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

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University of Groningen

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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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:

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22

Chapter 2

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,

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23

Introduction

The Gram-positive bacterium Bacillus subtilis was first isolated in the 19th_{century from hay,}

already suggesting its plant-associated ecological niche 1_{. 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 7,8_{. 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 61_{. The antiholin-like protein LrgA prevents dissipation of the proton}

motive force when the membrane potential is affected, reducing cell lysis under certain

conditions 58,60_._{lrgA transcription is induced by LytSR, a two-component regulator that}

senses changes in the membrane potential 80,81_{. The}_{cidABC-operon, regulated by CidR,}

induces cell death when the medium acidifies due to overproduction of acetate 59,82_{. 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 66,83_.

The Cid/Lrg network is widely conserved: 46% of all bacterial species for which

genome sequences are available possess homologues of this network 57_{. 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 67–69_{. 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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24

Chapter 2

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

represses ywbH transcription 84_{. The glucose level appears to influence}_{ysbAB expression,}

probably via CcpA 48,85,86_{, while CshA, an RNA helicase, also strongly affects the transcript}

level of ysbAB genes 87_{. 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 88_.

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 301_{(A). Predicted structures of YsbA and YwbH (B). Protein structures were}

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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 89,90_{. 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 91_{, 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 OD₆₀₀ in LB and the black dotted line in graph B represents the OD₆₀₀ in the presence of glucose.

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26

Chapter 2

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 48,85,86_{. 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_ysbA-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_ysbA-gfp in WT (A) and ∆lytS (B) background. Strains were grown under

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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 91_{. 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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28

Chapter 2

Figure 4. P_ysbA-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 OD₆₀₀ 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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30

Chapter 2

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 OD₆₀₀, 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 91_{. 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 59,92_{. 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. aureus62,81,93_{. 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 48,85,86,94_{. 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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32

Chapter 2

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 46,95_.

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 84_{. According to our results,}

this putative two-component regulatory system is mainly responsible for inducing ysbA

expression: when lytS is deleted, P_ysbA-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 23_{. 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 96_{. 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 91_.

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

In some species, such as in C. glutamicum and E. coli 97,98_{, 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 99_{. 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 41–43_{. 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 100–102_{. 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 103,104_._{E. coli excretes pyruvate when grown on succinate, and}

B. cereus on media containing glucose and yeast extract 105,106_{. Furthermore, pyruvate is}

excreted when B. subtilis is grown on medium containing malate 95_{. 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 88_{. It is known that several other pyruvate transporters}

are able to transport acetate as well, which might explain upregulation after acetate addition 97,107_.

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 LytT84_{. Furthermore, transcriptome analysis showed that}_{ywbH is expressed highly}

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34

Chapter 2

when malate and glucose are present in the medium 91_{, 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 88_{, 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 108_.

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) 109_{. When}

required, antibiotics were added to the medium in the following final concentrations: ampicillin (100 µg ml-1_{) for}_{E. coli, or tetracycline (6 µg ml}-1_{), kanamycin (5 µg ml}-1_),

spectinomycin (100 µg ml-1_{), chloramphenicol (5 µg ml}-1_{) 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) 110_{. PCRs}

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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 111_.

Table 1. Bacterial strains used in this study

Strain a _{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 112

Bacillus subtilis

168 1A700 trpC2 Kunst et al., 1997 12

168 P_xyl-comK amyE::P_xyl-comK, CmR _{168 variant based on Hahn et al., 1996}113

ME001 amyE::P_ysbA-gfp, CmR _{This study}

ME002 amyE::P_ywbH-gfp, CmR _{This study}

ME003 ysbA::tet, TcR _{This study}

ME005 ywbH::tet, TcR _{This study}

ME006 lytS::tet, TcR _{This study}

ME007 ywbI::tet, TcR _{This study}

ME008 amyE::P_hyperspank-ysbA, SpR _{This study}

ME009 amyE::P_hyperspank-ywbH, SpR _{This study}

ME010 ysbA::tet, TcR_{, amyE::P}

hyperspank-ywbH, SpR This study

ME011 ywbH::tet, TcR_{, amyE::P}

hyperspank-ysbA, SpR This study

ME012 ysbA::tet, TcR_{, amyE::ysbAB, Cm}R _{This study}

ME013 lytS::tet, TcR_{, amyE::lytST, Cm}R _{This study}

ME014 lytS::tet, TcR_{, amyE::P}

ysbA-gfp, CmR 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_rrnB-gfp+_{cat ‘amyE spec bla} _{Veening et al., 2009}115

P_ysbA-gfp amyE’ P_ysbA-gfp cat ‘amyE spec bla This study P_ywbH-gfp amyE’ P_ywbH-gfp cat ‘amyE spec bla This study

pBEST309 bla, tet Itaya et al., 1992 116

pDR111 bla amyE’ P_hyperspankspec lacI ‘amyE Gift of D. Rudner

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

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

pX bla amyE’ xylR P_xylAcm ‘amyE Kim et al., 1996 117

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

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

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Chapter 2

Single cell microscopy and flow cytometry

Strains were plated from -80°C stocks on LB plates containing antibiotics and grown overnight at 37°C. Subsequently, a colony was grown in LB medium + antibiotics for 8 hours, and diluted 1000x in LB or M9 medium containing proper antibiotics. This culture was grown overnight at 37°C, 220 rpm. The following morning, the culture was diluted to

an OD₆₀₀ of 0.05 or 0.1 in fresh medium, and morphology and/or fluorescence was assessed

using microscopy and flow cytometry at timepoints described in the results section. For microscopy, slides with 1% agarose in Phosphate-buffered Saline (PBS) were prepared. Bacterial cells were visualized using a Olympus DeltaVision microscope (Applied Precision) with a 300W Xenon Light source, CoolSNAP HQ2 camera (Princeton Instruments) and a 100x phase contrast objective (Olympus PlanApo 1.40 NA). A GFP filterset (Chroma, excitation at 470/40 nm, emission 525/50 nm) was used to detect green fluorescence. Microscopy data were stored using softWoRx 3.6.0 (Applied Precision) and

further processed and analyzed using ImageJ 143_.

For flow cytometry, fluorescence was measured using a BD FACSCanto Flow Cytometer (BD BioSciences) equipped with an argon laser (488 nm). For each sample, 50,000 cells were analyzed. Data containing fluorescent signals were collected by a FITC filter. The photomultiplier voltage was set at 700 V. Data were captured using BD FACSDiva software (BD Biosciences) and further analyzed using Flowing Software (http://www.flowingsoftware.com/).

Plasmid construction

Construction of the promoter-gfp fusion plasmids P_ysbA-gfp and P_ywbH-gfp was carried out

by amplifying the entire promoter region including the native RBS of ysbA and ywbH

using oligo PysbA-F+MfeI and PysbA-R+NheI, and PywbH-F+EcoRI and PywbH-R+NheI.

Subsequently, the PCR products were cleaved with NheI and EcoRI (ywbH) or NheI and

MfeI (ysbA), and ligated into the EcoRI and NheI sites of pGFP-rrnB, generating P_ysbA-gfp

and P_ywbH-gfp.

pDR111-ysbA and pDR111-ywbH were created by amplifying gene ysbA and ywbH

using primer ysbA-F+SalI+RBS and ysbA-R+NheI+2stop, and ywbH-F+SalI+RBS and ywbH-R+NheI+2stop. These primers contain perfect ribosomal binding sites in order to maximize the translation of the overexpressed proteins. PCR products were cleaved with NheI and SalI, and ligated into the corresponding sites of pDR111. This plasmid carries the

IPTG inducible hyperspank promoter, enabling overproduction of the proteins of interest.

Plasmids for complementation were created by cloning the entire lytST and ysbAB

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P_xylA and xylR, resulting in a plasmid containg the operons with their native promoters,

flanked by a chloramphenicol resistance cassette and amyE sites. First, ysbAB and lytST

were amplified using primer ysbAB-F-XbaI, ysbAB-R-XbaI and lytST-F-XbaI, lytST-R-XbaI. Subsequently, the PCR products were ligated into corresponding XbaI sites of plasmid

pX. Correct replacement of P_xylA and xylR by the operons of interest was confirmed by

PCR and restriction analysis.

Strain construction

Reporter strains carrying promoter-gfp fusions were generated by transforming P_ysbA-gfp

and P_ywbH-gfp to B. subtilis 168. Plasmids were integrated by double crossover into the

non-essential amyE locus of B. subtilis 168. Transformants were selected overnight on LB agar

plates containing chloramphenicol (cm). Double crossovers were selected by picking cm resistant, and spectinomycin (spec) sensitive clones, giving rise to ME001 and ME002.

pDR111-ysbA and pDR111-ywbH were also transformed into the amyE locus of B. subtilis,

and selected overnight on LB+spec, resulting in strains ME008 and ME009 respectively.

Integration into the amyE locus was verified for reporter and overexpression strains by

lack of α-amylase activity when strains were grown on LB+1% starch plates, and correct integration was confirmed by PCR.

To create strains ME003, ME005, ME006 and ME007, the genes of interest (ysbA,

ywbH, lytS and ywbI) were replaced with a tetracycline cassette from pBEST309. First,

flanking regions of ~1 kb were amplified using primer ysbA-F-up-BamHI + ysbA-R-up, ysbA-F-down + ysbA-R-down-BamHI; ywbH-F-up + ywbH-R-up-BamHI, ywbH-F-down-BamHI + ywbH-R-down; LytS-F-up-ywbH-F-down-BamHI + LytS-R-up, LytS-F-down + LytS-R-down-BamHI and YwbI-F-up-LytS-R-down-BamHI + YwbI-R-up, YwbI-F-down + YwbI-R-down-LytS-R-down-BamHI. The resulting PCR products and pBEST309 were digested with BamHI. After ligating the tetracycline cassette of pBEST309 to the upstream and downstream flanking region, the

mixture was concentrated in a SpeedVac concentrator and directly transformed to B.

subtilis 168 P_xyl-comK. This strain was used as an in-between host, because it reaches much

higher competence levels when induced with xylose after the first two hours of growth in MC-medium compared to the wild-type strain. In this case, fructose was added as carbon source to MC-medium, because glucose interferes with xylose and thereby disturbs proper induction of the xylose inducible promoter. Transformants were selected overnight on LB plates containing tetracycline and chloramphenicol. Subsequently, chromosomal DNA

was isolated and transformed into wild-type B. subtilis 168, and colonies were selected

that did not contain P_xyl-comK (cm sensitive colonies were picked) but did contain the

gene replacements (tc resistant).

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Chapter 2

Complementation strains were cloned by transforming pX-ysbAB and pX-lytST

into the ectopic amyE locus of ME003 and ME006, giving rise to ME012 and ME013.

Transformants were selected on tc and cm, and correct integration in the amyE locus of

the chromosome was verified as described above.

Combinations of strains were made by transforming chromosomal DNA of the reporter or overexpression strains into the deletion mutants. Transformants were selected on appropriate antibiotics (cm and tc, or spec and tc), and correct integration into the

amyE locus was verified as described before.

Growth curves and antibiotic/metabolic assays

Cultures were inoculated from -80°C stocks on LB plates containing antibiotics, and grown overnight at 37°C. The next day, single colonies were incubated and grown in LB medium with appropriate antibiotics for 8 hrs at 37°C, 220 rpm. After that, the culture was diluted in several ratios (ranging from 1:2000 – 1:30, depending on the medium used) in either LB or M9 medium and grown overnight. The next morning, the overnight culture

that was still in exponential growth phase was selected, and diluted to an OD₆₀₀ of 0.05

or 0.1 in M9 or LB medium (without antibiotics). When necessary, gene expression was induced by adding 1 mM IPTG in early exponential phase. Growth was followed by measuring the absorbance at 600 nm at different timepoints.

For antibiotic and metabolic assays, wild-type and deletion mutants or overexpression strains were grown to mid-exponential and/or stationary phase. Subsequently, the cultures were divided in equal volumes and compounds of interest (acetic acid 30mM; acetoin 30mM; nisin 50µg/ml; valinomycin 0.1-10µM; CCCP 100 µM;

gramicidin 25µg/ml) were added. As a control, the solvents of the antibiotics (destH₂O,

DMSO, ethanol) were added separately. Lysis was assessed by measuring the decrease in absorbance at 600 nm, and lysis behavior of the mutant strains was compared to that of

the wild-type. Changes in expression of ysbA and ywbH after the addition of antibiotic or

metabolic compounds was measured with the use of reporter strains ME001 and ME002. The strains were treated similarly as described above, and GFP expression of both strains was measured using flow cytometry and/or microscopy.

Autolysis assay

For autolysis assays, B. subtilis strains were grown overnight in LB medium (37°C, 220 rpm).

The following morning, cells were diluted in fresh LB to an OD₆₀₀of 0.05 and harvested

in late-exponential phase (OD₆₀₀=0.8) by centrifugation for 10 min at 4°C (14000 g). After

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Triton-X-100 in 50 mM Tris-HCl (pH 7.2) to an OD₆₀₀ of 1.6, and incubated at 37°C, 220

rpm. The decrease in the absorbance at 600 nm was measured every 30 min.

Plate reader experiments

Cultures were inoculated from -80°C stocks on LB agar plates with antibiotics, and grown overnight at 37°C. The following day, a single colony was picked and grown overnight in LB or SMM medium containing antibiotics when required, at 37°C, 220 rpm. After overnight growth, cultures were diluted in a 96-well microtitre plate by adding 2 µl of the culture in 200 µl fresh LB, MM or M9 medium containing pyruvate. Plates were incubated at 37°C and maximal shaking in either an Infinite 200 plate reader (Tecan) with a GFP filterset (Chroma, excitation at 485/20 nm, emission 535/25) and i-control 1.10

software (Tecan), or in a Biotek SynergyTM _{MX plate reader controlled by Gen5 software.}

Measurements were taken every 10 or 15 minutes. Absorbance values were collected at 600 nm, and GFP fluorescence was collected as top reading with a gain of 60 or 70. Data of all samples were collected in triplicates, and processed in Microsoft Excel by

calculating the average values as well as standard deviations. OD₆₀₀ values were corrected

for OD₆₀₀ background of the medium. GFP levels were corrected for OD₆₀₀ and background

fluorescence of the medium according to the following formula:

((GFP_reporter-GFP_LB)/(OD_reporter-OD_medium))-((GFP_wt-GFP_medium)/(OD_wt-OD_LB))

Determination of pyruvate levels

Extracellular pyruvate in M9+pyruvate was quantified by incubating a B. subtilis colony

for 8 hrs in LB medium (containing antibiotics when necessary). Subsequently, cells were grown overnight in M9+glucose with antibiotics in different dilutions, so that one of the cultures was still in exponential phase the following morning. The next day, cells were

washed in PBS, and diluted to an OD₆₀₀ of 0.05 in M9+pyruvate (6 g/L) and incubated

at 37°C, 220 rpm. At several time points (4, 6.5, 9, 12, 24 hrs) cells were removed by centrifuging 500 µl of the culture (10 min, 4°C, 14000 g), and filter sterilization of the

supernatant. For time course measurements of extracellular pyruvate in LB, wild-type B.

subtilis was grown overnight in LB, and the following morning diluted in fresh LB to an

OD₆₀₀ of 0.05. Samples were taken at 2 hr intervals, and cells were removed as described

before.

The concentration of pyruvate in the supernatants was determined in duplicate

with an EnzyChromTM_{pyruvate assay kit (BioAssay Systems, Hayward, CA), according}

to the manufacturer’s instructions. The total concentration of pyruvate consumed by the different strains was directly derived from extracellular pyruvate levels by subtracting

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Chapter 2

the concentration of pyruvate present in the medium after 24 hrs from the starting concentration of pyruvate in the medium.

Supplementary Information

Supplementary information can be downloaded at:

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