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

Processed on: 25-7-2018 PDF page: 103PDF page: 103PDF page: 103PDF page: 103 Part of this chapter was published in:

mBio 8 (6), e01963-17 (2017).

Summary and discussion

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Processed on: 25-7-2018 PDF page: 104PDF page: 104PDF page: 104PDF page: 104 104

The bacterial cytoplasm is separated from the environment by a sophisticated cell envelope that provides structure and rigidity to the cell, and fulfills many other functions, including signal transduction and nutrient transport. The cell envelope is a dynamic structure that constantly adapts to changing environmental conditions, and it is the first site of contact when interaction occurs between species. Although the structure of the Gram-positive bacterial cell wall is well known, the dynamics and precise functions of its components are not all completely understood. The research described in this PhD thesis was therefore aimed at investigating different processes occurring in or at the cell envelope of the Gram-positive model bacterium Bacillus subtilis, which is frequently used

by industry as enzyme producer or in agriculture as soil inoculant.

Our work primarily focused on the regulation of cell death by B. subtilis (chapter 2 and 3). We investigated the contribution of putative membrane-embedded holin-like proteins and wall teichoic acids to cell death. The direction of both studies altered after assembling initial data, in the end creating novel insights in the role of an antiholin-like protein and gene essentiality. Moreover, we studied the dynamics of the cell membrane and transcriptome during cold shock conditions (chapter 4), and characterized the YplP regulon that is activated during cold shock. Finally, we performed a dual transcriptome study of B. subtilis that attached to the fungus Aspergillus niger, and uncovered a

bacterial-Figure 1. Schematic overview of different cell-envelope related processes studied in this thesis, as described in the text (BFI: bacterial-fungal interaction).

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fungal interaction with potential benefits for both species (chapter 5). An overview of the research described in this thesis is given in Figure 1.

Thorough understanding of cell envelope-related processes and dynamics is essential to optimize growth and protein excretion. This is useful for industrial applications, for example to enhance enzyme secretion, which can be a bottleneck in

B. subtilis. Furthermore, growth yields can be improved by reducing cell lysis. Growth

fluctuates depending on nutritional and physical conditions, which can also influence the constitution of the cell envelope. The cell envelope is a major target for antibiotics, and fundamental knowledge of its functioning, and especially the regulation of cell lysis, can provide useful insights for the development of novel antibiotics, which is essential in this era of upcoming antibiotic resistance.

From cell death to metabolism: holin-antiholin homologues with new functions The cell envelope holds the cell together, and disruption of this structure results in cell lysis. Bacterial lysis was already described long ago, and depends on many external factors, such as osmotic shock and nutrient starvation. Remarkably, it appeared that bacteria can also commit suicide and kill themselves actively (active death, or programmed cell death; PCD) by following a genetic cell death program 55,61_{. One example includes biofilm}

formation in Staphylococcus aureus, where a small subpopulation lyses to provide DNA

that glues the extracellular matrix together 57_{. This process is regulated by the Cid/}

Lrg network, a genetic program that regulates cell death on a genetic level depending on environmental conditions and the metabolic status of the cell 275_{. The genome of}_B.

subtilis also contains genes encoding homologues of these genes, annotated as ywbH (cidA

homologue) and ysbA (lrgA homologue).

We initiated our research by investigating the role of these proteins in the regulation of cell death. However, in chapter 2 we demonstrated that YsbA and its two-component regulatory system (TCS) LytST do not play a role in programmed cell death of B. subtilis,

but instead have a metabolic function and are required for pyruvate utilization. The transcription of ysbA is controlled by two main regulators. Firstly, CcpA, the master

regulator of the carbon catabolite response (CCR) inhibits the expression of ysbA in the

presence of glucose, which is the preferred carbon source for B. subtilis. The second regulator

involved is the TCS LytST. The signal that LytS senses has not yet been experimentally elucidated, but it could respond to extracellular pyruvate levels or uptake fluxes. LytT subsequently activates ysbA transcription in the presence of pyruvate by binding to its

promoter region. The presumed localization of YsbA in the membrane suggests that it could function as transporter protein. A study that was recently published by Charbonnier

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et al. indeed confirmed that the ysbAB operon encodes for a hetero-oligomeric facilitated

pyruvate transporter 165_{. The operon was therefore renamed}_{pftAB (encoding a pyruvate}

facilitated transporter).

Although we did not focus on unraveling the entire LytT regulon, a heuristic microarray study of Kobayashi et al. (2001) revealed that pftAB was induced by LytT, while ywbH appeared to be repressed. The function of YwbH and its regulation by LytT have

not yet been experimentally explored, but YwbH is currently annotated as a putative holin-like protein, based on its sequence similarity to the prolytic protein CidA in S. aureus. So far, no connection to PCD has been found, but ywbH is expressed at high levels

in the presence of malate 164_{. Hence, repression by LytT, either directly or indirectly, might}

suggest a metabolic function of this gene that could potentially expand our knowledge on the pyruvate/malate metabolic pathway and its homeostasis.

LrgA and LytSR (or LytST) are present in various organisms, but few studies have been conducted that elucidate their direct role, among which the function of LytSR that has been best characterized in S. aureus57_{. LytS responds to changes in the membrane}

potential: upon dissipation, LytR induces expression of the antiholin-like protein LrgA, and thereby, the cell attempts to prevent total membrane permeabilization. In other organisms, the deletion of lytSR revealed distinct roles for this regulatory pathway: in Streptococcus mutans LytST governs lrgAB expression in response to glucose and reactive

oxygen species (ROS), while in Staphylococcus epidermidis, LytSR regulates cell death during

biofilm formation and pyruvate utilization 67,69_{. Thus, it appears that LytSR, although}

widely conserved, has variable roles in different organisms, all being connected to metabolism and cell death, or tentatively both (Fig. 2).

Notably, ubiquitous genes with high homology have diverged considerably in function throughout the course of evolution. However, the link between metabolism, ROS and PCD appears to be prevalent in nature. In eukaryotes, where apoptosis has been studied in far greater detail, networks regulating metabolism and PCD are deeply intertwined. The metabolic status of a cell determines its fate: whether a cell divides, grows or dies depends on the environment, nutrient availability and its energy status. A wide range of sensing mechanisms integrate the diverse signals and control gene expression accordingly. This is especially apparent in the mitochondria, which are thought to have a bacterial origin and where many of the cell’s key metabolic and signaling pathways are routed through. Specifically, the mitochondrial Bcl-2 protein family seems to play an important role in linking metabolism and PCD. This protein family has a dual function in signaling pathways governing metabolism and apoptosis, with certain members having both direct functions in nutrient utilization and apoptosis. In addition,

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Figure 2. Schematic representation of the LytST (LytSR) regulatory pathways in B. subtilis (A), S. aureus (B), S. mutans (C), and S. epidermidis (D). Arrows depict transcriptional activation, metabolic

conversions or transports, while red T-bars indicate negative regulation. Direct and indirect interactions are indicated with solid and dashed lines, respectively. Taken from van den Esker et

al. (2017)276_.

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Bcl-2 proteins also indirectly influence apoptosis by altering the metabolic status of the cell 277_{. The conserved nature of Bcl-2 like proteins suggests that the connection between}

metabolism and PCD is omnipresent.

The holin and antiholin-like proteins of S. aureus have been proposed to be functional

analogs of the Bcl-2 proteins: Bax is a proapoptotic protein causing mitochondrial outer membrane permeabilization and acts similar to CidA, while the anti-apoptotic Bcl-2 protein antagonizes this process analogously to LrgA. In S. aureus, CidA is transcribed

from the cidABC operon and is induced during overflow metabolism in the presence of

high pyruvate or acetate levels. The regulatory protein CidR influences both acetate formation by inducing CidC, a pyruvate oxidase, and acetoin formation via AlsSD. The

overall balance between acetate and acetoin formation in S. aureus makes the difference

between life and death, again showing a tie between metabolism and PCD. Certain features of eukaryotic apoptosis could be recognized within bacterial cell death, such as membrane permeabilization, DNA condensation and fragmentation. Furthermore, the metabolic state of a bacterial cell affects its antibiotic susceptibility 278_{. Therefore,}

it is plausible that bacteria possess networks with analogous function and metabolic dependencies that influence certain cells within a population to divide, to arrest growth or, when a metabolically unfavorable condition is met, to lyse. A future holistic research approach could unravel these links.

The essentiality of wall teichoic acids

Other molecules located in the cell envelope that influence cell death at different levels are wall teichoic acids (WTAs). WTAs reside in the cell wall and influence its charge, and thereby autolysis, but possibly also the secretion of charged compounds. Therefore, altering the amount of WTAs depending on the circumstances could be of great interest for industrial applications. In chapter 3, we intended to study the relation between the amount of wall teichoic acids in the cell wall and autolysin transcription. Although our generated strain had a similar phenotype as ∆tagO mutants published in previous studies,

and our transcriptome data corresponded with data published before 33,125,129_{, a big genomic}

region of ~207 kb was deleted partly overlapping with the SPβ-prophage region. This raises the debate of WTA essentiality in B. subtilis.

A gene is considered as essential if it is critical for survival in optimal growth conditions, for B. subtilis, this is in LB at 37°C. Essential genes are discovered by two main

strategies. Firstly, via targeted mutagenesis individual genes are precisely deleted and viability of the mutant strain is examined. Secondly, transposon-mediated mutagenesis results in a library of viable mutants, thereby revealing which genes could be indispensable

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as the transposon cannot inactivate those genes. In B. subtilis, 261 open reading frames of

4,100 in total are regarded as essential, among which 259 encode for proteins and two for RNAs 14_{. Indispensable genes are mainly housekeeping genes (genes that cover DNA}

replication, transcription and translation), genes involved in cell wall biosynthesis and metabolism 13,14_{. Growth in other media or conditions might require additional genes;}

these are ‘conditional essential’.

Until 2006, targeted inactivation of tag-genes did not result in viable strains and

WTAs were assumed to be indispensable 13,32,126,279,280_{. In 2006, D’Elia}_{et al. constructed}

a viable tagO deletion mutant via targeted mutagenesis, although the obtained colonies

were severely diminished in size and growth 33_{. The authors speculated that the small}

∆tagO colonies were missed before by other scientist. However, the deletion mutant was

never complemented, thus the possibility of rescue or other secondary site mutations was not excluded. The conditional mutant we created displayed a similar phenotype when depleted for tagO, but analysis of the transcriptome exposed a secondary site

mutation. Future experiments include replication studies by generating new tagO

de(p)letion mutants, and subsequent sequencing of the genomes of these strains to screen for possible mutations. Moreover, we suggest to sequence the genomes of other published ∆tagO deletion mutants as well in order to provide conclusive evidence on the

strains background. In order to avoid ambiguity, our study emphasizes the importance of complementation of published deletion strains, e.g. by expressing the gene from an ectopic locus.

Gene essentiality studies are fascinating for fundamental reasons, but these mutants are created in perfect laboratory circumstances and therefore, practical implications can be small. It has for example been shown that TarO, the TagO analogue in Staphylococcus aureus, is also dispensable, but in its natural niche, tarO knockout strains are not viable

281,282_{. In our experiments we also observed growth of the}_{tagO mutant in LB, but growth}

was ceased when the mutant was shifted to other media. Thus, even if it is possible to delete tagO without causing concomitant secondary site mutations, this gene is

conditionally essential, and ‘conditionally’ meaning all other conditions than rich broth media at an optimal temperature.

The cell envelope of Bacillus subtilis contributes to its temperature versatility

B. subtilis is a versatile bacterium that is able to survive in many different, sometimes

extreme, conditions. It has several differentiation programs contributing to adaptation, including biofilm formation and sporulation. By sensing changes in the outside environment, the cell regulates gene transcription accordingly and optimizes the

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production and modification of its individual components. The cell envelope is also continually adjusted to the external environment. Thereby, B. subtilis is able to proliferate

in many conditions and withstand large temperature variations ranging from 11 to 55°C 70_.

Adaptation of the cell envelope, and specifically the cytoplasmic membrane, is of great importance during cold shock and growth at low temperatures, and is the first response that enables cellular survival. Membrane fluidity is maintained by fatty acid desaturation of the phospholipids located in the plasma membrane. Subsequently, the transcription and translation machinery is rearranged to pursue growth at lower temperatures. To properly adjust to cold shock conditions, SigL is required in B. subtilis.

This RpoN-like sigma factor cooperates with bacterial enhancer binding proteins (bEBPs) to induce the expression of target genes. During cold shock, the bEBPs BkdR and YplP are expressed. BkdR regulates the utilization of branched-chain amino acid involved in fatty acid desaturation which are needed to maintain membrane fluidity 160_{. YplP is also}

required for optimal cold adaptation, but the regulon of this putative bEBP had not yet been characterized 79_.

In chapter 4, we performed a computational and transcriptomic study to elucidate the SigL-YplP regulon. During our work in chapter 2 we already noticed the presence of a predicted SigL box in the ysbA promoter (DBTBS), which implies that ysbA is activated

by SigL under specific conditions 94_{. Our RNA sequencing experiment demonstrated that}

ysbAB expression is induced by SigL-YplP under cold shock conditions. In chapter 2, we

showed that YsbA is involved in pyruvate metabolism, and Charbonnier et al. confirmed

that YsbAB acts as membrane-embedded pyruvate transporter 165_{. The results of this}

chapter therefore indicate that pyruvate transport alters during cold shock, and gives novel insights into ysbAB regulation.

Knowledge on specific bacterial pyruvate transporters is scarce, but proteins involved in pyruvate formation and metabolism are known to be induced in other bacteria under cold shock conditions as well 183,283–285_{. Mesophilic bacteria have an enhanced}

substrate requirement at low growth temperatures, and transport is therefore expected to be induced 286,287_{. Metabolic routes are shifted towards alternative carbon pathways}

in many bacteria upon cold shock, such as the fast, anaerobic generation of lactate 287_.

Pyruvate metabolism is at the core of many cellular pathways, and the induction of pyruvate transport could aid in the adaptation of carbon metabolism and the optimization of amino acid synthesis.

Our general RNA sequencing experiment investigated the cold shock on the short and long-term (chapter 4). The advantage of RNA sequencing over microarray studies include the exploration of novel open reading frames (ORFs) and RNA species, while

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in microarrays only spotted known ORFs can be detected. Indeed, multiple tRNAs and few miscRNAs were induced, which were not discovered before in microarray studies. Moreover, RNA sequencing delivers low background levels since hybridization is eliminated, thereby avoiding problems related to cross-hybridization and non-optimal hybridization. RNA sequencing does not give relative, but rather absolute values resulting in a broader dynamic range. The fold changes in our study were much larger compared to previous microarrays (e.g. des: 88 vs 10x induced, desKR 123/107 vs 11.9/18.5x) 78_{. Thus, the}

elaborate results of our RNA sequencing experiment complement previous microarray studies.

The ability to adapt to low temperatures is of great importance for soil organisms such as B. subtilis, as temperatures can rapidly decline in this environment. Acclimatization

occurs at multiple levels, and in this study we have expanded the knowledge on the short and long-term reaction of B. subtilis to cold shock. Moreover, we created insights into

the YplP regulon, although these results need further validation. It appears that YsbAB, a pyruvate transporter protein, is induced under cold shock conditions. Thus, next to adaptation of fatty acids in the cell membrane, the expression of membrane proteins is adjusted to optimize nutrient uptake and growth at low temperatures.

Bacterial-Fungal interactions: From bench to nature

B. subtilis is a popular Gram-positive model organism, and strain 168 has since decades

been most frequently used due to its high competence rates, making it easy to genetically modify. This increased genetic competence likely results from UV-irradiation of its parent strain, and irradiation, combined with growth for many cycles under laboratory settings, generated a fast-growing domesticated strain. Amendable strains are invaluable to industry, as it is possible to optimize growth and secretion processes, thereby increasing the yield. However, after decades of research it was recognized that domesticated strains have lost several traits that are common in more ‘wild’ strains, such as the capability of swarming and the formation of complex biofilms 288_{. This also appeared from our study}

in chapter 5 where we discovered that B. subtilis can interact with the filamentous fungus Aspergillus niger. The bacterial-fungal interaction (BFI) was strain specific, as attachment

between B. subtilis 168 or Aspergillus oryzae has not been observed under the conditions

tested. This study thus shows major behavioral differences between laboratory strains and strains occurring in nature, as was demonstrated before for e.g. fruiting body formation

219,289_{. Therefore, the extrapolation of laboratory results obtained by studying domesticated}

strains under artificial conditions to natural situations is not always straightforward.

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B. subtilis is originally a plant-associated bacterium that in its natural environment

encounters many different species that are also present in the soil 1_{. The microscale}

distribution of this bacterium in the soil has rarely been studied. Soil consists of open spaces and aggregates formed by minerals (e.g. clay or sand) through which water moves and nutrients diffuse. Within these open pores microorganisms reside. Research has indicated that B. subtilis colonies are relatively small, and count on average five cells 290_.

Increasing the knowledge of the B. subtilis micro-habitat is essential for understanding the

soil ecosystem and to overcome the gap between bench and nature 291_{. The possibility to}

mimic environmental conditions in the laboratory will enable issue-driven research, in which environmental problems can be systematically studied 292_.

This will also aid in developing novel applications of BFIs. BFIs can affect plant growth at different levels, for example by solubilizing phosphorus and nitrogen present in the soil that are otherwise inaccessible for plants 293,294_{. Understanding of BFIs could}

therefore be relevant for agricultural applications. B. subtilis is already frequently used

in agriculture, e.g as soil inoculant to promote plant growth, and as biocontrol agent

11,207,295–297_{. The interaction between}_{B. subtilis and other species, such as fungi, could alter}

or reinforce the beneficial effects, e.g. by changing enzyme or secondary metabolite production. Both B. subtilis and A. niger can be isolated from soil, so it is possible that

interaction also occurs in nature. The BFI in our study altered the metabolism of both organisms involved and caused a decrease in both the bacterial stress response and surfactin production, suggesting this interaction is advantageous, at least for B. subtilis.

Therefore, examining this interaction under environmental conditions might be useful for agricultural applications. Both organisms are used as working horses by industry as well. It appears that, at least B. subtilis, benefits from this interaction, and it will be interesting to

determine the effect of co-growth with A. niger on relevant features such as protein yield,

secondary metabolite excretion and growth to optimize industrial conditions in this way. Hence, this study can be seen as a starting point for future, more elaborate investigations.

Concluding remarks

The cell envelope of B. subtilis is an intricate structure that contributes to the versatility

of this organism. It is required for all facets of cellular functioning, such as growth, differentiation and adaptation to changing conditions. The work performed in this thesis focused on different processes related to the cell envelope, thereby identifying novel functions of its components and reactions to variable conditions. Next to providing novel fundamental insights, the reported work gives directions to future studies aimed at more

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practical industrial or agricultural implications including the optimization of growth conditions and enzyme or secondary metabolite secretion.

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