein CidA induces

lysis by pore formation, whereas the antiholin-like protein LrgA inhibits this. CidR induces cidABC transcription during overflow metabolism, probably in response to accumulation of acetate and corresponding acifidication of the medium. Induction of the alsSD operon by CidR is not shown. LytSR is a two-component regulatory system that induces lrgA transcription after a decrease in the membrane potential. Reproduced from Sadykov and Bayles (2012) 300_.

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The phospholipids in the cell membrane of B. subtilis consist of at least five different

types of head groups, with attached a hydrophobic tail composed of lipids 75_{. In many} species, these lipids are straight-chained, but in B. subtilis, they consist of a large portion

of iso- and anteiso-branched fatty acid (FA) species belonging to the branched chain FA family 76_{. The iso-branched FAs are directly derived from valine and leucine, and for the} anteiso-branched FA’s, isoleucine is required. This shows that amino acid metabolism and FA synthesis are tightly coupled in B. subtilis77_.

When the temperature decreases, the composition of the membrane changes in order to maintain its fluidity. This happens via two main mechanisms. On the long term, lipids of different composition are incorporated into the cell membrane 77_{. In cell membranes} consisting of straight-chained FAs, the saturation is changed whereas in membranes with branched FAs, the 12- and 13 -methyltetradecanoic acid content is important 76_{. For}

B. subtilis, on the long-term a switch is made from iso- to anteiso-branched FAs because

the melting point for anteiso-branched FAs is significantly lower 70,74,77_{. How the ratio} between iso- and anteisobranched FA’s is regulated is not yet understood.

The second mechanism occurs much faster, and is regulated by the DesKR regulon 78_{. This two-component regulatory system regulates the desaturation of FAs, with the} sensor DesK probably sensing the membrane fluidity 74_{. DesKR induces the transcription} of des, a phospholipid desaturase that modifies incorporated branched saturated FA’s

by introducing a double bound in the membrane lipids. This results in the presence of unsaturated FA’s in the membrane 70_._{des transcription is inhibited by the presence of} unsaturated FA’s in the medium, and by the presence of anteiso-branched FA’s in the membrane 77_{. When extracellular isoleucine is available, anteiso-branched FA’s are already} incorporated in the membrane at 37°C 77_{. Hence, the}_{des response is less relevant in this} situation, and thus the CSR depends on the medium in which the bacterium is growing.

4. Scope of this thesis

In this thesis, we focus on gaining a greater understanding of the cell envelope of B. subtilis.

Although the structure of the envelope is largely known, the dynamics of the cell envelope in varying conditions and the molecular mechanisms behind the diverse functions it has, are far from understood.

As described in this chapter, the Cid/Lrg network regulates autolysin activity and cell death in the bacterium S. aureus. It is already known for decades that eukaryotic cells

have specialized apoptotic programs, but bacterial programmed cell death is a rather unexplored topic. The genome of B. subtilis encodes for proteins that are similar to the

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Cid/Lrg network: YwbH is annotated as putative holin-like protein, whereas YsbA is assumed to act as antiholin-like protein. However, this inference is only based on gene similarity, and experiments have never confirmed the roles of these genes. Therefore, in

chapter 2, we initially investigated the influence of YsbA and YwbH on cell death. We

did not find any connection to the regulation of murein hydrolase activity, but instead established that the antiholin-like protein YsbA and its regulator LytST are involved in pyruvate utilization and hence, have a metabolic function.

Next to the Cid/Lrg network, WTAs control autolysin activity at both transcriptional and post-transcriptional level. Deletion of tagO results in a strain where

WTAs are absent from the cell wall, leading to altered morphology, division and growth. Although the phenotype of this mutant has been described, it is not clear what happens intracellularly. Therefore, in chapter 3 we examined the effect of WTA depletion and

repletion on the transcriptome of B. subtilis. The effect of high and low tagO gene dosages

on transcriptome level was studied using RNA sequencing. Mapping of transcriptome data of the tagO mutant revealed a big secondary site mutation, raising the debate of tagO

essentiality.

As described above, the cell envelope is a dynamic structure. It is for example crucial that the cell envelope adapts properly after a cold shock to enable survival of the cell and maintains the fluidity of the lipid bilayer. Although the cold shock response (CSR) of B. subtilis is already generally understood, more detailed studies are lacking.

Some microarrays have for instance identified that yplP mutants are more sensitive to

cold-shock, but the reason is unknown 78,79_{. Furthermore, all studies focusing on the} CSR have been performed with microarrays. In chapter 4, we expanded the knowledge

regarding the CSR utilizing RNA sequencing. Additionally, expression of yplP deletion

mutants on the short- and long term was compared to the wild-type, thereby defining the YplP regulon.

In chapter 5, a detailed study on a bacterial-fungal interaction is described. This

study shows that B. subtilis is able to interact with the fungus Aspergillus niger, a fungi that

is also commonly found in the soil. B. subtilis can attach to A. niger, and using a specific

dual-transcriptome approach, we reveal that interaction affects the metabolim of both organisms, and that this BFI could be beneficial to both organisms.

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