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

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

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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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Mariëlle van den Esker 2018

Cell envelope related processes in Bacillus subtilis

Cell death, transport and cold shock

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University of Groningen, the Netherlands. The work was funded by the Simon Stevin Meester price awarded to O.P. Kuipers by Stichting Technische Wetenschappen (STW).

Printing of this thesis was financially supported by the Groningen Graduate School of Sciences and the University of Groningen.

Printed by Ipskamp Printing, Enschede, the Netherlands

Cover design by Marielle van den Esker and Lara Leijtens (persoonlijkproefschrift.nl) Layout: Lara Leijtens (persoonlijkproefschrift.nl)

© 2018 M.H. van den Esker, The Netherlands All rights reserved

ISBN (printed version): 978-94-034-0899-6

ISBN (electronic version): 978-94-034-0898-9

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Prof. dr. O. P. Kuipers

Beoordelingscommissie

Prof. dr. L. W. Hamoen

Prof. dr. M. Heinemann

Prof. dr. D.J. Scheffers

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Chapter 1

General introduction p. 8

Chapter 2

YsbA and LytST are essential for pyruvate utilization in Bacillus subtilis p. 20

Chapter 3

Are wall teichoic acids essential or not? RNA sequencing reveals a large secondary mutation in a Bacillus subtilis tagO mutant

p. 42

Chapter 4

The cold shock response of Bacillus subtilis analyzed by RNA-sequencing: YplP regulates pyruvate transport at low temperatures

p. 60

Chapter 5

Bacillus subtilis attachment to Aspergillus niger hyphae results in mutually altered metabolism

p. 80

Chapter 6

Summary and general discussion p. 102

Chapter 7

References p. 114

Addendum

Nederlandse samenvatting voor de leek Dankwoord

p. 134

p. 144

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General introduction

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1. Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has been isolated in the 19

century from hay, already suggesting its plant-associated niche

. The presence of genes involved in colonizing plant roots and consuming plant material further indicates that the rhizosphere is the natural habitat of B. subtilis

. Some B. subtilis strains are even able to enter the plant, making it an endophyte

. B. subtilis is well adjusted to surviving in this environment, as it is able to swim and swarm, and form complex biofilms

. 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

. 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

. Apart from direct contact, excretion of secondary metabolites allow organisms to send and receive signals and in that way, communicate with the outside world

. 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

.

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

. 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

. Most essential

genes encode for proteins involved in information processing, cell envelope synthesis,

shape maintenance, division and energetics

. 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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11 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

.

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, Gram-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

. 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)

.

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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

.

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

. 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

.

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

. 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

. 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

. The initial steps of PG synthesis take place at the cytoplasmic

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13 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)

. 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

. 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)

. 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)

. 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

. 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

.

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

.

Although it was thought for a long time that WTAs are essential, a recent study revealed that it is possible to delete tagO

. This results in the absence of WTAs in the cell wall, and aberrant morphology as cells become rounded and cripple

. 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

. 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

. WTAs and LTAs cannot be deleted together, demonstrating the

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redundancy of both polymers

. The abundance of TAs already show their importance in cellular functioning, but the molecular mechanisms behind their roles are not well understood

.

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. subtilis

. Picture is adapted from Sewell

and Brown (2014)

.

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15 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

. Besides, the cell constantly has to adapt to varying available nutrient sources

. 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

. Summarized, specific uptake proteins are expressed that act either passively or actively

. 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

.

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

. 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

. 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

. Among these 300 genes are transport proteins required for carbon uptake

. 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

. 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

. 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

.

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

. 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

. 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

.

The Cid/Lrg network consists of the cid and lrg operons that play an important role in controlling murein hydrolase activity

. 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

. 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

. 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

. 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

. 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

between cell death and metabolism

. A model of this network is given in Figure 3.

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17 Interestingly, it was found that 46% of all bacterial species for which genome sequences are available, possess homologues of the Cid/Lrg regulatory network

. 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

. 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

. 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

. 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

. 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

.

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)

.

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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

. 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

. 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. subtilis

.

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

. 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

. 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

. 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

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. This two-component regulatory system regulates the desaturation of FAs, with the sensor DesK probably sensing the membrane fluidity

. 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

. 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

. When extracellular isoleucine is available, anteiso-branched FA’s are already incorporated in the membrane at 37°C

. 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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19 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

. 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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Marielle H. van den Esker, Ákos T. Kovács, Oscar P. Kuipers

YsbA and LytST are essential for pyruvate utilization in Bacillus subtilis

This chapter was published in:

Environmental Microbiology 19 (1), 83-94 (2017).

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Abstract

The genome of Bacillus subtilis encodes homologues of the Cid/Lrg network. In other bacterial species, this network consists of holin- and antiholin-like proteins that regulate cell death by controlling murein hydrolase activity. The YsbA protein of B. subtilis is currently annotated as a putative antiholin-like protein that possibly impedes cell death, whereas YwbH is thought to act as holin-like protein. However, the actual functions of YsbA and YwbH in B. subtilis have never been characterized. Therefore, we examined the impact of these proteins on growth and cell death in B. subtilis. We did not find a connection to the regulation of programmed cell death, but instead, our experiments reveal that YsbA and its two-component regulator LytST are essential for growth on pyruvate. Moreover, deletion of ysbA and lytS significantly reduces pyruvate consumption.

Our findings suggest that LytST induces ysbA transcription in the presence of pyruvate,

and that YsbA is involved in pyruvate utilization presumably by functioning as pyruvate

uptake system. We show that B. subtilis excretes pyruvate as overflow metabolite in rich

medium, indicating that pyruvate could be a common nutrient in the environment. Hence,

YsbA and LytST might play a major role in environmental growth of B. subtilis.

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Introduction

The Gram-positive bacterium Bacillus subtilis was first isolated in the 19

century from hay, already suggesting its plant-associated ecological niche

. Its genome encodes many genes associated with adaptation to this environment as it is able to swim, swarm and form complex biofilms. Furthermore, B. subtilis is capable of utilizing a wide variety of carbon sources including plant material composed of rhamnose and arabinose

. Since its first discovery, B subtilis has become a widely used model organism, and many gene functions have been unraveled. However, the functions of nearly one thousand genes are still poorly characterized or completely unknown.

The roles of the YwbH and YsbA proteins of B. subtilis are also unknown, but they are homologous to CidA and LrgA of Staphylococcus aureus and their function is therefore assumed to be similar. CidA and LrgA are involved in the regulation of cell death, presumably by controlling murein hydrolase activity comparable to holin- and antiholin like proteins

. The antiholin-like protein LrgA prevents dissipation of the proton motive force when the membrane potential is affected, reducing cell lysis under certain conditions

. lrgA transcription is induced by LytSR, a two-component regulator that senses changes in the membrane potential

. The cidABC-operon, regulated by CidR, induces cell death when the medium acidifies due to overproduction of acetate

. The balance between acetic acid and acetoin produced during growth on high glucose levels appears to play a major role in regulating cell death: acidification of the medium by acetic acid excretion results in the induction of the cell death pathway, whereas the neutral overflow metabolite acetoin counteracts cell death

.

The Cid/Lrg network is widely conserved: 46% of all bacterial species for which genome sequences are available possess homologues of this network

. However, only a few studies have been performed to identify the true function of these genes in other bacterial species. These studies indicate that the Cid/Lrg network mainly regulates cell death, but also plays a role in other pathways

. In B. subtilis, homologues of the cid/lrg operon (ywbHG/ysbAB), lytSR (lytST) and cidR (ywbI) are present as well, and the location and orientation of the genes in B. subtilis is similar to those of the genes in S. aureus (Fig. 1A).

The ysbAB operon, encoding the putative antiholin YsbA (69% similarity to LrgA) and the putative endolysin YsbB, is located next to the two-component regulatory system LytST.

The ywbHG operon, encoding the putative holin-like protein YwbH (60% similarity to CidA) and a protein with unknown function, YwbG, is located downstream of the gene encoding for the LysR-type regulator ywbI.

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A previous microarray study has shown that LytST induces ysbAB expression, but represses ywbH transcription

. The glucose level appears to influence ysbAB expression, probably via CcpA

, while CshA, an RNA helicase, also strongly affects the transcript level of ysbAB genes

. Recently, it has been shown that in B. subtilis NCIB 3610, ysbAB and ywbH are both important for biofilm formation as well, since deletion of them results in delayed biofilm formation. Furthermore, expression of ysbA and ywbH is induced in the presence of acetic acid in B. subtilis NCIB 3610

.

Although these studies suggest that YsbA and YwbH might act similarly in B. subtilis as LrgA and CidA in S. aureus, it has not yet been examined whether these proteins are really expressed and functional in regulating cell death. Therefore, we examined the

Figure 1. Operon structure of the Cid/Lrg network in Staphylococcus aureus and Bacillus subtilis

according to MGcV

(A). Predicted structures of YsbA and YwbH (B). Protein structures were

predicted by TMHMM

, using the amino acid sequences of both proteins.

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25 function of the YsbA and YwbH proteins of B. subtilis. No relation to programmed cell death was found, but we did discover that ysbA and lytS are essential for growth on pyruvate.

We therefore propose that YsbA does not act as a conventional antiholin-like protein, but instead has a metabolic function and is required for pyruvate transport or utilization.

Results

Characterization and expression of YwbH and YsbA

LrgA and CidA are located in the membrane, where they influence murein hydrolase activity. The structures of the putative holin YwbH and putative antiholin YsbA of B.

subtilis were predicted with TMHMM and SWISS-MODEL

. Both software programs predicted that YwbH and YsbA are membrane proteins with a very similar structure, comprising four transmembrane domains with the C- and N-terminus located in the cytosol (Fig. 1B).

In order to get more insight in the in vivo function of YsbA and YwbH, we searched for a condition in which both proteins are expressed simultaneously. Therefore, reporter strains were created by fusing the promoters of both genes with their native RBS to a gene coding for a green fluorescent protein. It is known that both genes are expressed in LB medium

, and growth of our reporter strains in a plate reader showed that both genes are maximally expressed in the transition phase, which occurs after the logarithmic growth phase in which cells divide 0.93 times per hour (Fig. 2A). ysbA expression was approximately ten times higher compared to ywbH expression. At single cell level, ysbA expression appeared to be very heterogeneous at the onset of stationary phase: some cells were non-fluorescent while others were very bright (Fig. 3A). Other in-between states

Figure 2. Promoter activity of ywbH and ysbA in LB (A) and of ysbA in LB and LB+1% glucose (B). Cells were grown in a plate reader and growth and fluorescence of the reporter strains were measured at 10 minute intervals. Averages of three experiments with the SD are plotted. The grey dotted line indicates the transition phase after ~4 hrs of growth, the black line shows the OD

in LB and the black dotted line in graph B represents the OD

in the presence of glucose.

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were also witnessed. Since both genes show a similar expression pattern in LB, this medium was chosen to perform follow-up experiments in.

Previous research revealed that metabolism, and especially the presence of glucose, regulates expression of CidA and LrgA in S. aureus. ysbA contains a putative cre-site in its promoter region, and microarrays indicated that expression of this gene is regulated by CcpA

. To verify this, GFP fluorescence of ME001 was quantified in LB+1% glucose.

Glucose addition indeed reduced ysbA transcription, confirming repression by CcpA due to the presence of a cre-box (Fig. 2B). The presence of glucose did not alter GFP expression of strain ME002 (data not shown).

Subsequently, regulation of the ysbAB operon by its putative regulator LytST was examined by combining reporter strain ME001 with the deletion marker from strain ME005, resulting in strain ME014 (∆lytS P

-gfp). Fluorescence of ME014 was quantified using flow cytometry and microscopy, and compared to ME001. No GFP fluorescence was observed in the ∆lytS background in LB in any of the growth phases, while fluorescence was detected in the wild-type background (Fig. 3B). This indicates that expression of ysbA was mainly induced by LytST under the conditions tested.

Figure 3. GFP fluorescence of P

-gfp in WT (A) and ∆lytS (B) background. Strains were grown under

the same conditions (LB to transition phase, OD~1.5). Scale bar represents 5 µm.

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27 YwbH and YsbA do not function as a conventional holin- and antiholin pair in B.

subtilis

If YwbH and YsbA are truly functional as a conventional holin and antiholin pair, deletion or overexpression of one of them would, at least under certain conditions, result in altered murein hydrolase activity, and hence, different rates of cell death. Deletion of the holin or its inducer YwbI is expected to result in less cell death, whereas loss of the antiholin or its regulator LytST would supposedly cause an increase in cell death. Furthermore, LrgA and CidA of S. aureus are involved in regulating the tolerance towards certain antimicrobial compounds, and in controlling cell death in response to acidification of the medium. To examine whether YsbA and YwbH have similar functions, deletion mutants of ysbA and ywbH and their regulators ywbI and lytS were created, as well as strains in which ysbA and ywbH could be overexpressed.

However, deletion or overexpression of these genes did not change growth in LB or chemically defined medium supplemented with glucose (Fig. S1 and S2). Furthermore, the expression of ysbA and ywbH did not change in response to the addition of the antimicrobial compounds carbonyl cyanide m-chlorophenyl hydrazone (CCCP), valinomycin, gramicidin or by-products of metabolism (acetoin and acetic acid) (Fig. S3A and Fig. S4).

Also, susceptibility to the membrane dissipating compounds CCCP, valinomycin and gramicidin was not altered in the strains where ysbA and ywbH were overexpressed or deleted (Fig. S3B). Finally, we tested the sensitivity of the deletion strains to Triton-X-100 in an autolysis assay, but no differences could be detected between wild-type and mutant strains (Fig. S5). This indicates that the mutants were not more prone to lysis than the wild-type. Hence, none of our experiments provide evidence that the genes of the putative Cid/Lrg network of B. subtilis are functional as conventional holin/antiholin pair.

YsbA and LytS are essential for pyruvate utilization

Although no direct relation between the putative Cid/Lrg network of B. subtillis and the regulation of cell death was found, our data demonstrates that ysbA is expressed in LB, and is repressed in the presence of glucose by CcpA. Therefore, we hypothesized that this gene might play a role in metabolism. Previous experiments have shown that ysbA is highly expressed after glucose exhaustion, and when B. subtilis grows in M9 medium with pyruvate as only carbon source

. We became interested in the cause of high gene expression in the presence of pyruvate. We first confirmed that ysbA is expressed in M9 + pyruvate medium by growing ME001 in M9 medium containing pyruvate in a plate reader.

Cultures were pre-grown in LB, and after inoculating the cells in M9 with pyruvate, cells expressed ysbA to a high, constant level (Fig. 4A). Furthermore, microscopy experiments

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Figure 4. P

-gfp expression in M9 medium containing pyruvate. Growth and fluorescence was measured for 29 hours and averages of three experiments with the SD are plotted. The black line represents the absorbance at 600 nm, and the grey line shows GFP fluorescence (A). GFP expression at single cell level in M9 medium with pyruvate. Scale bare is 5 µm (B). Growth curves of wild-type,

∆ysbA and ∆lytS in M9P (C). Two biological replicates are plotted.

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29 revealed that expression of ysbA is homogeneous in the presence of pyruvate, with all cells expressing the ysbAB operon (Fig. 4B). Deletion of lytS again completely abolished ysbA expression in M9 medium containing pyruvate as fluorescence was reduced to background levels in strain ME014 (Fig. S6).

To gain more insight in the function of YsbA, growth of the ∆ysbA and ∆lytS knockout mutant strains were compared to growth of the wild-type in M9 medium supplemented with pyruvate. Remarkably, ∆ysbA and ∆lytS were unable to grow in the presence of pyruvate, whereas the wild-type reached an OD

of 1.5-2 after 24 hrs incubation, with a growth rate of 0.23 divisions per hour (Fig. 4C). Upon switching from M9 containing glucose to M9 with pyruvate, the morphology of ∆ysbA and ∆lytS changed from rod-shaped to coccoid and cells became significantly smaller (average length of 1.5 µm), whereas the wild-type maintained its regular shape with an average length of 2.5 µm (Fig. 5). We also followed growth of ∆ywbH in the same medium, but no growth defect was observed (data not shown). Complementation of ∆ysbA and ∆lytS was accomplished by expressing

Figure 5. Cell size and morphology of the B. subtilis 168 wild-type, ∆ysbA and ∆lytS in M9 medium containing pyruvate. Boxplots represent the cell length. Dots represent the 5th and 95th percentile, and the whiskers show the 10th and 90th percentile of the data. Microscopy pictures show the morphology of the strains in M9 medium containing pyruvate. Scale bar represents 5µm.

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the genes under the control of the native promoter ectopically in the amyE locus, which restored growth back to wild-type levels (Fig. S7). These results indicate that YsbA and LytS are required for growth in pyruvate, and therefore probably play a role in pyruvate metabolism.

The inability of ∆ysbA and ∆lytS to grow in M9+pyruvate might either result from the inability of these strains to metabolize pyruvate, or from excessive cell death triggered in these circumstances by deletion of the putative antiholin ysbA and its regulator lytS.

To assess whether ∆ysbA and ∆lytS are able to utilize pyruvate, the amount of pyruvate consumption was determined over time using a pyruvate assay kit. The amount of pyruvate consumed after 24 hrs in the wild-type and complementation cultures was ~80 mM, whereas pyruvate uptake in the ∆ysbA and ∆lytS was <10 mM (Fig. 6). Hence, ysbA and lytS deletion significantly reduced pyruvate utilization. The wild-type phenotype was restored when the ysbA and lytS genes were introduced into the amyE locus in the mutants. This demonstrates that YsbA and LytS are required for pyruvate consumption.

Our previous experiments revealed that ysbA is expressed heterogeneously in LB medium in the transition from exponential to stationary phase. To assess whether this could relate to pyruvate consumption as well, we checked whether pyruvate is secreted when B. subtilis is grown in LB. After 2 hrs of growth (logarithmic growth phase), 35 µM

Figure 6. Growth and levels of pyruvate consumed by B. subtilis 168 wild-type, ∆ysbA, ∆lytS and the

complemented strains during 24 hrs growth in M9+pyruvate. Dotted lines indicate OD

, while solid

lines show the levels of pyruvate uptake. Experiments were performed in duplicate, but for clarity,

only one representative line is shown.

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31 pyruvate was present in the medium, which completely disappeared in early stationary phase, meaning that the secreted pyruvate was utilized between mid-exponential and early stationary phase. Hence, pyruvate seems to be excreted as overflow metabolite and is consumed after other favorable nutrients are depleted in the transition phase, when ysbA expression is maximal.

According to the transcriptome data of Nicolas et al, ywbH is expressed to a high level in M9 medium containing malate and glucose, but not pyruvate

. We confirmed this by growing ME002 in M9M/G and assessing fluorescence micro-scopically. Indeed, all bacteria expressed ywbH homogeneously (Fig. S8). Subsequently, we measured growth of deletion mutant ME005 and compared it to growth of the wildtype, but no growth defects were found (data not shown). Also, the morphology of ME005 was similar to the rod-shaped wild-type. Hence, the reason of ywbH expression in malate/glucose remains unclear.

Discussion

In this study, the function of the B. subtilis Cid/Lrg homologues was investigated. Due to the high similarity of this network to the Cid/Lrg network of S. aureus, it has been generally assumed that this network is active in a similar way, namely in controlling murein hydrolase activity, and thereby regulating cell death as holin- and antiholin-like proteins. However, we could not find a relation between the Cid/Lrg homologues in B.

subtilis and regulation of cell death. Deletion or overexpression of any of the genes of the network does not result in a change in growth or lysis of B. subtilis, while this is the case in S. aureus

. Furthermore, antibiotic or Triton-X-100 susceptibility is not altered in the mutant strains compared to the wild-type, and the addition of secondary metabolites or antimicrobial compounds does not change ysbA and ywbH expression in B. subtilis, in contrast to S. aureus

. Hence, no direct evidence was found that the putative Cid/Lrg homologues of B. subtilis act as holin-antiholin like network.

However, we did gain new insights in the functioning of YsbA, which is currently annotated as putative antiholin. Our experiments revealed that addition of glucose to the medium represses ysbA expression, indicating regulation by CcpA due the presence of a cre-box in the promoter region of ysbA. Thus, we experimentally verified the in silico prediction that has suggested the occurrence of a cre-site in the ysbA promoter, and the microarray studies indicating that ysbA is regulated by CcpA

. Catabolite responsive elements (cre-sites) are regions in promoters that can be bound by the master regulator of carbon metabolism CcpA. CcpA ensures that glucose, the most favorable

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carbon source for B. subtilis, is used first by either up- or downregulating the expression of genes required for glucose metabolism and the uptake of other carbon sources. Malate is the second preferred carbon source, and also causes catabolite repression via CcpA

. Repression of ysbA by CcpA indicates that it is in some way connected to growth on other carbon sources than glucose or malate.

Next to CcpA, LytST also regulates ysbA expression

. According to our results, this putative two-component regulatory system is mainly responsible for inducing ysbA expression: when lytS is deleted, P

-gfp fluorescence diminishes to undetectable levels.

LytS is annotated as histidine kinase that, after sensing a specific environmental stimulus, phosphorylates the cognate response regulator LytT, which subsequently induces ysbA transcription. Bacterial two-component regulatory systems are used in specific conditions to induce the transcription of genes that are normally not expressed so that bacteria can quickly adapt to changes in their environment

. LytST induces ysbA transcription to a high, homogeneous level in M9 medium containing pyruvate as carbon source. When either the regulator lytS or the affected gene ysbA are deleted, B. subtilis loses its ability to grow on pyruvate. Both mutants are disturbed in pyruvate consumption, while expressing the genes in an ectopic locus completely restores growth and utilization of pyruvate in the mutants. Hence, both proteins are essential for growth on this monocarboxylate.

This, together with the fact that ysbA is repressed by CcpA in the presence of glucose, suggests that, instead of acting as an antiholin-like protein, YsbA is involved in a process leading to proper pyruvate utilization. Pyruvate is a major metabolite, since it is the end- product of the Embden-Meyerhof-Parnas pathway, a process in which glucose is converted into two pyruvate molecules. Subsequently, pyruvate can enter the citric acid cycle, or, in excess glucose, overflow metabolites are formed from pyruvate such as lactate, acetoin or acetate

. Pyruvate is also a precursor for various amino acids, such as alanine. Several bacterial species are able to use extracellular pyruvate as sole carbon source, which is also the case for B. subtilis

.

However, how prokaryotes import pyruvate from the medium is largely unexplored.

In some species, such as in C. glutamicum and E. coli

, pyruvate transporters have been

identified but for many, including B. subtilis, nothing is known about the uptake of this

monocarboxylate. Although the uncharged molecule pyruvic acid can freely diffuse

through the lipid bilayer of the cell membrane, the pKa of 2.5 makes it likely that under

physiological conditions most molecules are in a dissociated, charged state

. Therefore,

in most bacterial species that are able to metabolize pyruvate, transport proteins would

be required to import this compound.

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33 Since enzymes involved in intracellular metabolism of pyruvate have already been well-determined in B. subtilis, and because YsbA is –according to our predictions- located in the cell membrane, we hypothesize that YsbA might be involved in the uptake of pyruvate. Two-component regulatory systems are widely used by bacteria to regulate the expression of metabolite transporters

. The lytST operon is constituvely expressed

91

. We envision that LytS senses the presence of pyruvate and phosphorylates LytT, after which ysbAB transcription is induced. When glucose and pyruvate are both present in the medium, CcpA represses transcription of ysbAB to prevent pyruvate from entering the cell, thereby ensuring glucose is metabolized first. This also explains the reduced size of ∆ysbA and ∆lytS, as it is known that cell size is primarily determined by nutrient availability; bacteria in nutrient-poor environments are generally smaller than bacteria in richer habitats

. However, At this stage we cannot yet exclude the possibility that there is an indirect effect of ysbA and lytS deletion on pyruvate utilization, for example a blockage of an intracellular metabolic pathway leading to reduced pyruvate consumption.

Little is known about the presence of pyruvate in the environment. Some bacterial species excrete pyruvate as overflow metabolite, for example, luminous and halophilic bacteria create a lot of pyruvate during growth on glucose, which they consume after other nutrients are depleted

. E. coli excretes pyruvate when grown on succinate, and B. cereus on media containing glucose and yeast extract

. Furthermore, pyruvate is excreted when B. subtilis is grown on medium containing malate

. Thus, it might well be possible that pyruvate is a general, but poorly characterized overflow metabolite and therefore it might be a common nutrient in the environment of microbes.

This could also explain why ysbA is expressed heterogeneously in LB medium. Our experiments reveal that pyruvate is excreted as overflow metabolite, and taken up at the onset of stationary phase. If pyruvate is excreted together with other compounds, only part of the population consumes pyruvate, while other cells metabolize different nutrients. Previously, it has been described that ysbAB is also induced in the presence of acetate in B. subtilis NCIB 3610

. It is known that several other pyruvate transporters are able to transport acetate as well, which might explain upregulation after acetate addition

.

Although it is likely that YsbA plays a role in pyruvate uptake or metabolism, other scenarios cannot be excluded. No role for YwbH and YsbA as conventional holin- and antiholin was detected in our study, but it does seem that the regulation of these genes is in some way coupled. A microarray in which the regulon of LytST was determined by overexpressing lytT has shown that ysbA is upregulated by LytST, while ywbH is repressed by LytT

. Furthermore, transcriptome analysis showed that ywbH is expressed highly

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when malate and glucose are present in the medium

, which was confirmed by our experimental data. Although no growth defects on this medium were observed, it might indicate that ywbH is regulated by metabolism as well, but in an opposite manner of ysbA.

Summarized, our results provide no evidence that YsbA or YwbH are involved in regulating cell lysis, but instead indicate that these genes have a metabolic function. YsbA has a role in the pyruvate utilization, and is regulated by LytST and CcpA. In the future, it would be interesting to verify that YsbA is a pyruvate transporter, and to investigate how it is exactly regulated by LytST on a molecular level. Furthermore, the function of YwbH remains elusive and additional experiments should provide more understanding of the role of this gene. According to Chen et al., YwbH and YsbA both play a role during biofilm formation because deletion of them results in delayed biofilm formation

, but as our experiments suggest, this could also be due to metabolic effects. Perhaps, the undomesticated strain B. subtilis NCIB 3610 could be used in prospective experiments to examine the connection of pyruvate and biofilm formation, as this strain is able to form robust biofilms, while B. subtilis 168 shows reduced biofilm formation and requires distinct cultivating conditions for biofilm development

.

Experimental procedures

Bacterial strains, plasmids and media

Strains and plasmids used in this study are shown in Table 1 and Table 2. E.coli MC1061 and Bacillus subtilis 168 1A700 were grown in Lysogeny Broth (LB-Lennox: 1% Bacto- Tryptone, 0.5% Bacto-yeast extract, 0.5 % NaCl), SMM supplemented with 0.5% glucose and tryptophan (50 µg/ml), or M9 medium supplemented with tryptophan (50 µg/ml) and either glucose (3 g/L), glucose + malate (2 g/L + 3 g/L) or pyruvate (6 g/L)

. When required, antibiotics were added to the medium in the following final concentrations:

ampicillin (100 µg ml

) for E. coli, or tetracycline (6 µg ml

), kanamycin (5 µg ml

), spectinomycin (100 µg ml

), chloramphenicol (5 µg ml

) for B. subtilis. For induction, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the medium. Strains were incubated at 37°C, 220 rpm unless stated otherwise. Solid media were prepared by adding 1.5% (wt/vol) agar.

Molecular cloning

All primers used in this study are listed in Table S1. DNA purification, restriction, ligation

and transformation to E. coli was carried out as described by Sambrook et al. (1989)

. PCRs

were performed using chromosal DNA of B. subtilis 168 as a template. Phusion polymerase,

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35 restriction enzymes and T4 ligase were purchased from Fermentas. All constructs were verified by PCR and sequencing (Macrogen). B. subtilis transformations were performed as previously described

.

Table 1. Bacterial strains used in this study

Strain

Relevant features Reference or source

E. coli

MC1061 F-, araD139, ∆(ara-leu)7696, ∆(lac)X74, galU, galK, hsdR2, mcrA, mcrB1, rspL

Wertman, Wyman, & Botstein, 1986

Bacillus subtilis

168 1A700 trpC2 Kunst et al., 1997

168 P

-comK amyE::P

-comK, Cm

168 variant based on Hahn et al., 1996

ME001 amyE::P

-gfp, Cm

This study

ME002 amyE::P

-gfp, Cm

This study

ME003 ysbA::tet, Tc

This study

ME005 ywbH::tet, Tc

This study

ME006 lytS::tet, Tc

This study

ME007 ywbI::tet, Tc

This study

ME008 amyE::P

-ysbA, Sp

This study ME009 amyE::P

-ywbH, Sp

This study ME010 ysbA::tet, Tc

, amyE::P

-ywbH, Sp

This study ME011 ywbH::tet, Tc

, amyE::P

-ysbA, Sp

This study ME012 ysbA::tet, Tc

, amyE::ysbAB, Cm

This study ME013 lytS::tet, Tc

, amyE::lytST, Cm

This study ME014 lytS::tet, Tc

, amyE::P

-gfp, Cm

This study

a

all Bacillus strains constructed in this study are derivatives of strain 168 1A700

Table 2. Plasmids used in this study

Plasmid Relevant features Reference or source

pGFP-rrnB amyE’ P

-gfp

cat ‘amyE spec bla Veening et al., 2009

P

-gfp amyE’ P

-gfp cat ‘amyE spec bla This study

P

-gfp amyE’ P

-gfp cat ‘amyE spec bla This study

pBEST309 bla, tet Itaya et al., 1992

pDR111 bla amyE’ P

spec lacI ‘amyE Gift of D. Rudner pDR111-ysbA bla amyE’ P

– ysbA spec lacI ‘amyE This study pDR111-ywbH bla amyE’ P

– ywbH spec lacI ‘amyE This study pX bla amyE’ xylR P

cm ‘amyE Kim et al., 1996

pX-ysbAB bla amyE’ ysbAB cm ‘amyE This study

pX-lytST bla amyE’ lytST cm ‘amyE This study

2