Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis

Accepted Manuscript Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis Sébastien Gélis-Jeanvoine, Alexis Canette, Michel Gohar, Thibault Caradec, Christelle Lemy, Myriam Gominet, Philippe Jacques, Didier Lereclus, Leyla Slamti PII:

S0923-2508(16)30062-6

DOI:

10.1016/j.resmic.2016.06.002

Reference:

RESMIC 3512

To appear in:

Research in Microbiology

Received Date: 21 January 2016 Revised Date:

23 May 2016

Accepted Date: 6 June 2016

Please cite this article as: S. Gélis-Jeanvoine, A. Canette, M. Gohar, T. Caradec, C. Lemy, M. Gominet, P. Jacques, D. Lereclus, L. Slamti, Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis, Research in Microbiologoy (2016), doi: 10.1016/ j.resmic.2016.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1⁄

ACCEPTED MANUSCRIPT Genetic⁄and⁄functional⁄analyses⁄of⁄krs,⁄a⁄locus⁄encoding⁄kurstakin,⁄

2⁄

a⁄lipopeptide⁄produced⁄by⁄Bacillus⁄thuringiensis⁄⁄

3⁄ ⁄ Sébastien⁄Gélis-Jeanvoinea,⁄Alexis⁄Canettea,⁄Michel⁄Gohara,⁄Thibault⁄Caradecb,⁄

5⁄

Christelle⁄Lemya,⁄Myriam⁄Gominetc,⁄Philippe⁄Jacquesb,⁄Didier⁄Lereclusa*,⁄Leyla⁄

6⁄

Slamtia*⁄

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4⁄

a

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7⁄ ⁄

Micalis⁄Institute,⁄INRA,⁄AgroParisTech,⁄Université⁄Paris-Saclay,⁄78350⁄Jouy-en-Josas,⁄

9⁄

France⁄

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8⁄

10⁄

⁄

b

University⁄of⁄Lille,⁄EA⁄7394,⁄ICV-Institut⁄Charles⁄Viollette,⁄ProBioGEM⁄team,⁄

12⁄

Polytech'Lille,⁄avenue⁄Langevin,⁄59655⁄Villeneuve⁄d'Ascq,⁄France⁄

13⁄

⁄

c

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11⁄

Institut⁄Pasteur,⁄CNRS⁄URA⁄2172,⁄Unité⁄de⁄Biologie⁄des⁄Bactéries⁄Pathogènes⁄à⁄Gram⁄

15⁄

positif,⁄75015⁄Paris,⁄France⁄

16⁄ ⁄

[email protected],⁄[email protected],⁄[email protected],⁄[email protected],⁄ [email protected],⁄[email protected],⁄⁄[email protected],⁄ [email protected]⁄ *Correspondence⁄and⁄reprints⁄ ⁄

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17⁄ 18⁄ 19⁄ 20⁄

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14⁄

ACCEPTED MANUSCRIPT 21⁄ Abstract 22⁄

Bacteria of the Bacillus genus are able to synthesize several families of lipopeptides. These

23⁄ small molecules are the product of non-ribosomal peptide synthetases. In 2000, it was found that 24⁄ Bacillus thuringiensis, an entomopathogenic bacterium of the Bacillus cereus group, produced a

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25⁄ previously unknown lipopeptide: kurstakin. Genomic analyses reveal that the krs locus, encoding 26⁄ the kurstakin synthetases, is specific to the B. cereus group, but is unevenly distributed within this 27⁄ group. Previous work showed that krs transcription requires the necrotrophism quorum-sensor

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28⁄ NprR. Here, we demonstrated that the genes of the krs locus form an operon and we defined its 29⁄ transcription start site. Following krs transcription at the population and single-cell levels in

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30⁄ multiple culture conditions, we depicted a condition-dependent transcription pattern, indicating that 31⁄ production of kurstakin is subject to environmental regulation. Consistent with this idea, we found 32⁄ krs transcription to be regulated by another master regulator, Spo0A, suggesting that krs expression 33⁄ is fine-tuned by integrating multiple signals. We also reported an unknown DNA palindrome in the

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34⁄ krs promoter region that modulates krs expression. Due to their surfactant properties, lipopeptides 35⁄ could play several physiological roles. We showed that the krs locus was required for proper biofilm

37⁄ ⁄

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36⁄ structuration.⁄

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38⁄ Keywords:⁄Biofilm;⁄Transcriptional⁄regulation;⁄Spo0A;⁄Bacillus⁄cereus⁄group⁄ ⁄

ACCEPTED MANUSCRIPT 39⁄ 40⁄ ⁄

1. ⁄Introduction⁄ Introduction Lipopeptides⁄are⁄well-known⁄secondary⁄metabolites⁄produced⁄by⁄bacteria⁄of⁄

41⁄ the⁄ Bacillus⁄ and⁄ Pseudomonas⁄ species.⁄ They⁄ are⁄ the⁄ products⁄ of⁄ big⁄ enzymatic⁄ 42⁄ complexes,⁄the⁄so-called⁄non-ribosomal⁄peptide⁄synthetases⁄(NRPSs).⁄Lipopeptides⁄

44⁄ fengycins,⁄ iturins⁄ and⁄ kurstakins⁄ [1]

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43⁄ produced⁄ by⁄ Bacillus⁄ belong⁄ to⁄ four⁄ structurally⁄ diverse⁄ families:⁄ surfactins,⁄ .⁄ These⁄ molecules⁄ present⁄ physical⁄

46⁄ biosurfactants,⁄

emulsifiers,⁄

antifungals,⁄

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45⁄ properties⁄ of⁄ interest⁄ for⁄ various⁄ industrial⁄ sectors⁄ and⁄ are⁄ used⁄ as⁄ antibacterials,⁄

.⁄ This⁄ has⁄ led⁄ to⁄

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47⁄ antitumorals,⁄ immunosuppressants⁄ and⁄ plant⁄ elicitors⁄ [2 4]

antivirals,⁄

48⁄ extensive⁄ studies⁄ on⁄ lipopeptide⁄ properties,⁄ but⁄ little⁄ is⁄ known⁄ about⁄ their⁄ 49⁄ biological⁄role⁄and⁄the⁄regulation⁄of⁄their⁄production⁄(see⁄[5]⁠ ⁄for⁄a⁄review).⁄ 50⁄ Most⁄data⁄currently⁄available⁄show⁄that⁄transcription⁄of⁄lipopeptide⁄synthetases⁄ .⁄

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51⁄ is⁄ often⁄ regulated⁄ by⁄ two-component⁄ and/or⁄ quorum-sensing⁄ systems⁄ [5]

52⁄ Transcription⁄ of⁄ srfA,⁄ the⁄ locus⁄ responsible⁄ for⁄ the⁄ synthesis⁄ of⁄ the⁄ most⁄

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53⁄ studied⁄lipopeptide,⁄surfactin,⁄is⁄regulated⁄by⁄the⁄ComXPA⁄quorum-sensing⁄system⁄ 54⁄ in⁄ Bacillus⁄ subtilis⁄ [6,7]

.⁄ Interestingly,⁄ despite⁄ being⁄ quorum-regulated,⁄

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55⁄ srfA⁄ was⁄ shown⁄ to⁄ be⁄ transcribed⁄ in⁄ only⁄ a⁄ small⁄ part⁄ of⁄ the⁄ cells⁄ in⁄ the⁄ 56⁄ population⁄ [8]

.⁄ Surfactin⁄ is⁄ then⁄ perceived⁄ as⁄ a⁄ signal⁄ by⁄ the⁄ non-producer⁄

57⁄ bacteria,⁄which⁄increases⁄phosphorylation⁄of⁄Spo0A⁄via⁄the⁄kinase⁄KinC.⁄Spo0A~P,⁄ 58⁄ the⁄active⁄form⁄of⁄the⁄master⁄regulator⁄of⁄differentiation⁄in⁄ B.⁄subtilis,⁄will⁄ 59⁄ then⁄trigger⁄biofilm⁄formation,⁄cannibalism⁄and⁄sporulation⁄[9] 60⁄ ⁄

.⁄

Bacillus⁄thuringiensis⁄is⁄an⁄entomopathogenic⁄bacterium⁄from⁄the⁄ Bacillus⁄

61⁄ cereus⁄ group⁄ [10]

.⁄ B.⁄thuringiensis⁄ kurstaki⁄ HD-1⁄ was⁄ shown⁄ to⁄ produce⁄

ACCEPTED MANUSCRIPT 62⁄ kurstakins,⁄ molecules⁄ that⁄ constitute⁄ a⁄ new⁄ class⁄ of⁄ Bacilli⁄ lipopeptides⁄ 63⁄ [11]

.⁄ ⁠ur⁄ model⁄ strain⁄ B.⁄thuringiensis⁄ 407⁄ also⁄ harbors⁄ a⁄ krs⁄ locus⁄ and⁄

64⁄ produces⁄ kurstakin⁄ [12,13]

.⁄ The⁄ krs⁄ locus⁄ (Fig.⁄ S1)⁄ encodes⁄ the⁄ NRPS⁄

65⁄ responsible⁄ for⁄ kurstakin⁄ synthesis⁄ and⁄ contains⁄ 6⁄ genes:⁄ krsE,⁄ predicted⁄ to⁄

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66⁄ encode⁄a⁄permease⁄potentially⁄involved⁄in⁄the⁄efflux⁄of⁄kurstakin⁄[14,15];⁄the⁄3⁄ 67⁄ kurstakin⁄synthetases⁄ krsA,⁄ krsB⁄and⁄ krsC;⁄ sfp,⁄a⁄phosphopantheteine transferase; and 68⁄ krsD, a type II thioesterase. It was shown that krs transcription is activated at the onset of

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69⁄ sporulation by the quorum sensor NprR, a major regulator required for the necrotrophic stage 70⁄ during the life cycle of B. thuringiensis (i.e. its capacity to survive in the cadaver of an insect host)

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71⁄ [13]⁠ . In contrast, SinR was found to negatively affect krs transcription in a biofilm-promoting 72⁄ medium [16]⁠ . 73⁄

Lipopeptides⁄were⁄shown⁄to⁄be⁄involved⁄in⁄various⁄bacterial⁄processes⁄that⁄

75⁄ (see⁄ [5]

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74⁄ often⁄appear⁄to⁄be⁄linked⁄to⁄the⁄adaptation⁄of⁄a⁄bacterium⁄to⁄a⁄particular⁄niche⁄ ⁄ for⁄ a⁄ review).⁄ In⁄ the⁄ case⁄ of⁄ B.⁄thuringiensis,⁄ kurstakin⁄ could⁄ be⁄

76⁄ involved⁄in⁄necrotrophism⁄by⁄increasing⁄the⁄spreading⁄ability⁄of⁄the⁄population,⁄

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77⁄ by⁄ promoting⁄ biofilm⁄ formation⁄ ⁄ or⁄ by⁄ protecting⁄ B.⁄thuringiensis⁄ against⁄ host⁄

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78⁄ innate⁄ immunity⁄ or⁄ the⁄ host⁄ microbiota.⁄ The⁄ two⁄ latter⁄ proposed⁄ functions⁄ of⁄ 79⁄ kurstakin⁄ rely⁄ on⁄ its⁄ putative⁄ ability⁄ to⁄ destabilize⁄ biological⁄ membranes⁄ of⁄ 80⁄ both⁄ prokaryotes⁄ and⁄ eukaryotes,⁄ as⁄ ⁄ is⁄ the⁄ case⁄ for⁄ all⁄ other⁄ known⁄ 81⁄ lipopeptides⁄ (reviewed⁄ in⁄ [5]

).⁄ It⁄ was⁄ notably⁄ shown⁄ that⁄ two⁄ lipopeptides⁄

82⁄ induced⁄ the⁄ lysis⁄ of⁄ amoeba,⁄ protozoans⁄ that⁄ strongly⁄ resemble⁄ phagocytes,⁄ the⁄ 83⁄ main⁄actors⁄of⁄eukaryote⁄⁄innate⁄immunity⁄[17] 84⁄

.

The potential involvement of kurstakin in the life cycle of B. thuringiensis and its potential

85⁄ industrial interest prompted us to investigate its regulation and roles. Here, we demonstrated that the

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86⁄ krs locus is an operon that is unevenly distributed within the B. cereus group. We showed that, in 87⁄ addition to being activated by NprR, the transcription of krs is affected by Spo0A and a sequence in 88⁄ the upstream intergenic region of krs encompassing a DNA palindrome. Finally, we bring evidence

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89⁄ that kurstakin is involved in proper biofilm structuration.⁄ ⁄

ACCEPTED MANUSCRIPT 90⁄ 2.⁄Materials⁄and⁄methods 2.⁄Materials⁄and⁄methods⁄ 91⁄ 2.1.Bacterial⁄strains⁄and⁄growth⁄conditions⁄ 92⁄

B.⁄thuringiensis⁄str.⁄407⁄Cry-⁄(referred⁄to⁄as⁄B.⁄thuringiensis⁄407⁄in⁄the⁄

93⁄ text)⁄ is⁄ an⁄ acrystalliferous⁄ strain⁄ cured⁄ of⁄ its⁄ cry⁄ plasmid⁄ [18]

⁄and⁄TG1⁄of⁄ Escherichia⁄coli⁄were⁄used⁄as⁄hosts⁄in⁄the⁄process⁄of⁄plasmid⁄

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94⁄ DH5

.⁄ Strains⁄

95⁄ construction.⁄ Plasmid⁄ DNA⁄ was⁄ then⁄ prepared⁄ from⁄ the⁄ dam⁄ dcm⁄ Escherichia⁄ coli⁄

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96⁄ strain⁄ ET12567⁄ (Stratagene,⁄ La⁄ Jolla,⁄ CA,⁄ USA)⁄ and⁄ transformed⁄ by⁄ 97⁄ electroporation⁄ of⁄ B.⁄thuringiensis⁄ 407⁄ as⁄ described⁄ previously⁄ [18] .⁄

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98⁄ B.⁄thuringiensis⁄ 407⁄ derivative⁄ strains⁄ used⁄ in⁄ this⁄ study⁄ are⁄ listed⁄ in⁄ Table⁄ 99⁄ S1.⁄ E.⁄ coli⁄ strains⁄ were⁄ grown⁄ in⁄ shaken⁄ LB⁄ at⁄ 37°C⁄ [19] .⁄ B.⁄thuringiensis⁄ 100⁄ 407⁄was⁄grown⁄in⁄LB,⁄BHI⁄(adapted⁄from⁄[20,21]

),⁄LBP⁄or⁄HCT⁄at⁄30°C.⁄LBP⁄and⁄

101⁄ HCT⁄ are⁄ known⁄ to⁄ promote⁄ B.⁄thuringiensis⁄ differentiation⁄ [22,23]

.⁄ ⁠hen⁄

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102⁄ required,⁄ the⁄ medium⁄ was⁄ supplemented⁄ with⁄ antibiotics⁄ at⁄ the⁄ following⁄ 103⁄ concentrations:⁄ 100⁄µg/mL⁄ ampicillin⁄ (for⁄ E.⁄coli),⁄ 200⁄µg/mL⁄ kanamycin,⁄

105⁄ ⁄

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104⁄ 200⁄µg/mL⁄spectinomycin⁄and⁄10⁄µg/mL⁄erythromycin⁄(for⁄B.⁄thuringiensis⁄407).⁄

107⁄ ⁄

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106⁄ 2.2.DNA⁄manipulations⁄

Plasmid⁄ DNA⁄ was⁄ extracted⁄ from⁄ E.⁄ coli⁄ by⁄ the⁄ standard⁄ alkaline⁄ lysis⁄

108⁄ method⁄ and⁄ followed⁄ by⁄ standard⁄ purification⁄ on⁄ a⁄ silica⁄ column,⁄ using⁄ the⁄ 109⁄ PureYield⁄ Plasmid⁄ Miniprep⁄ System⁄ (Promega,⁄ Madison,⁄ ⁠I,⁄ USA).⁄ Restriction⁄ 110⁄ enzymes⁄and⁄T4⁄DNA⁄ligase⁄(New⁄England⁄Biolabs,⁄USA)⁄were⁄used⁄according⁄to⁄the⁄ 111⁄ manufacturer's⁄instructions.⁄⁠ligonucleotide⁄primers⁄(Table⁄S3)⁄were⁄synthesized⁄ 112⁄ by⁄Sigma-Proligo⁄(Paris,⁄France).⁄PCRs⁄were⁄performed⁄in⁄thin-walled⁄tubes⁄in⁄a⁄

ACCEPTED MANUSCRIPT 113⁄ 2720⁄Thermak⁄cycler⁄(Applied⁄Biosystem,⁄USA).⁄Amplified⁄fragments⁄were⁄purified⁄ 114⁄ on⁄ silica⁄ columns⁄ using⁄ the⁄ ⁠izard⁄ SV⁄ Gel⁄ and⁄ PCR⁄ Clean-Up⁄ System⁄ (Promega,⁄ 115⁄ Madison,⁄ ⁠I,⁄ USA).⁄ Digested⁄ fragments⁄ were⁄ separated⁄ on⁄ a⁄ 0.8⁠⁄ TAE-agarose⁄ gel⁄ 116⁄ containing⁄ethidium⁄bromide⁄and⁄purified⁄with⁄the⁄same⁄kit.⁄Sanger⁄sequencing⁄of⁄

118⁄ ⁄ 119⁄ 2.3.Directed⁄plasmid⁄mutagenesis⁄ 120⁄ ⁄

To⁄ construct⁄ the⁄ pHT304-18 Pkrs(FL)

-lacZ⁄ plasmid⁄ (Table⁄ S2),⁄ the⁄

-lacZ⁄ plasmid⁄ was⁄ used⁄ as⁄ the⁄ template⁄ of⁄ a⁄ PCR⁄ with⁄ the⁄

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121⁄ pHT304-18

Pkrs(0A*)

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117⁄ plasmid⁄and⁄PCR⁄DNA⁄was⁄done⁄by⁄Beckman⁄Coulter⁄Genomics⁄(Takeley,⁄UK).⁄

122⁄ high-fidelity⁄ Phusion⁄ polymerase⁄ (NEB)⁄and⁄ ⁄ ⁄ SG74⁄ and⁄ SG75⁄ primers⁄ (Table⁄ S3).⁄ 123⁄ In⁄this⁄plasmid,⁄the⁄putative⁄Spo0A⁄box⁄in⁄the⁄ krs⁄promoter⁄region⁄was⁄mutated⁄ 124⁄ from⁄TTTGACGAA⁄to⁄TTTcAgGAA⁄to⁄prevent⁄Spo0A⁄binding⁄[24]

.⁄The⁄whole⁄plasmid,⁄

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125⁄ integrating⁄ the⁄ two⁄ point⁄ mutations,⁄ was⁄ synthesized⁄ during⁄ the⁄ reaction.⁄ The⁄ 126⁄ template⁄ plasmid⁄ was⁄ subsequently⁄ degraded⁄ by⁄ addition⁄ of⁄ DpnI,⁄ a⁄ restriction⁄

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127⁄ enzyme⁄ that⁄ recognizes⁄ methylated⁄ GATC⁄ motifs.⁄ The⁄ PCR⁄ product⁄ was⁄ transformed⁄ 128⁄ in⁄ E.⁄coli⁄ TG1⁄ and⁄ the⁄ recombinant⁄ clones⁄ were⁄ selected⁄ for⁄ resistance⁄ to⁄

130⁄ ⁄

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129⁄ ampicillin.⁄The⁄presence⁄of⁄the⁄mutations⁄was⁄verified⁄by⁄Sanger⁄sequencing.⁄

131⁄ 2.4.Construction⁄of⁄the⁄B.⁄thuringiensis⁄407⁄recombinant⁄strain⁄ 132⁄ ⁄

The⁄ krsC⁄ gene,⁄ encoding⁄ KrsC,⁄ the⁄ largest⁄ of⁄ the⁄ three⁄ kurstakin⁄

133⁄ synthetases,⁄was⁄disrupted⁄by⁄insertion⁄of⁄the⁄promoter-less⁄and⁄terminator-less⁄ 134⁄ lacZ⁄ gene,⁄ by⁄ homologous⁄ recombination⁄ (as⁄ described⁄ previously⁄ [25] 135⁄ the⁄ thermosensitive⁄ plasmid⁄ pRN5101

),⁄ using⁄

krsC::lacZ⁄ ⁄ (Table⁄ S2).⁄ This⁄ strain⁄ was⁄

ACCEPTED MANUSCRIPT 136⁄ verified⁄by⁄PCR⁄and⁄by⁄Sanger⁄sequencing⁄of⁄the⁄regions⁄used⁄for⁄recombination.⁄ 137⁄ This⁄ strain⁄ was⁄ used⁄ both⁄ to⁄ monitor⁄ krs⁄ transcription⁄ from⁄ the⁄ chromosomal⁄ 138⁄ promoter⁄and⁄as⁄a⁄

krsC⁄deletion⁄mutant.⁄This⁄mutation⁄most⁄likely⁄has⁄a⁄polar⁄

139⁄ effect⁄ on⁄ the⁄ two⁄ downstream⁄ genes⁄ sfp⁄ and⁄ krsD.⁄ These⁄ genes⁄ are⁄ predicted⁄ to⁄

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140⁄ encode⁄a⁄phosphopantheteine⁄transferase⁄and⁄a⁄type⁄II⁄thioesterase⁄respectively,⁄ 141⁄ two⁄ enzymes⁄ known⁄ to⁄ be⁄ involved⁄ in⁄ non-ribosomal⁄ peptide⁄ synthesis.⁄ It⁄ is⁄

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142⁄ therefore⁄not⁄surprising⁄to⁄find⁄them⁄within⁄the⁄ krs⁄locus⁄and⁄it⁄is⁄reasonable⁄ 143⁄ to⁄ think⁄ that⁄ the⁄ products⁄ of⁄ these⁄ genes⁄ are⁄ involved⁄ in⁄ (albeit⁄ not⁄

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144⁄ necessarily⁄exclusively⁄in)⁄kurstakin⁄synthesis.⁄ 145⁄ ⁄

146⁄ 2.5.RT-PCR⁄analysis⁄of⁄the⁄krs⁄locus⁄ 147⁄ ⁄

B.⁄thuringiensis⁄ cells⁄ were⁄ grown⁄ in⁄ shaking⁄ LB⁄ at⁄ 30°C⁄ until⁄ 5⁄ h⁄ after⁄

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148⁄ the⁄ onset⁄ of⁄ transition⁄ phase⁄ (t5)⁄ and⁄ a⁄ 2⁄mL⁄ sample⁄ was⁄ mixed⁄ with⁄ 2⁄mL⁄ of⁄ 149⁄ RNAlater⁄(Life⁄Technologies⁄Ambion,⁄USA)⁄before⁄storage⁄at⁄-20°C.⁄After⁄gentle⁄

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150⁄ thawing,⁄ the⁄ sample⁄ was⁄ centrifuged⁄ and⁄ the⁄ pellet⁄ was⁄ resuspended⁄ in⁄ 1⁄mL⁄ of⁄ 151⁄ Trizol⁄(Life⁄Technologies⁄Ambion,⁄USA).⁄Resuspended⁄cells⁄were⁄disrupted⁄by⁄bead⁄

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152⁄ beating⁄ (Mini⁄ 0.1⁄mm⁄ zirconia/silica⁄ beads,⁄ Biospec⁄ Products)⁄ for⁄ 2⁄ x⁄ 45⁄s⁄ at⁄ 153⁄ 6.5⁄M/s⁄in⁄a⁄Fastprep⁄24⁄(MP⁄Biomedicals).⁄The⁄supernatant⁄was⁄transferred⁄to⁄a⁄ 154⁄ clean⁄ tube⁄ and⁄ 100⁄µL⁄ of⁄ 1-bromo-3-chloropropane⁄ (Sigma)⁄ were⁄ added.⁄ The⁄ 155⁄ suspension⁄ was⁄ vortexed,⁄ incubated⁄ for⁄ 10⁄min⁄ at⁄ room⁄ temperature⁄ and⁄ 156⁄ centrifuged⁄ for⁄ 15⁄min⁄ at⁄ 12000⁄x⁄g⁄ at⁄ 4°C.⁄ The⁄ aqueous⁄ phase⁄ was⁄ then⁄ 157⁄ transferred⁄to⁄a⁄clean⁄tube⁄to⁄which⁄0.1⁄volume⁄of⁄sodium⁄acetate⁄and⁄0.7⁄volume⁄ 158⁄ of⁄ isopropanol⁄ were⁄ added.⁄ The⁄ suspension⁄ was⁄ mixed⁄ and⁄ incubated⁄ at⁄ 4°C⁄ for⁄

ACCEPTED MANUSCRIPT 159⁄ 30⁄min⁄ to⁄ precipitate⁄ nucleic⁄ acids.⁄ Nucleic⁄ acids⁄ were⁄ then⁄ pelleted⁄ by⁄ 160⁄ centrifugation⁄ (20⁄min⁄ at⁄ 12000⁄x⁄g⁄ at⁄ 4°C),⁄ washed⁄ twice⁄ with⁄ 75⁠⁄ ethanol,⁄ 161⁄ air-dried⁄ and⁄ resuspended⁄ in⁄ water.⁄ Possible⁄ traces⁄ of⁄ contaminating⁄ DNA⁄ were⁄ 162⁄ removed⁄ by⁄ DNase⁄ treatment⁄ (TURB⁠⁄ DNase,⁄ Life⁄ Technologies⁄ Ambion,⁄ USA)⁄

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163⁄ according⁄ to⁄ the⁄ manufacturer's⁄ instructions.⁄ RNA⁄ quantity⁄ and⁄ integrity⁄ was⁄ 164⁄ assessed⁄ via⁄ A260/A280⁄ and⁄ A260/A230⁄ (NanoDrop⁄ 2000,⁄ Thermo⁄ Scientific)⁄ and⁄ RNA⁄ .⁄

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165⁄ Integrity⁄ Number⁄ (Nano⁄ chip,⁄ BioAnalyzer⁄ 2100,⁄ Agilent⁄ Technologies)⁄ [26]

166⁄ Total⁄ RNA⁄ was⁄ reverse-transcribed⁄ into⁄ cDNA⁄ with⁄ the⁄ AffinityScript⁄ qPCR⁄ cDNA⁄

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167⁄ synthesis⁄kit⁄(Agilent),⁄using⁄the⁄provided⁄random⁄primers;⁄2⁄µL⁄of⁄the⁄reaction⁄ 168⁄ were⁄ used⁄ as⁄ a⁄ template⁄ in⁄ a⁄ classic⁄ 20⁄µL⁄ 30⁄ cycle⁄ ⁄ PCR⁄ with⁄ Taq⁄ polymerase⁄ 169⁄ (NEB).⁄ Primers⁄ pairs⁄ were⁄ designed⁄ to⁄ amplify⁄ over⁄ intergenic⁄ regions,⁄ except⁄ 170⁄ for⁄ the⁄ internal⁄ krsB⁄ positive⁄ control.⁄ RT-PCR⁄ primers⁄ are⁄ listed⁄ in⁄ Table⁄ S3.⁄

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171⁄ Primers⁄ pairs⁄ are⁄ numbered⁄ according⁄ to⁄ Fig.⁄ 2A⁄ (i.e.⁄ RT1_F⁠⁄ and⁄ RT1_REV⁄ 172⁄ correspond⁄to⁄reaction⁄1⁄in⁄the⁄Fig.).⁄

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173⁄ ⁄ 174⁄ 2.6.Primer⁄extension⁄

Total⁄ RNA⁄ for⁄ the⁄ primer⁄ extension⁄ assay⁄ was⁄ prepared⁄ from⁄ a⁄ shaken⁄ HCT⁄

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175⁄ ⁄

176⁄ culture⁄ at⁄ 37°C⁄ at⁄ t2⁄ as⁄ described⁄ previously⁄ [27]

.⁄ To⁄ detect⁄ the⁄

177⁄ transcription⁄start⁄site⁄of⁄the⁄ krs⁄locus,⁄primer⁄extension⁄was⁄performed⁄using⁄ 178⁄ the⁄oligonucleotide⁄tss_bc2450⁄reverse⁄complementary⁄to⁄the⁄5

⁄end⁄of⁄the⁄ krsE⁄

179⁄ gene⁄ (Table⁄ S2).⁄ The⁄ same⁄ oligonucleotide⁄ was⁄ used⁄ to⁄ prime⁄ dideoxy⁄ sequencing⁄ 180⁄ reactions⁄from⁄the⁄B.⁄thuringiensis⁄407⁄chromosome.⁄ 181⁄ ⁄

ACCEPTED MANUSCRIPT 182⁄ 2.7.

-Galactosidase⁄assay⁄

183⁄ ⁄

Cells⁄ were⁄ cultured⁄ in⁄ shaking⁄ LB⁄ at⁄ 30°C⁄ and⁄

184⁄ was⁄ assayed⁄ as⁄ described⁄ previously⁄ [28] 185⁄ as⁄

-galactosidase⁄ activity⁄

.⁄ Specific⁄ activities⁄ were⁄ expressed⁄

-galactosidase⁄ units⁄ per⁄ milligram⁄ of⁄ total⁄ protein.⁄ Means⁄ and⁄ standard⁄

187⁄ appropriate,⁄Student

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186⁄ deviations⁄ of⁄ at⁄ least⁄ three⁄ independent⁄ experiments⁄ are⁄ presented.⁄ ⁠hen⁄ s⁄t-Test⁄(unpaired⁄samples,⁄two-sided,⁄unequal⁄variances,⁄

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188⁄ confidence⁄ interval⁄ =⁄ 0.95)⁄ was⁄ used⁄ to⁄ assess⁄ the⁄ significance⁄ of⁄ observed⁄ 189⁄ differences.⁄ Resulting⁄ p⁄ values⁄ were⁄ corrected⁄ using⁄ Benjamini,⁄ Hochberg,⁄ and⁄ s⁄method.⁄

191⁄ ⁄ 192⁄ 2.8.Air-liquid⁄biofilm⁄formation⁄ 193⁄ ⁄

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190⁄ Yekutieli

Biofilms⁄ were⁄ allowed⁄ to⁄ form⁄ in⁄ HCT⁄ in⁄ 6⁄mL⁄ glass⁄ tubes⁄ and⁄ 48-well⁄

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194⁄ polyvinylchloride⁄ (PVC)⁄ plates⁄ (Costar⁄ 3548,⁄ Cambridge,⁄ USA)⁄ as⁄ described⁄ 195⁄ previously⁄ [29];⁄ 24-h⁄ microtiter⁄ plate⁄ incubations⁄ at⁄ 30°C⁄ allowed⁄ for⁄ best⁄

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196⁄ biofilm⁄ structure⁄ ⁄ observation,⁄ while⁄ 48-h⁄ glass⁄ tube⁄ incubations⁄ at⁄ 30°C⁄ 197⁄ allowed⁄ for⁄ formation⁄ of⁄ robust⁄ and⁄ more⁄ easily⁄ manipulated⁄ pellicles.⁄ To⁄

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198⁄ quantify⁄ biofilm⁄ formation,⁄ the⁄ planktonic⁄ phase⁄ was⁄ discarded⁄ with⁄ a⁄ Pasteur⁄ 199⁄ pipette⁄and⁄the⁄total⁄biofilm⁄(ring⁄and⁄pellicle)⁄was⁄resuspended⁄in⁄1⁄mL⁄of⁄PBS⁄ 200⁄ to⁄measure⁄the⁄⁠D600.⁄To⁄test⁄the⁄resistance⁄of⁄pellicles⁄to⁄pressure,⁄we⁄melted⁄ 201⁄ the⁄tip⁄of⁄a⁄Pasteur⁄pipette⁄until⁄it⁄formed⁄a⁄ball⁄of⁄5⁄millimeter⁄of⁄diameter⁄ 202⁄ and⁄used⁄it⁄to⁄push⁄on⁄the⁄pellicles⁄(see⁄Movie⁄S1).⁄ 203⁄ ⁄ 204⁄ 2.9.Biofilm⁄formation⁄on⁄solid⁄medium⁄⁄

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Biofilms⁄ on⁄ solid⁄ medium⁄ were⁄ grown⁄ as⁄ follows⁄ (adapted⁄ from⁄ [30]

).⁄

206⁄ Strains⁄ were⁄ streaked⁄ on⁄ LB⁄ agar⁄ plates⁄ and⁄ incubated⁄ at⁄ 30°C⁄ overnight.⁄ The⁄ 207⁄ next⁄ day,⁄ a⁄ few⁄ colonies⁄ were⁄ scraped⁄ and⁄ resuspended⁄ in⁄ PBS⁄ to⁄ an⁄ ⁠D600⁄ of⁄ 1,⁄ 208⁄ and⁄2⁄µL⁄of⁄this⁄suspension⁄was⁄spotted⁄at⁄the⁄center⁄of⁄the⁄plate.⁄The⁄droplet⁄

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209⁄ was⁄allowed⁄to⁄penetrate⁄the⁄agar⁄(~5⁄min)⁄before⁄incubation⁄for⁄72⁄h⁄at⁄30°C.⁄ 210⁄ After⁄ incubation,⁄ the⁄ colony⁄ was⁄ scraped⁄ and⁄ resuspended⁄ thoroughly⁄ in⁄ 1⁄mL⁄ of⁄

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211⁄ 4⁠⁄ formaldehyde⁄ in⁄ PBS.⁄ Cells⁄ were⁄ incubated⁄ ~5⁄min⁄ at⁄ room⁄ temperature⁄ and⁄ 212⁄ centrifuged⁄for⁄30⁄s⁄⁄at⁄13.000⁄x⁄g.⁄The⁄pellet⁄was⁄resuspended⁄in⁄1⁄mL⁄of⁄PBS⁄

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213⁄ and⁄ centrifuged⁄ 5⁄min⁄ at⁄ 600⁄x⁄g⁄ to⁄ wash⁄ out⁄ formaldehyde.⁄ The⁄ pellet⁄ was⁄ 214⁄ finally⁄resuspended⁄in⁄100⁄µL⁄of⁄GTE⁄buffer⁄[30] 215⁄ ⁄ 216⁄ 2.10.Flow⁄cytometry⁄analyses⁄

Flow⁄ cytometry⁄ analyses⁄ were⁄ adapted⁄ from⁄ [31]

.⁄ Fluorescence⁄ was⁄

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217⁄ ⁄

.⁄

218⁄ measured⁄ on⁄ a⁄ CyFlow⁄ Space⁄ cytometer⁄ (Partec,⁄ France).⁄ GFP⁄ fluorescence⁄ driven⁄

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219⁄ by⁄ the⁄ krs⁄ promoter⁄ was⁄ recovered⁄ after⁄ excitation⁄ by⁄ a⁄ solid⁄ blue-laser⁄ 220⁄ emitting⁄at⁄488⁄nm⁄combined⁄with⁄a⁄500-nm⁄long⁄pass⁄dichroic⁄mirror⁄and⁄a⁄527-nm⁄

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221⁄ band⁄ pass⁄ filter⁄ (512 542)⁄ (FL1⁄ Channel).⁄ Measures⁄ were⁄ acquired⁄ using⁄ 222⁄ logarithmic⁄ gains⁄ and⁄ detector⁄ settings,⁄ adjusted⁄ on⁄ a⁄ sample⁄ of⁄ reporter-less⁄ 223⁄ cells,⁄ to⁄ define⁄ cellular⁄ autofluorescence.⁄ Gating⁄ on⁄ FSC SSC⁄ was⁄ used⁄ to⁄ 224⁄ discriminate⁄bacteria⁄from⁄background⁄particles.⁄For⁄each⁄sample,⁄100.000⁄gated⁄ 225⁄ events⁄ were⁄ acquired.⁄ Data⁄ were⁄ collected⁄ with⁄ the⁄ FloMax⁄ software⁄ (Partec,⁄ 226⁄ France)⁄and⁄figures⁄were⁄generated⁄with⁄⁄⁄⁠easel⁄2.0⁄software⁄(⁠EHI,⁄USA).⁄ 227⁄ ⁄

ACCEPTED MANUSCRIPT 228⁄ 2.11.Confocal⁄laser⁄scanning⁄spectral⁄microscopy⁄ 229⁄ ⁄

⁠vernight⁄ HCT⁄ precultures⁄ (30°C)⁄ were⁄ used⁄ to⁄ inoculate⁄ fresh⁄ HCT⁄

230⁄ incubated⁄at⁄30°C⁄to⁄an⁄⁠D600⁄of⁄0.05.⁄⁠hen⁄the⁄cultures⁄reached⁄an⁄⁠D600⁄of⁄~1,⁄ 231⁄ they⁄were⁄diluted⁄to⁄an⁄⁠D600⁄of⁄0.01⁄in⁄10⁄mL⁄fresh⁄HCT⁄in⁄30⁄mL⁄vials;⁄0.4⁄µm⁄

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232⁄ polycarbonate⁄ isopore⁄ filters⁄ (Millipore)⁄ were⁄ punched⁄ out⁄ to⁄ an⁄ appropriate⁄ 233⁄ diameter,⁄ UV-sterilized⁄ and⁄ set⁄ to⁄ float⁄ in⁄ the⁄ 30⁄mL⁄ vials.⁄ Samples⁄ were⁄ then⁄

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234⁄ incubated⁄for⁄48⁄h⁄at⁄30°C.⁄The⁄filters⁄were⁄gently⁄transferred⁄onto⁄35⁄mm⁄low⁄ 235⁄ µ-dishes⁄ (Ibidi)⁄ containing⁄ 800⁄ µl⁄ of⁄ HCT⁄ medium⁄ supplemented⁄ with⁄ fluorescent⁄

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236⁄ markers.⁄ The⁄ nucleic⁄ acid⁄ dye⁄ Syto9⁄ (Life⁄ Technologies)⁄ was⁄ used⁄ at⁄ a⁄ final⁄ 237⁄ concentration⁄of⁄6.67⁄nM⁄and⁄the⁄lectin⁄stain⁄Concanavalin⁄A⁄(ConA)⁄Alexa⁄Fluor⁄ 238⁄ 633-conjugated⁄ (glycoconjugates⁄ detection,⁄ Life⁄ Technologies)⁄ was⁄ used⁄ at⁄ a⁄ 239⁄ final⁄ concentration⁄ of⁄ 200⁄ µg/mL.⁄ Samples⁄ were⁄ incubated⁄ in⁄ the⁄ dark⁄ at⁄ room⁄

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240⁄ temperature⁄for⁄at⁄least⁄10⁄min.⁄The⁄dishes⁄were⁄then⁄mounted⁄on⁄the⁄motorized⁄ 241⁄ stage⁄ of⁄ an⁄ inverted⁄ confocal⁄ microscope⁄ (Leica⁄ TCS⁄ SP8⁄ A⁠BS,⁄ Leica⁄

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242⁄ Microsystems)⁄at⁄the⁄MIMA2⁄platform⁄(http://www.jouy.inra.fr/mima2/).⁄The⁄dishes⁄ 243⁄ were⁄scanned⁄using⁄a⁄40×/0.8⁄N.A.⁄water⁄immersion⁄objective⁄lens⁄because⁄of⁄its⁄

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244⁄ interesting⁄working⁄distance⁄(3,300⁄µm)⁄allowing⁄thick⁄z-stacks⁄at⁄a⁄convenient⁄ 245⁄ magnification⁄ level.⁄ Syto9⁄ was⁄ excited⁄ at⁄ 488⁄ nm⁄ using⁄ an⁄ argon⁄ laser⁄ (output⁄ 246⁄ power⁄at⁄30⁠,⁄A⁠TF⁄at⁄0.5⁠)⁄and⁄the⁄emitted⁄

green

⁄fluorescence⁄was⁄recorded⁄

247⁄ from⁄498⁄to⁄600⁄nm⁄with⁄a⁄PMT⁄detector⁄with⁄a⁄750⁄V⁄gain.⁄Simultaneously,⁄ConA⁄ 248⁄ was⁄excited⁄at⁄633⁄nm⁄using⁄an⁄He-Ne⁄laser⁄(A⁠TF⁄at⁄5⁠)⁄and⁄the⁄emitted⁄

red

⁄

249⁄ fluorescence⁄ was⁄ recorded⁄ from⁄ 643⁄ to⁄ 750⁄ nm⁄ with⁄ a⁄ hybrid⁄ detector⁄ with⁄ 20⁠⁄ 250⁄ digital⁄gain.⁄2D⁄sections⁄were⁄acquired⁄at⁄600⁄Hz⁄with⁄numerical⁄zoom⁄1,⁄512⁄x⁄

ACCEPTED MANUSCRIPT 251⁄ 512⁄xy⁄image⁄definition⁄and⁄a⁄z-step⁄of⁄1⁄ 252⁄ biofilms⁄were⁄then⁄reconstructed⁄with⁄the⁄

m.⁄Three-dimensional⁄projections⁄of⁄ easy⁄3D⁄blend

⁄module⁄of⁄the⁄IMARIS⁄

253⁄ 7.7.2⁄software⁄(Bitplane).⁄

255⁄ 2.12.⁠hole⁄bacterial⁄cell⁄MALDI-T⁠F⁄mass⁄spectrometry⁄ 256⁄ ⁄

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254⁄ ⁄

Bacteria⁄ were⁄ plated⁄ on⁄ LB⁄ agar⁄ and⁄ incubated⁄ at⁄ 30⁄ °C⁄ for⁄ 72⁄ h.⁄

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257⁄ Individual⁄bacterial⁄colonies⁄were⁄carefully⁄removed⁄from⁄the⁄agar⁄surface⁄with⁄ 258⁄ a⁄ tungsten⁄ wire⁄ loop⁄ and⁄ immediately⁄ suspended⁄ in⁄ a⁄ tube⁄ containing⁄ a⁄ matrix⁄

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259⁄ solution⁄(10⁄mg/mL⁄cyano-4-hydroxycinnamic⁄acid⁄in⁄70⁠⁄water,⁄30⁠⁄acetonitrile,⁄ 260⁄ and⁄ 0.1⁠⁄ TFA).⁄ Samples⁄ were⁄ vortexed⁄ and⁄ centrifuged⁄ at⁄ 5,000⁄ rpm.⁄ For⁄ routine⁄ 261⁄ analyses,⁄1⁄

L⁄of⁄sample⁄solution⁄was⁄spotted⁄onto⁄a⁄MALDI-T⁠F⁄MTP⁄384⁄target⁄

262⁄ plate⁄(Bruker⁄Daltonik⁄GmbH,⁄Leipzig,⁄Germany)⁄according⁄to⁄the⁄procedure⁄of⁄the⁄

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263⁄ dried-droplet⁄ preparation.⁄ Mass⁄ profile⁄ ⁄ experiments⁄ were⁄ analyzed⁄ with⁄ an⁄ 264⁄ Ultraflex⁄ MALDI-ToF/ToF⁄ mass⁄ spectrometer⁄ (Bruker,⁄ Bremen,⁄ Germany)⁄ equipped⁄

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265⁄ with⁄ a⁄ smartbeam⁄ laser.⁄ Samples⁄ were⁄ analyzed⁄ using⁄ an⁄ accelerating⁄ voltage⁄ of⁄ 266⁄ 25⁄kV⁄and⁄matrix⁄suppression⁄in⁄deflexion⁄mode⁄at⁄ m/z⁄750.⁄The⁄laser⁄power⁄was⁄

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267⁄ set⁄ to⁄ just⁄ above⁄ the⁄ threshold⁄ of⁄ ionization⁄ (around⁄ 35⁠).⁄ Spectra⁄ were⁄ 268⁄ acquired⁄ in⁄ reflector-positive⁄ mode⁄ in⁄ the⁄ range⁄ of⁄ 800⁄ to⁄ 3000⁄ Da.⁄ Each⁄ 269⁄ spectrum⁄ was⁄ the⁄ result⁄ of⁄ 2,000⁄ laser⁄ shots⁄ per⁄ m/z⁄ segment⁄ per⁄ sample⁄ 270⁄ delivered⁄in⁄10⁄sets⁄of⁄50⁄shots⁄distributed⁄in⁄three⁄different⁄locations⁄on⁄the⁄ 271⁄ surface⁄of⁄the⁄matrix⁄spot.⁄The⁄instrument⁄was⁄externally⁄calibrated⁄in⁄positive⁄ 272⁄ reflector⁄ mode⁄ using⁄ Bradykinin⁄ (1-7)⁄ [M+H]+⁄ 757.3991,⁄ Angiotensin⁄ II⁄ [M+H]+⁄

ACCEPTED MANUSCRIPT

273⁄ 1046.5418,⁄Angiotensin⁄I⁄[M+H]+⁄1296.6848,⁄Substance⁄P⁄[M+H]+⁄1347.7354,⁄Bombesin⁄ 274⁄ [M+H]+⁄1619.8223,⁄ACTH⁄(1-17)⁄[M+H]+⁄2093,0862.⁄ 275⁄ ⁄ 276⁄ 2.13.Infection⁄of⁄Galleria⁄mellonella⁄larvae⁄ Larvae⁄infections⁄were⁄carried⁄out⁄as⁄described⁄in⁄[13]

278⁄ inoculum⁄was⁄set⁄to⁄2.105⁄CFU.⁄

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279⁄ ⁄ 280⁄ 2.14.Bioinformatics⁄analyses⁄

Non-ribosomal⁄peptide⁄synthetases⁄were⁄detected⁄in⁄the⁄reference⁄genome⁄of⁄

282⁄ B.⁄thuringiensis⁄ 407⁄ [32]

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281⁄ ⁄

⁄ (chromosome:⁄ INSDC⁄ CP003889.1,⁄ plasmids:⁄ INSDC⁄

283⁄ CP003890.1⁄to⁄CP003898.1)⁄with⁄antiSMASH⁄3⁄[33,34] 284⁄ ⁄

,⁄except⁄that⁄the⁄

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277⁄

.⁄

The⁄presence⁄of⁄all⁄krs⁄genes⁄was⁄investigated⁄in⁄all⁄completely⁄sequenced⁄

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285⁄ species⁄ with⁄ BLASTp⁄ (v.⁄ 2.2.30+)⁄ on⁄ the⁄ unfiltered⁄ nr⁄ database⁄ of⁄ NCBI⁄ (as⁄ of⁄ 286⁄ 2015-06-01)⁄[35],⁄using⁄B.⁄thuringiensis⁄407⁄sequences⁄as⁄queries.⁄ Distribution⁄ of⁄ kurstakin⁄ synthetases⁄ among⁄ the⁄ B.⁄cereus⁄ group⁄ was⁄

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287⁄ ⁄

288⁄ assessed⁄ with⁄ an⁄ in-house⁄ Python⁄ script⁄ (BLASTats,⁄ https://github.com/seb-

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289⁄ ksl/BLASTats/).⁄ Briefly,⁄ this⁄ script⁄ submits⁄ a⁄ protein⁄ sequence⁄ to⁄ NCBI's⁄ 290⁄ tBLASTn⁄ using⁄ the⁄ nr/nt⁄ database.⁄ Hits⁄ whose⁄ best⁄ high⁄ scoring⁄ segment⁄ pair⁄ 291⁄ (HSP)⁄was⁄over⁄80⁠⁄sequence⁄identity⁄and⁄over⁄85⁠⁄query⁄coverage⁄were⁄considered⁄ 292⁄ significant.⁄ These⁄ thresholds⁄ allowed⁄ detection⁄ of⁄ krs⁄ genes⁄ with⁄ no⁄ false293⁄ positive⁄ on⁄ other⁄ NRPS⁄ loci.⁄ The⁄ name⁄ of⁄ the⁄ strain(s)⁄ generating⁄ each⁄ 294⁄ significant⁄hit⁄was⁄then⁄parsed⁄from⁄hit⁄titles.⁄Strains⁄were⁄grouped⁄by⁄species⁄ 295⁄ and⁄ compared⁄ to⁄ the⁄ number⁄ of⁄ completely⁄ sequenced⁄ genomes⁄ available⁄ for⁄ each⁄

ACCEPTED MANUSCRIPT 296⁄ species,⁄ giving⁄ the⁄ relative⁄ presence⁄ of⁄ query⁄ protein⁄ among⁄ selected⁄ species.⁄ 297⁄ Significant⁄ hits⁄ for⁄ each⁄ gene⁄ of⁄ the⁄ krs⁄ locus⁄ were⁄ further⁄ aligned⁄ with⁄ 298⁄ Clustal

⁄ (v.⁄ 1.2.1)⁄ [36]⁄ and⁄ alignments⁄ were⁄ concatenated.⁄ The⁄ resulting⁄

299⁄ alignment⁄ was⁄ curated⁄ with⁄ Gblocks⁄ (v.⁄ 0.91b)⁄ [37,38]⁄ ⁄ and⁄ an⁄ approximately-

301⁄ [39,40]

.⁄

Trees⁄

were⁄

then⁄

edited⁄

303⁄ ⁄

FigTree⁄

SC

302⁄ (http://tree.bio.ed.ac.uk/software/figtree/).⁄

with⁄

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300⁄ maximum-likelihood⁄ phylogenetic⁄ tree⁄ was⁄ built⁄ with⁄ FastTree⁄ (v.⁄ 2.1.7-1)⁄ 1.4-2)⁄

Upstream⁄ intergenic⁄ sequences⁄ of⁄ all⁄ found⁄ krs⁄ loci⁄ were⁄ retrieved⁄ from⁄

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304⁄ Genbank⁄ using⁄ an⁄ in-house⁄ Python⁄ script⁄ and⁄ aligned⁄ with⁄ Clustal

⁄ (v.⁄ 1.2.1)⁄

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.⁄The⁄alignment⁄was⁄then⁄edited⁄with⁄Jalview⁄(v.⁄2.9.0b2)⁄[41]⁄ .⁄ ⁄

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305⁄ [36]

(v.⁄

ACCEPTED MANUSCRIPT 306⁄ 3.Results 3.Results⁄ 307⁄ 3.1.The⁄krs⁄locus⁄is⁄not⁄evenly⁄distributed⁄within⁄the⁄B.⁄cereus⁄group⁄ 308⁄ ⁄

Database⁄searches⁄of⁄the⁄ krs⁄gene⁄products⁄with⁄all⁄the⁄available⁄GenBank⁄

309⁄ sequences⁄ did⁄ not⁄ yield⁄ any⁄ significant⁄ hit⁄ outside⁄ the⁄ B.⁄cereus⁄ group,⁄

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310⁄ suggesting⁄that⁄ krs⁄is⁄specific⁄to⁄this⁄group.⁄However,⁄not⁄all⁄strains⁄contain⁄ 311⁄ an⁄intact⁄krs⁄locus.⁄The⁄B.⁄thuringiensis⁄species⁄exhibits⁄the⁄highest⁄frequency⁄

SC

312⁄ of⁄ strains⁄ containing⁄ an⁄ intact⁄ krs⁄ locus⁄ (Krs+⁄ strains),⁄ with⁄ 52⁠⁄ (12/23)⁄ 313⁄ positive⁄ strains,⁄ vs.⁄ only⁄ 12⁠⁄ (3/26)⁄ for⁄ B.⁄cereus⁄ and⁄ 0⁠⁄ (0/31)⁄ for⁄

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314⁄ B.⁄anthracis⁄(Table⁄1,⁄Table⁄S4).⁄The⁄absence⁄of⁄krs⁄in⁄all⁄B.⁄anthracis⁄strains⁄ 315⁄ is⁄explained⁄by⁄the⁄high⁄monomorphism⁄of⁄this⁄species⁄[42]

.⁄It⁄should⁄also⁄be⁄

316⁄ noted⁄ that⁄ the⁄ 23⁄ B.⁄thuringiensis⁄ genomes⁄ encompassed⁄ the⁄ Bacillus⁄ 317⁄ bombysepticus⁄ str.⁄ ⁠ang⁄ genome⁄ [43]

.⁄ Indeed,⁄ although⁄ B.⁄bombysepticus⁄ was⁄

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318⁄ originally⁄described⁄as⁄a⁄separate⁄species,⁄its⁄16S⁄RNA⁄and⁄genomic⁄phylogenies,⁄ 319⁄ its⁄ ability⁄ to⁄ form⁄ parasporal⁄ inclusions,⁄ as⁄ well⁄ as⁄ its⁄ entomopathogenicity⁄

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320⁄ towards⁄ Bombyx⁄ mori⁄ strongly⁄ indicates⁄ that⁄ it⁄ is⁄ most⁄ likely⁄ to⁄ be⁄ a⁄ 321⁄ misclassified⁄strain⁄of⁄the⁄B.⁄thuringiensis⁄species.⁄ Among⁄ the⁄ 23⁄ completely⁄ sequenced⁄ B.⁄thuringiensis⁄ genomes,⁄ three⁄ are⁄

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322⁄ ⁄

323⁄ redundant⁄(genome⁄alignments⁄point⁄to⁄them⁄as⁄the⁄same⁄strain):⁄Bt407,⁄CT-43⁄and⁄ 324⁄ IS5056.⁄It⁄is⁄therefore⁄not⁄surprising⁄to⁄find⁄that⁄all⁄three⁄are⁄Krs+,⁄and⁄the⁄ 325⁄ frequency⁄ within⁄ B.⁄ thuringiensis⁄ should⁄ be⁄ corrected⁄ to⁄ 10⁄ out⁄ of⁄ 21.⁄ Two⁄ 326⁄ sequences⁄of⁄ B.⁄thuringiensis⁄ serovar⁄kurstaki⁄str.⁄YBT-1520⁄are⁄available,⁄but⁄ 327⁄ surprisingly,⁄ one⁄ of⁄ the⁄ two⁄ sequences⁄ (INSDC⁄ CP004858.1)⁄ seems⁄ to⁄ have⁄ a⁄ 328⁄ truncated⁄ non-functional⁄ krsC,⁄ while⁄ the⁄ other⁄ ⁄ ⁄ (INSDC⁄ CP007607.1)⁄ has⁄ a⁄ Krs+⁄

ACCEPTED MANUSCRIPT 329⁄ locus.⁄It⁄should⁄also⁄be⁄noted⁄that⁄ B.⁄thuringiensis⁄ serovar⁄galleriae⁄str.⁄HD330⁄ 29⁄ harbors⁄ all⁄ krs⁄ genes,⁄ but⁄ its⁄ krsB⁄ gene⁄ seems⁄ disrupted,⁄ making⁄ the⁄ locus⁄ 331⁄ Krs-.⁄This⁄event⁄is⁄likely⁄recent,⁄because⁄sequences⁄of⁄the⁄other⁄ krs⁄genes⁄have⁄ 332⁄ not⁄ yet⁄ diverged⁄ from⁄ those⁄ of⁄ B.⁄thuringiensis⁄ 407⁄ (>90⁠⁄ overall⁄ identity).⁄

334⁄ cannot⁄exclude⁄the⁄possibility⁄of⁄sequencing⁄errors.⁄

A⁄ tree⁄ was⁄ built⁄ from⁄ all⁄ Krs+⁄ loci⁄ and⁄ reveals⁄ the⁄ high⁄ conservation⁄ of⁄

SC

335⁄ ⁄

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333⁄ Although⁄ these⁄ last⁄ two⁄ strains⁄ (YBT-1520⁄ and⁄ HD-29)⁄ were⁄ counted⁄ as⁄ Krs-,⁄ we⁄

336⁄ this⁄locus⁄(Fig.⁄1).⁄A⁄phylogenetic⁄tree⁄of⁄the⁄ B.⁄cereus⁄group⁄was⁄proposed⁄by⁄ ⁄ and⁄ further⁄ refined⁄ by⁄ Tourasse⁄ et⁄ al.,⁄ based⁄ on⁄

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337⁄ Guinebretière⁄ et⁄ al.⁄ [44]

338⁄ available⁄ MLST/AFLP/MLEE⁄ data⁄ [45]

.⁄ These⁄ trees⁄ contain⁄ seven⁄ clusters.⁄ The⁄

339⁄ topology⁄ of⁄ the⁄ krs⁄ tree⁄ is⁄ consistent⁄ with⁄ the⁄ existing⁄ trees⁄ and⁄ shows⁄ that⁄ 340⁄ most⁄ Krs+⁄ strains⁄ belong⁄ to⁄ cluster⁄ II⁄ of⁄ the⁄ B.⁄cereus⁄ group.⁄

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341⁄ B.⁄weihenstephanensis⁄ KBAB4⁄ and⁄ B.⁄mycoides⁄ strain⁄ ATCC⁄ 6462⁄ are⁄ the⁄ only⁄ Krs+⁄ 342⁄ strains⁄ that⁄ fall⁄ into⁄ cluster⁄ III,⁄ and⁄ they⁄ are⁄ also⁄ the⁄ only⁄ two⁄ strains⁄ in⁄

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343⁄ which⁄the⁄krs⁄locus⁄is⁄located⁄on⁄a⁄plasmid.⁄In⁄B.⁄weihenstephanensis⁄KBAB4,⁄the⁄ 344⁄ krs⁄ locus⁄ lies⁄ on⁄ the⁄ 400⁄kb⁄ plasmid⁄ pB⁠B401,⁄ flanked⁄ by⁄ two⁄ putative⁄ IS240⁄

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345⁄ insertion⁄ elements.⁄ In⁄ B.⁄mycoides⁄ ATCC⁄ 6462,⁄ the⁄ krs⁄ locus⁄ is⁄ found⁄ on⁄ the⁄ 346⁄ 360⁄kb⁄ plasmid⁄ pBMX_1,⁄ flanked⁄ by⁄ putative⁄ phage⁄ genes.⁄ To⁄ date,⁄ these⁄ are⁄ the⁄ 347⁄ only⁄sequenced⁄plasmids⁄containing⁄krs.⁄ 348⁄ ⁄ 349⁄ 3.2.The⁄krs⁄locus⁄forms⁄a⁄six-gene⁄operon⁄ 350⁄ ⁄

The⁄ krs⁄locus⁄of⁄ B.⁄thuringiensis⁄407⁄consists⁄of⁄six⁄genes⁄spanning⁄over⁄

351⁄ 30⁄kb⁄on⁄the⁄chromosome⁄(Fig.⁄2A).⁄Very⁄short⁄(2⁄and⁄13⁄nt),⁄or⁄the⁄absence⁄of,⁄

ACCEPTED MANUSCRIPT 352⁄ intergenic⁄ spaces⁄ suggested⁄ that⁄ the⁄ first⁄ four⁄ genes⁄ were⁄ co-transcribed.⁄ To⁄ 353⁄ verify⁄ this⁄ hypothesis⁄ and⁄ examine⁄ whether⁄ the⁄ last⁄ two⁄ genes⁄ belonged⁄ to⁄ the⁄ 354⁄ same⁄transcriptional⁄unit,⁄we⁄investigated⁄⁄⁄transcripts⁄of⁄the⁄krs⁄locus.⁄Total⁄ 355⁄ RNA⁄ extracted⁄ from⁄ B.⁄thuringiensis⁄ 407⁄ cells⁄ was⁄ used⁄ to⁄ perform⁄ RT-PCR⁄ with⁄

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356⁄ primer⁄pairs⁄amplifying⁄all⁄intergenic⁄sequences⁄(Fig.⁄2A⁄and⁄2B,⁄Table⁄S3).⁄⁠e⁄ 357⁄ amplified⁄fragments⁄of⁄the⁄expected⁄size⁄with⁄each⁄primer⁄pair,⁄indicating⁄that⁄

SC

358⁄ consecutive⁄ genes⁄ were⁄ co-transcribed.⁄ This⁄ result,⁄ combined⁄ with⁄ recent⁄ 359⁄ unpublished⁄ RNA-seq⁄ data⁄ from⁄ our⁄ team⁄ and⁄ in⁄ silico⁄ analyses⁄ showing⁄ no⁄

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360⁄ detection⁄ of⁄ internal⁄ terminators,⁄ strongly⁄ suggests⁄ that⁄ krs⁄ is⁄ an⁄ operon.⁄ 361⁄ However,⁄ based⁄ on⁄ the⁄ data⁄ presented⁄ here,⁄ we⁄ cannot⁄ rule⁄ out⁄ the⁄ presence⁄ of⁄ 362⁄ alternative⁄promoters⁄and/or⁄internal⁄attenuators.⁄ 363⁄ ⁄

365⁄ ⁄

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364⁄ 3.3.Analysis⁄of⁄the⁄krs⁄operon⁄promoter⁄region⁄ ⁠e⁄then⁄performed⁄primer⁄extension⁄experiments⁄using⁄primer⁄tss_bc2450⁄and⁄

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366⁄ RNA⁄ extracted⁄ from⁄ B.⁄thuringiensis⁄ 407⁄ cells⁄ to⁄ identify⁄ transcription⁄ start⁄ 367⁄ sites⁄(TSSs)⁄upstream⁄from⁄ krsE.⁄Fig.⁄3A⁄shows⁄a⁄single⁄TSS⁄327⁄bp⁄upstream⁄of⁄

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368⁄ the⁄ krsE⁄ translation⁄ start⁄ site.⁄ Putative⁄ -10⁄ and⁄ -35⁄ boxes⁄ resembling⁄ the⁄ 369⁄ TTGACA⁄ and⁄ TATAAT⁄ consensus⁄ of⁄ B.⁄ subtilis⁄ [46]⁄ ⁄ ⁄ ⁄ suggest⁄ that⁄ transcription⁄ 370⁄ from⁄this⁄promoter⁄depends⁄on⁄ 371⁄ ⁄

A

.⁄

In⁄ silico⁄ analysis⁄ of⁄ the⁄ intergenic⁄ region⁄ upstream⁄ of⁄ krsE⁄ revealed⁄

372⁄ three⁄ notable⁄ features:⁄ two⁄ head-to-tail⁄ oriented⁄ potential⁄ SinR⁄ binding⁄ 373⁄ sequences,⁄ a⁄ potential⁄ Spo0A⁄ binding⁄ site⁄ and⁄ an⁄ exact⁄ DNA⁄ palindrome⁄ composed⁄ 374⁄ of⁄ two⁄ 15⁄ nt⁄ inverted⁄ repeats⁄ spaced⁄ by⁄ 6⁄ nt⁄ (Fig.⁄ 3B).⁄ Conservation⁄ of⁄ these⁄

ACCEPTED MANUSCRIPT 375⁄ features⁄was⁄assessed⁄by⁄aligning⁄upstream⁄intergenic⁄sequences⁄of⁄all⁄found⁄krs⁄ 376⁄ operons⁄ in⁄ the⁄ nr/nt⁄ database⁄ (Fig.⁄ S2).⁄ ⁠verall,⁄ this⁄ region⁄ seems⁄ very⁄ well⁄ 377⁄ conserved⁄ in⁄ 15⁄ of⁄ the⁄ 17⁄ aligned⁄ sequences,⁄ with⁄ the⁄ 2⁄ plasmidic⁄ sequences⁄ 378⁄ clearly⁄forming⁄an⁄out-group.⁄⁠ithin⁄the⁄15⁄chromosomal⁄sequences,⁄the⁄putative⁄

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379⁄ -10⁄ and⁄ -35⁄ boxes,⁄the⁄ TSS,⁄ the⁄ downstream⁄ potential⁄ SinR⁄ binding⁄ sequence⁄ and⁄ 380⁄ the⁄putative⁄Spo0A⁄binding⁄site⁄are⁄fully⁄conserved.⁄Two⁄alleles⁄of⁄the⁄upstream⁄

SC

381⁄ potential⁄ SinR⁄ binding⁄ sequence⁄ are⁄ found,⁄ both⁄ compatible⁄ with⁄ the⁄ published⁄ 382⁄ SinR⁄ box⁄ [47].⁄ The⁄ palindrome⁄ is⁄ the⁄ least⁄ conserved⁄ feature,⁄ but⁄ is⁄ still⁄

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383⁄ present⁄in⁄11⁄out⁄of⁄the⁄15⁄chromosomal⁄sequences.⁄⁠ithin⁄these⁄11⁄sequences,⁄it⁄ 384⁄ is⁄ interesting⁄ to⁄ note⁄ that⁄ two⁄ alleles⁄ are⁄ found,⁄ both⁄ forming⁄ an⁄ exact⁄ 385⁄ palindrome.⁄Indeed,⁄in⁄the⁄sequences⁄of⁄ B.⁄thuringiensis⁄strains⁄HD-1,⁄HD73⁄and⁄ 386⁄ YBT-1520,⁄G5⁄and⁄its⁄cognate⁄C32⁄transitioned⁄to⁄A5⁄and⁄T32,⁄repectively.⁄ To⁄ assess⁄ the⁄ functionality⁄ of⁄ these⁄ features,⁄ we⁄ measured⁄

-

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387⁄ ⁄

388⁄ galactosidase⁄ activity⁄ generated⁄ by⁄ transcriptional⁄ fusions⁄ between⁄ various⁄

EP

389⁄ fragments⁄ amplified⁄ from⁄ this⁄ intergenic⁄ region⁄ and⁄ the⁄ reporter⁄ gene⁄ lacZ⁄ on⁄ 390⁄ the⁄pHT304-18Z⁄plasmid⁄3⁄h⁄after⁄the⁄end⁄of⁄exponential⁄phase⁄(t3)⁄in⁄HCT⁄(Fig.⁄

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391⁄ 3C).⁄In⁄this⁄medium,⁄maximal⁄expression⁄of⁄krs⁄is⁄reached⁄at⁄t3⁄(Fig.⁄4A),⁄hence⁄ 392⁄ its⁄use⁄as⁄a⁄reference⁄point.⁄⁠ur⁄reference⁄full-length⁄fusion⁄(FL)⁄spanned⁄from⁄ 393⁄ -739⁄to⁄+84⁄relative⁄to⁄the⁄TSS,⁄covering⁄the⁄entire⁄upstream⁄intergenic⁄region⁄ 394⁄ and⁄the⁄beginning⁄of⁄krsE.⁄All⁄consecutive⁄fusions⁄share⁄the⁄same⁄3'⁄end⁄at⁄+84.⁄ 395⁄ Transcription⁄ of⁄ the⁄ FL⁄ fusion⁄ followed⁄ the⁄ same⁄ pattern⁄ as⁄ the⁄ chromosomal⁄ 396⁄ fusion,⁄ albeit⁄ at⁄ higher⁄ levels⁄ (Fig.⁄ S3).⁄ The⁄ map1⁄ fragment,⁄ in⁄ which⁄ both⁄ 397⁄ putative⁄ SinR⁄ binding⁄ sequences⁄ were⁄ removed,⁄ presented⁄ approximately⁄ the⁄ same⁄

ACCEPTED MANUSCRIPT 398⁄ activity⁄ as⁄ the⁄ full-length⁄ fragment⁄ (0.83⁄ +/-⁄ 0.06),⁄ suggesting⁄ that⁄ these⁄ 399⁄ sequences⁄ are⁄ not⁄ functional⁄ in⁄ the⁄ conditions⁄ used⁄ in⁄ this⁄ experiment⁄ (Fig.⁄ 400⁄ 3D).⁄ These⁄ results⁄ are⁄ supported⁄ by⁄ data⁄ showing⁄ that,⁄ in⁄ our⁄ conditions,⁄ 401⁄ Pkrs(FL)⁄ activity⁄ is⁄ unchanged⁄ in⁄ a⁄ B.⁄thuringiensis⁄ 407⁄

sinIR⁄ mutant⁄ strain⁄

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402⁄ compared⁄ to⁄ the⁄ ⁠T⁄ strain⁄ (Fig.⁄ S4).⁄ Removing⁄ an⁄ additional⁄ 290⁄nt⁄ region⁄ 403⁄ encompassing⁄ the⁄ palindrome⁄ (fusion⁄ map2)⁄ resulted⁄ in⁄ a⁄ twofold⁄ increase⁄ in⁄⁄⁄ -galactosidase⁄ activity⁄ (1.98⁄ +/-⁄ 0.08),⁄ indicating⁄ that⁄ this⁄ sequence⁄

SC

404⁄

405⁄ negatively⁄ affects⁄ ⁄ ⁄ transcription⁄ of⁄ krs.⁄ Removing⁄ 44⁄ and⁄ 130⁄ additional⁄

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406⁄ nucleotides⁄ (fusions⁄ map3⁄ and⁄ map4,⁄ respectively)⁄ induced⁄ a⁄ threefold⁄ drop⁄ 407⁄ compared⁄ to⁄ ⁄ ⁄ map2⁄ fusion,⁄ suggesting⁄ the⁄ presence⁄ of⁄ an⁄ activating⁄ region⁄ 408⁄ immediately⁄downstream⁄of⁄the⁄palindrome.⁄Removing⁄32⁄more⁄nucleotides⁄from⁄the⁄ 409⁄ 5'⁄ end,⁄ leaving⁄ only⁄ 45⁄ bp⁄ upstream⁄ of⁄ the⁄ TSS⁄ (fusion⁄ map5),⁄ generated⁄ an⁄

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410⁄ almost⁄ inactive⁄ fusion⁄ (0.03⁄ +/-⁄ 0.01).⁄ Altogether,⁄ these⁄ results⁄ point⁄ to⁄ the⁄ 411⁄ palindrome⁄ and⁄ its⁄ upstream⁄ region⁄ as⁄ playing⁄ a⁄ negative⁄ role⁄ in⁄ transcription⁄

413⁄ ⁄

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412⁄ from⁄this⁄promoter.⁄

415⁄ ⁄

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414⁄ 3.4.krs⁄Expression⁄is⁄modulated⁄by⁄Spo0A⁄ ⁄The⁄presence⁄of⁄a⁄predicted⁄Spo0A⁄box⁄between⁄the⁄TSS⁄and⁄the⁄translation⁄

416⁄ start⁄ codon⁄ suggested⁄ that⁄ Spo0A~P⁄ could⁄ act⁄ as⁄ a⁄ direct⁄ repressor⁄ of⁄ krs⁄ 417⁄ transcription⁄ [48]

.⁄ To⁄ assess⁄ the⁄ involvement⁄ of⁄ Spo0A⁄ in⁄ krs⁄ transcription,⁄

418⁄ we⁄first⁄monitored⁄transcription⁄of⁄the⁄FL⁄fusion⁄in⁄a⁄

spo0A⁄strain⁄between⁄t2⁄

419⁄ and⁄ t5,⁄ a⁄ time⁄ frame⁄ within⁄ which⁄ an⁄ effect⁄ of⁄ Spo0A⁄ on⁄ krs⁄ transcription⁄ was⁄ 420⁄ most⁄ likely⁄ to⁄ occur⁄ (Fig.⁄ 3E).⁄ Transcription⁄ of⁄ the⁄ fusion⁄ was⁄ upregulated⁄in⁄

ACCEPTED MANUSCRIPT 421⁄ this⁄ strain,⁄ by⁄ factors⁄ increasing⁄ from⁄ 1.3⁄ to⁄ 2.3⁄ over⁄ time,⁄ indicating⁄ that⁄ 422⁄ Spo0A⁄ negatively⁄ affects⁄ krs⁄ transcription.⁄ To⁄ discriminate⁄ between⁄ direct⁄ and⁄ 423⁄ indirect⁄repression,⁄we⁄compared⁄ lacZ⁄expression⁄generated⁄by⁄the⁄FL⁄fusion⁄and⁄ 424⁄ a⁄fusion⁄covering⁄the⁄same⁄region,⁄but⁄where⁄the⁄putative⁄Spo0A⁄box⁄was⁄mutated⁄

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425⁄ from⁄ TTTGACGAA⁄ to⁄ TTTcAgGAA⁄ to⁄ prevent⁄ Spo0A⁄ binding⁄ (fusion⁄ 0A*)⁄ [24]

.⁄

426⁄ Transcription⁄ from⁄ this⁄ fusion⁄ was⁄ not⁄ significantly⁄ higher⁄ than⁄ that⁄ from⁄ the⁄

SC

427⁄ FL⁄fusion⁄(Fig.⁄3D).⁄Although⁄we⁄cannot⁄rule⁄out⁄a⁄possible⁄role⁄for⁄the⁄Spo0A⁄ 428⁄ box,⁄ our⁄ results⁄ suggest⁄ that⁄ the⁄ effect⁄ of⁄ Spo0A⁄ on⁄ krs⁄ transcription⁄ is⁄

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429⁄ indirect.⁄ 430⁄ ⁄

431⁄ 3.5.krs⁄expression⁄responds⁄to⁄growth⁄conditions⁄ 432⁄ ⁄

The⁄involvement⁄of⁄Spo0A⁄in⁄⁄⁄expression⁄of⁄the⁄ krs⁄operon⁄prompted⁄us⁄to⁄

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433⁄ assess⁄ whether⁄ ⁄ ⁄ transcription⁄ of⁄ this⁄ locus⁄ was⁄ affected⁄ by⁄ various⁄ 434⁄ environmental⁄ conditions.⁄ ⁠e⁄ constructed⁄ a⁄ strain⁄ harboring⁄ the⁄ chromosomal⁄

EP

435⁄ transcriptional⁄fusion⁄krsC::lacZ⁄to⁄measure⁄its⁄activity⁄in⁄different⁄media⁄and⁄ 436⁄ conditions.⁄ ⁠e⁄ first⁄ measured⁄ krs⁄ transcription⁄ in⁄ shaken⁄ liquid⁄ LB,⁄ BHI,⁄ LB-

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437⁄ peptone⁄(LBP)⁄or⁄HCT.⁄All⁄four⁄media⁄are⁄rich⁄and⁄complex;⁄however,⁄LBP⁄and⁄HCT,⁄ 438⁄ unlike⁄ LB⁄ and⁄ BHI,⁄ are⁄ known⁄ to⁄ promote⁄ efficient⁄ B.⁄thuringiensis⁄ 439⁄ differentiation⁄like⁄sporulation⁄and⁄biofilm⁄formation⁄[22,23]

.⁄Interestingly,⁄

440⁄ we⁄could⁄show⁄that⁄ krs⁄transcription⁄exhibited⁄different⁄patterns⁄in⁄LB⁄and⁄BHI⁄ 441⁄ versus⁄LBP⁄and⁄HCT⁄(Fig.⁄4A).⁄Transcription⁄started⁄1-2⁄h⁄earlier⁄in⁄HCT⁄and⁄LBP⁄ 442⁄ than⁄ in⁄ LB⁄ and⁄ BHI,⁄ respectively.⁄ In⁄ HCT⁄ and⁄ LBP,⁄ maximal⁄ 443⁄ activity⁄ was⁄ measured⁄ at⁄ t3,⁄ while⁄ in⁄ LB⁄ and⁄ BHI⁄

-galactosidase⁄

-galactosidase⁄ activity⁄

ACCEPTED MANUSCRIPT 444⁄ increased⁄ at⁄ least⁄ up⁄ to⁄ t5.⁄ The⁄ delayed⁄ expression⁄ of⁄ the⁄ krs⁄ operon⁄ in⁄ LB⁄ 445⁄ medium⁄ compared⁄ to⁄ HCT⁄ is⁄ in⁄ agreement⁄ with⁄ its⁄ activation⁄ by⁄ NprR,⁄ as⁄ 446⁄ previously⁄described⁄for⁄nprA,⁄another⁄NprR-regulated⁄gene⁄[28] 447⁄ ⁄

.⁄

To⁄ gain⁄ deeper⁄ insight⁄ into⁄ krs⁄ transcription,⁄ we⁄ used⁄ gfp⁄ as⁄ a⁄ reporter⁄

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448⁄ to⁄ assess⁄ krs⁄ transcription⁄ at⁄ the⁄ single⁄ cell⁄ level.⁄ ⁠e⁄used⁄ ⁄ transcriptional⁄ 449⁄ fusion⁄between⁄the⁄full-length⁄krs⁄promoter⁄region⁄and⁄the⁄sgfp⁄reporter⁄gene⁄on⁄

SC

450⁄ the⁄pHT304⁄plasmid⁄to⁄overcome⁄low⁄fluorescence⁄issues.⁄⁠e⁄measured⁄fluorescence⁄ 451⁄ levels⁄of⁄a⁄B.⁄thuringiensis⁄strain⁄containing⁄our⁄plasmid⁄in⁄shaken⁄LB⁄and⁄in⁄a⁄

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452⁄ colony⁄grown⁄on⁄LB⁄agar⁄(Fig.⁄4B).⁄In⁄shaken⁄medium,⁄the⁄fluorescence⁄signal⁄was⁄ 453⁄ maximal⁄ at⁄ t20⁄ (data⁄ not⁄ shown)⁄ and⁄ was⁄ evenly⁄ distributed⁄ around⁄ a⁄ single⁄ 454⁄ median,⁄ reflecting⁄ ⁄ unimodal⁄ distribution⁄ of⁄ krs⁄ transcription⁄ (Fig.⁄ 4B,⁄ top⁄ 455⁄ panel).⁄In⁄a⁄colony⁄grown⁄on⁄LB⁄agar,⁄the⁄fluorescence⁄signal⁄was⁄maximal⁄72⁄h⁄

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456⁄ post-inoculation⁄ (data⁄ not⁄ shown)⁄ and⁄ was⁄ distributed⁄ around⁄ two⁄ medians,⁄ 457⁄ reflecting⁄ a⁄ bi-modal⁄ transcription⁄ pattern⁄ (Fig.⁄ 4B,⁄ bottom⁄ panel).⁄ The⁄

EP

458⁄ leftmost⁄peak⁄overlapped⁄with⁄background⁄auto-fluorescence,⁄suggesting⁄that⁄the⁄ 459⁄ colony⁄contained⁄two⁄distinct⁄populations,⁄of⁄which⁄only⁄one⁄transcribed⁄ krs⁄at⁄

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460⁄ relatively⁄ homogeneous⁄ levels.⁄ This⁄ heterogeneity⁄ could⁄ result⁄ from⁄⁄⁄ 461⁄ differential⁄expression⁄of⁄krs⁄in⁄cells⁄at⁄different⁄developmental⁄stages⁄within⁄ 462⁄ the⁄biofilm.⁄ 463⁄ ⁄ 464⁄ 3.6.The⁄krs⁄operon⁄is⁄not⁄essential⁄for⁄necrotrophism⁄ 465⁄ ⁄

It⁄ was⁄ initially⁄ published⁄ that⁄ kurstakin⁄ was⁄ essential⁄ for⁄ the⁄ survival⁄

466⁄ of⁄ B.⁄thuringiensis⁄ in⁄ the⁄ insect⁄ cadaver⁄ [13]

,⁄ however,⁄ we⁄ recently⁄ showed⁄

ACCEPTED MANUSCRIPT 467⁄ that⁄ it⁄ was⁄ incorrect⁄ and⁄ resulted⁄ from⁄ a⁄ mistake⁄ (erratum⁄ pending).⁄ The⁄ krs⁄ 468⁄ locus⁄ was⁄ intended⁄ to⁄ be⁄ deleted⁄ from⁄ the⁄ B.⁄thuringiensis⁄ 407⁄ strain,⁄ but⁄ 469⁄ instead⁄was⁄mistakenly⁄deleted⁄from⁄ B.⁄cereus⁄ ATCC⁄14579,⁄a⁄strain⁄in⁄which⁄the⁄ 470⁄ nprR⁄gene⁄is⁄interrupted⁄by⁄a⁄transposon.⁄As⁄NprR⁄was⁄shown⁄to⁄be⁄required⁄for⁄

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471⁄ necrotrophism,⁄ the⁄ loss⁄ of⁄ necrotrophism⁄ of⁄ the⁄ B.⁄cereus⁄ ATCC⁄ 14579⁄ krsABC⁄ 472⁄ strain⁄ compared⁄ to⁄ the⁄ B.⁄thuringiensis⁄ 407⁄ ⁠T⁄ strain⁄ most⁄ likely⁄ results⁄ from⁄

SC

473⁄ the⁄absence⁄of⁄NprR.⁄Indeed,⁄new⁄results⁄indicate⁄that⁄a⁄ B.⁄thuringiensis⁄ krsC⁄ 474⁄ mutant,⁄ unable⁄ to⁄ produce⁄ kurstakin⁄ (Fig.⁄ 5),⁄ was⁄ actually⁄ not⁄ affected⁄ in⁄ its⁄

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475⁄ necrotrophic⁄ ability⁄ (Fig.⁄ S5).⁄ However,⁄ repeated⁄ experiments⁄ (data⁄ not⁄ shown)⁄ 476⁄ confirm⁄ that⁄ constitutive⁄ expression⁄ of⁄ the⁄ krs⁄ operon⁄ in⁄ a⁄ nprR-nprX⁄ mutant⁄ 477⁄ partially⁄ restores⁄ ⁄ ⁄ survival⁄ of⁄ bacteria⁄ in⁄ the⁄ insect⁄ cadaver,⁄ as⁄ ⁄ was⁄ 478⁄ initially⁄ shown⁄ [13]

.⁄ Together,⁄ these⁄ results⁄ suggest⁄ that⁄ kurstakin⁄ could⁄

480⁄ ⁄

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479⁄ play⁄a⁄non-essential⁄role⁄in⁄necrotrophism.⁄⁄

482⁄ ⁄

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481⁄ 3.7.The⁄krs⁄operon⁄is⁄involved⁄in⁄biofilm⁄formation⁄ The⁄ involvement⁄ of⁄ the⁄ krs⁄ operon⁄ in⁄ biofilm⁄ formation⁄ was⁄ already⁄

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483⁄ investigated⁄by⁄Dubois⁄and⁄coll.,⁄but⁄suffered⁄the⁄same⁄strain⁄mistake⁄described⁄ 484⁄ in⁄the⁄previous⁄paragraph⁄[13].⁄However,⁄the⁄potential⁄biophysical⁄properties⁄of⁄ 485⁄ kurstakin⁄ and⁄ its⁄ probable⁄ secretion⁄ via⁄ KrsE⁄ suggested⁄ that⁄ this⁄ lipopeptide⁄ 486⁄ could⁄ still⁄ play⁄ a⁄ role⁄ in⁄ biofilm⁄ formation⁄ [5,14,15] 487⁄ formation⁄of⁄the⁄ B.⁄thuringiensis⁄407⁄

.⁄ ⁠e⁄ compared⁄ biofilm⁄

krsC⁄strain⁄to⁄that⁄of⁄the⁄⁠T⁄strain⁄in⁄

488⁄ various⁄ conditions.⁄ Absorbance⁄ measures⁄ showed⁄ that⁄ biofilms⁄ formed⁄ by⁄ the⁄ 489⁄

krsC⁄mutant⁄strain⁄in⁄glass⁄tubes⁄were⁄40⁠⁄less⁄dense⁄than⁄the⁄⁠T⁄strain⁄after⁄

ACCEPTED MANUSCRIPT 490⁄ 48⁄hs,⁄while⁄biofilms⁄formed⁄in⁄PVC⁄plate⁄wells⁄had⁄the⁄same⁄density⁄after⁄24⁄h⁄ 491⁄ (Fig.⁄6A).⁄However,⁄surface⁄pellicles⁄formed⁄by⁄the⁄

krsC⁄mutant⁄strain⁄in⁄the⁄

492⁄ PVC⁄plate⁄wells⁄exhibited⁄an⁄ultrastructure⁄different⁄from⁄that⁄of⁄the⁄⁠T⁄strain⁄ 493⁄ (Fig.⁄ 6B).⁄ Pellicles⁄ formed⁄ by⁄ the⁄ ⁠T⁄ strain⁄ seemed⁄ organized⁄ into⁄ dense⁄

krsC⁄ mutant⁄ strain⁄ were⁄

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494⁄ networks⁄ of⁄ alveoli,⁄ while⁄ pellicles⁄ formed⁄ by⁄ the⁄

495⁄ made⁄of⁄larger⁄petals,⁄loosely⁄organized.⁄These⁄pellicles⁄were⁄also⁄different⁄in⁄

SC

496⁄ their⁄ resistance⁄ to⁄ pressure.⁄ ⁠e⁄ used⁄a⁄ homemade⁄ sealed⁄ tip⁄ Pasteur⁄ pipette⁄to⁄ 497⁄ apply⁄ pressure⁄ on⁄ top⁄ of⁄ the⁄ pellicles⁄ (Movie⁄ S1).⁄ ⁠hile⁄ we⁄ could⁄ stab⁄ the⁄

499⁄ well,⁄ suggesting⁄ that⁄ the⁄

krsC⁄pellicle⁄sunk⁄to⁄the⁄bottom⁄of⁄the⁄

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498⁄ pellicle⁄formed⁄by⁄the⁄⁠T⁄strain,⁄the⁄

krsC⁄ pellicle⁄ was⁄ more⁄ loosely⁄ attached⁄ to⁄ the⁄

500⁄ surrounding⁄material⁄than⁄its⁄⁠T⁄equivalent.⁄Confocal⁄microscopy⁄observations⁄of⁄ 501⁄ biofilms⁄formed⁄on⁄filters⁄floating⁄at⁄the⁄surface⁄of⁄static⁄cultures⁄shows⁄that⁄

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502⁄ the⁄ krsC⁄mutant⁄biofilm⁄architecture⁄differed⁄from⁄that⁄of⁄the⁄⁠T⁄strain⁄(Fig.⁄ 503⁄ 6C).⁄ ⁠rganization⁄ of⁄ the⁄ cells⁄ in⁄ the⁄ ⁠T⁄ pellicle⁄ appears⁄ homogeneous⁄ and⁄

505⁄

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504⁄ compact,⁄while⁄cells⁄seem⁄to⁄form⁄scattered⁄patches⁄of⁄various⁄densities⁄in⁄the⁄

krsC⁄ pellicle.⁄ Most⁄ of⁄ the⁄ time,⁄

krsC⁄ pellicles⁄ were⁄ also⁄ much⁄ thinner⁄ and⁄

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506⁄ contained⁄ much⁄ fewer⁄ cells⁄ than⁄ ⁠T⁄ ones⁄ (Fig.⁄ S6).⁄ Unfortunately⁄ we⁄ could⁄ not⁄ 507⁄ complement⁄ krsC⁄ deletion⁄ with⁄ our⁄ methodology,⁄ due⁄ to⁄ the⁄ length⁄ of⁄ this⁄ gene⁄ 508⁄ (14.880⁄nt).⁄ ⁄

ACCEPTED MANUSCRIPT 509⁄ 4.Discussion 4.Discussion⁄ Discussion 510⁄ ⁄

⁄Kurstakin⁄ belongs⁄ to⁄ a⁄ new⁄ class⁄ of⁄ lipopeptides⁄ that⁄ has⁄ been⁄ recently⁄

511⁄ discovered.⁄Transcription⁄of⁄the⁄krs⁄locus,⁄responsible⁄for⁄kurstakin⁄synthesis,⁄ 512⁄ is⁄ activated⁄ at⁄ the⁄ onset⁄ of⁄ sporulation⁄ by⁄ NprR~NprX,⁄ a⁄quorum⁄ sensing⁄ system⁄ .⁄ Here⁄ we⁄ report⁄

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513⁄ involved⁄ in⁄ the⁄ necrotrophism⁄ of⁄ B.⁄thuringiensis⁄ [13]

514⁄ additional⁄ data⁄ on⁄ the⁄ transcriptional⁄ regulation⁄ of⁄ this⁄ locus⁄ and⁄ describe⁄ a⁄

516⁄ ⁄

SC

515⁄ function⁄of⁄this⁄lipopeptide⁄in⁄a⁄model⁄B.⁄thuringiensis⁄strain.⁄

The⁄ krs⁄locus⁄contains⁄the⁄genes⁄encoding⁄the⁄three⁄kurstakin⁄synthetases⁄

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517⁄ (krsA,⁄ krsB⁄ and⁄ krsC),⁄ a⁄ phosphopatheteinyl⁄ transferase⁄ (sfp),⁄ a⁄ type⁄ II⁄ 518⁄ thioesterase⁄ (krsD)⁄ and⁄ a⁄ membrane⁄ protein⁄ (krsE)⁄ potentially⁄ involved⁄ in⁄ the⁄ 519⁄ export⁄of⁄kurstakin⁄[14,15]

.⁄Considering⁄the⁄function⁄of⁄other⁄NRPS⁄loci,⁄Sfp⁄

520⁄ and⁄KrsD⁄are⁄thought⁄to⁄be⁄involved⁄in⁄⁄⁄turnover⁄of⁄the⁄kurstakin⁄synthetases⁄

522⁄ [49]

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521⁄ as⁄ phosphopantheteinyl⁄ transferase⁄ and⁄ type⁄ II⁄ thioesterase,⁄ respectively⁄ .⁄ The⁄ krs⁄ locus⁄ was⁄ found⁄ only⁄ in⁄ genomes⁄ of⁄ the⁄ B.⁄cereus⁄ group,⁄

EP

523⁄ revealing⁄that⁄kurstakin⁄could⁄be⁄specific⁄to⁄this⁄group.⁄Interestingly,⁄krs⁄was⁄ 524⁄ found⁄mostly⁄in⁄the⁄B.⁄thuringiensis⁄species.⁄This⁄could⁄imply⁄that⁄kurstakin⁄is⁄

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525⁄ involved⁄in⁄the⁄ecology⁄of⁄this⁄organism,⁄potentially⁄in⁄the⁄infectious⁄cycle⁄of⁄ 526⁄ host⁄larvae.⁄Two⁄complete⁄ krs⁄loci⁄were⁄also⁄found⁄on⁄big⁄plasmids,⁄raising⁄the⁄ 527⁄ possibility⁄for⁄krs⁄to⁄be⁄transferred⁄between⁄strains.⁄ 528⁄ ⁄

⁄The⁄ krs⁄ locus⁄ consists⁄ of⁄ 6⁄ genes⁄ spanning⁄ 30⁄ kb.⁄ ⁠e⁄ showed⁄ that⁄ all⁄ 6⁄

529⁄ genes⁄in⁄the⁄ krs⁄locus⁄form⁄an⁄operon,⁄and⁄we⁄identified⁄a⁄transcription⁄start⁄ 530⁄ site⁄upstream⁄of⁄ krsE,⁄327⁄bp⁄upstream⁄of⁄its⁄translation⁄start⁄codon.⁄Analysis⁄ 531⁄ of⁄ krs⁄expression⁄kinetics⁄in⁄various⁄media⁄indicated⁄that⁄the⁄transcription⁄of⁄

ACCEPTED MANUSCRIPT 532⁄ this⁄ locus⁄ was⁄ influenced⁄ by⁄ environmental⁄ cues.⁄ ⁠e⁄ also⁄ showed⁄ that⁄ krs⁄ 533⁄ transcription⁄was⁄negatively⁄affected⁄by⁄Spo0A,⁄probably⁄indirectly.⁄However,⁄we⁄ 534⁄ were⁄unable⁄to⁄show⁄⁄regulation⁄of⁄ krs⁄expression⁄by⁄SinR,⁄either⁄by⁄monitoring⁄ 535⁄ krs⁄ expression⁄ in⁄ a⁄ B.⁄thuringiensis⁄ 407⁄

-lacZ⁄ fusion⁄ to⁄ a⁄ fusion⁄ lacking⁄ both⁄ putative⁄ SinR⁄

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536⁄ comparing⁄ a⁄ reference⁄ Pkrs

sinIR⁄ strain⁄ (Fig.⁄ S4)⁄ or⁄ by⁄

537⁄ boxes⁄(Fig.⁄3D).⁄These⁄results⁄do⁄not⁄corroborate⁄microarray⁄analyses⁄reporting⁄ .⁄ This⁄ discrepancy⁄

SC

538⁄ a⁄ possible⁄ repression⁄ of⁄ krs⁄ by⁄ SinR⁄ in⁄ LBP⁄ medium⁄ [16]

539⁄ might⁄ result⁄ from⁄ the⁄ different⁄ conditions⁄ and⁄ methodologies⁄ used⁄ in⁄ these⁄ two⁄

541⁄ ⁄

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540⁄ studies.⁄

⁠e⁄ found⁄ krs⁄ to⁄ be⁄ transcribed⁄ earlier⁄ in⁄ media⁄ favorable⁄ to⁄ sporulation⁄

542⁄ than⁄ in⁄ rich⁄ sporulation-delaying⁄ media.⁄ This⁄ is⁄ in⁄ agreement⁄ with⁄ the⁄ 543⁄ regulation⁄of⁄krs⁄transcription⁄by⁄NprR⁄and⁄it⁄is⁄notable⁄that⁄the⁄expression⁄of⁄

545⁄ ⁄

.⁄

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544⁄ nprA,⁄another⁄NprR-regulated⁄gene,⁄was⁄upregulated⁄in⁄HCT⁄compared⁄to⁄LB⁄[28]

⁠e⁄ also⁄ showed⁄ that⁄ a⁄ sequence⁄ encompassing⁄ the⁄ DNA⁄ palindrome⁄ spanning⁄

EP

546⁄ the⁄ -497⁄ to⁄ -207⁄ region,⁄ relative⁄ to⁄ the⁄ TSS,⁄ negatively⁄ affected⁄ krs⁄ 547⁄ expression.⁄ This⁄ palindrome⁄ was⁄ not⁄ found⁄ elsewhere⁄ in⁄ the⁄ genome⁄ of⁄

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548⁄ B.⁄thuringiensis⁄ 407,⁄ even⁄ with⁄ permissive⁄ settings.⁄ This⁄ suggests⁄ that⁄ 549⁄ regulation⁄from⁄this⁄element⁄is⁄specific⁄to⁄ krs,⁄though⁄we⁄cannot⁄rule⁄out⁄the⁄ 550⁄ presence⁄ in⁄ the⁄ genome⁄ of⁄ different⁄ sequences⁄ exhibiting⁄ the⁄ same⁄ function.⁄ 551⁄ ⁠hether⁄ this⁄ region⁄ requires⁄ a⁄ trans⁄ regulatory⁄ element⁄ or⁄ acts⁄ in⁄ cis⁄ remains⁄ 552⁄ to⁄be⁄determined.⁄Recent⁄RNA-seq⁄data⁄from⁄our⁄laboratory⁄(unpublished)⁄confirm⁄ 553⁄ the⁄ position⁄ of⁄ the⁄ TSS⁄ and⁄ show⁄ no⁄ apparent⁄ transcription⁄ of⁄ the⁄ region⁄ 554⁄ including⁄the⁄palindrome,⁄ruling⁄out⁄the⁄hypotheses⁄of⁄a⁄small⁄antisense⁄RNA⁄or⁄

ACCEPTED MANUSCRIPT 555⁄ a⁄riboswitch.⁄Further⁄studies⁄are⁄required⁄to⁄precisely⁄determine⁄the⁄molecular⁄ 556⁄ function⁄ of⁄ the⁄ palindrome,⁄ beginning⁄ with⁄ the⁄ determination⁄ of⁄ a⁄ potential⁄ 557⁄ protein-DNA⁄interaction.⁄ 558⁄ ⁄

The⁄ product⁄ of⁄ the⁄ krs⁄ locus,⁄ kurstakin,⁄ could⁄ be⁄ involved⁄ at⁄ different⁄

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559⁄ stages⁄ of⁄ the⁄ life⁄ cycle⁄ of⁄ B.⁄thuringiensis.⁄ Here⁄ we⁄ showed⁄ that⁄ krs⁄ was⁄ 560⁄ required⁄ for⁄ proper⁄ structuration⁄ and⁄ resistance⁄ of⁄ B.⁄thuringiensis⁄ biofilms.⁄

SC

561⁄ ⁠hen⁄ formed⁄ in⁄ the⁄ absence⁄ of⁄ krs⁄ transcription,⁄ the⁄ biofilm⁄ exhibited⁄ a⁄ 562⁄ different⁄ ultra-⁄ and⁄ microstructure⁄ compared⁄ to⁄ that⁄ of⁄ the⁄ ⁠T⁄ strain,⁄ and⁄ the⁄

M AN U

563⁄ pellicle⁄ behaved⁄ differently⁄ when⁄ subjected⁄ to⁄ pressure.⁄ Two⁄ non-mutually⁄ 564⁄ exclusive⁄hypotheses⁄could⁄explain⁄this⁄phenotype:⁄the⁄

krsC⁄pellicle⁄could⁄be⁄

565⁄ less⁄ adherent⁄ to⁄ the⁄ surrounding⁄ surface⁄ ⁄ and/or⁄ it⁄ could⁄ be⁄ more⁄ robust⁄ than⁄ 566⁄ the⁄ ⁠T⁄ strain.⁄ Both⁄ cases⁄ would⁄ produce⁄ the⁄ same⁄ outcome:⁄ the⁄ forces⁄ attaching⁄

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567⁄ the⁄ pellicle⁄ to⁄ the⁄ surrounding⁄ material⁄ are⁄ lower⁄ than⁄ the⁄ forces⁄ structuring⁄ 568⁄ the⁄ pellicle,⁄ which⁄ is⁄ precisely⁄ what⁄ we⁄ observe⁄ when⁄ applying⁄ it⁄ on⁄ the⁄

EP

569⁄ pellicle.⁄It⁄should⁄be⁄noted⁄that⁄the⁄involvement⁄of⁄the⁄ krs⁄operon⁄in⁄biofilm⁄ 570⁄ formation⁄ was⁄ investigated⁄ only⁄ in⁄ B.⁄thuringiensis⁄ 407.⁄ Some⁄ strains⁄ lacking⁄

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571⁄ the⁄ operon⁄ are⁄ known⁄ to⁄ form⁄ biofilms,⁄ and⁄ it⁄ would⁄ be⁄ interesting⁄ to⁄ compare⁄ 572⁄ their⁄structure⁄to⁄biofilms⁄formed⁄by⁄B.⁄thuringiensis⁄407⁄⁠T⁄and⁄ 573⁄ ⁄

krsC.⁄

Extracting⁄ and⁄ purifying⁄ kurstakin⁄ would⁄ allow⁄ further⁄ experiments.⁄ For⁄

574⁄ instance,⁄rheology⁄studies⁄on⁄biofilms⁄with⁄purified⁄kurstakin⁄could⁄help⁄assess⁄ 575⁄ its⁄ precise⁄ implication⁄ in⁄ the⁄ biofilm⁄ formation⁄ process.⁄ Exogenous⁄ purified⁄ 576⁄ kurstakin⁄could⁄also⁄be⁄used⁄to⁄try⁄to⁄rescue⁄the⁄phenotype⁄of⁄a⁄biofilm⁄formed⁄ 577⁄ by⁄the⁄kurstakin-defective⁄mutant.⁄

ACCEPTED MANUSCRIPT 578⁄ Conflict⁄of⁄interest⁄ Conflict⁄of⁄interest

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The⁄authors⁄declare⁄that⁄they⁄have⁄no⁄conflict⁄of⁄interest.⁄ ⁄

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579⁄

ACCEPTED MANUSCRIPT 580⁄ Acknowledgments⁄ Acknowledgments 581⁄

⁠e⁄ are⁄ indebted⁄ to⁄ Agnès⁄ Réjasse,⁄ Francis⁄ Repoila⁄ and⁄ Françoise⁄ ⁠essner⁄

582⁄ for⁄ their⁄ help⁄ setting⁄ up⁄ the⁄ RNA⁄ purification⁄ protocol.⁄ ⁠e⁄ are⁄ grateful⁄ to⁄ 583⁄ Stéphane⁄Perchat⁄and⁄Rafael⁄Patiño-Navarrete⁄for⁄fruitful⁄discussions.⁄⁠e⁄thank⁄

585⁄ Nationale⁄de⁄la⁄Recherche

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584⁄ the⁄referees⁄for⁄thoughtful⁄advice.⁄This⁄study⁄was⁄funded⁄by⁄the⁄French⁄

⁄(Bt-Surf,⁄ANR-12-EMMA-0005).⁄S.⁄Gélis-Jeanvoine⁄was⁄

.⁄ ⁄

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587⁄ supérieur⁄et⁄de⁄la⁄recherche

Ministère⁄ de⁄ l

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586⁄ supported⁄ by⁄ a⁄ fellowship⁄ from⁄ the⁄ French⁄

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Agence⁄

enseignement⁄

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Springer⁄Berlin⁄Heidelberg;⁄2011,⁄p.⁄57 91.⁄doi:10.1007/978-3-642-14490-5.⁄

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594⁄

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626⁄

thuringiensis.⁄PLoS⁄Pathog⁄2012;8:e1002629.⁄

627⁄

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625⁄

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631⁄

doi:10.1007/s00253-012-4181-2.⁄

SC

630⁄

al.⁄Structure,⁄biosynthesis,⁄and⁄properties⁄of⁄kurstakins,⁄nonribosomal⁄ lipopeptides⁄from⁄Bacillus⁄spp.⁄Appl⁄Microbiol⁄Biotechnol⁄2012;95:593 600.⁄

629⁄

632⁄ [15]⁄ Li⁄X,⁄Yang⁄H,⁄Zhang⁄D,⁄Li⁄X,⁄Yu⁄H,⁄Shen⁄Z.⁄⁠verexpression⁄of⁄specific⁄ proton⁄motive⁄force-dependent⁄transporters⁄facilitate⁄the⁄export⁄of⁄

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633⁄

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638⁄

Biofilms.⁄PLoS⁄⁠ne⁄2014;9:e87532.⁄doi:10.1371/journal.pone.0087532.⁄

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639⁄ [17]⁄ Mazzola⁄M,⁄de⁄Bruijn⁄I,⁄Cohen⁄MF,⁄Raaijmakers⁄JM.⁄Protozoan-induced⁄ 640⁄

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641⁄

defense⁄mechanism⁄for⁄Pseudomonas⁄fluorescens.⁄Appl⁄Environ⁄Microbiol⁄

642⁄

2009;75:6804 11.⁄doi:10.1128/AEM.01272-09.⁄

EP

643⁄ [18]⁄ Lereclus⁄D,⁄Arantes⁄⁠,⁄Chaufaux⁄J,⁄Lecadet⁄M.⁄Transformation⁄and⁄ expression⁄of⁄a⁄cloned⁄delta-endotoxin⁄gene⁄in⁄Bacillus⁄thuringiensis.⁄

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ACCEPTED MANUSCRIPT 654⁄ [22]⁄ Lecadet⁄MM,⁄Blondel⁄M⁠,⁄Ribier⁄J.⁄Generalized⁄transduction⁄in⁄Bacillus⁄ 655⁄

thuringiensis⁄var.⁄berliner⁄1715⁄using⁄bacteriophage⁄CP-54Ber.⁄J⁄Gen⁄

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659⁄

doi:10.1128/AEM.72.1.937-941.2006.⁄

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660⁄ [24]⁄ York⁄K,⁄Kenney⁄TJ,⁄Satola⁄S,⁄Moran⁄CP,⁄Poth⁄H,⁄Youngman⁄P.⁄Spo0A⁄controls⁄ 661⁄

the⁄sigma⁄A-dependent⁄activation⁄of⁄Bacillus⁄subtilis⁄sporulation-specific⁄

662⁄

transcription⁄unit⁄spoIIE.⁄J⁄Bacteriol⁄1992;174:2648 58.⁄

663⁄ [25]⁄ Lereclus⁄D,⁄Vallade⁄M,⁄Chaufaux⁄J,⁄Arantes⁄⁠,⁄Rambaud⁄S.⁄Expansion⁄of⁄ insecticidal⁄host⁄range⁄of⁄Bacillus⁄thuringiensis⁄by⁄in⁄vivo⁄genetic⁄

665⁄

recombination.⁄Bio/Technology⁄1992;10:418 21.⁄

SC

664⁄

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666⁄ [26]⁄ Schroeder⁄A,⁄Mueller⁄⁠,⁄Stocker⁄S,⁄Salowsky⁄R,⁄Leiber⁄M,⁄Gassmann⁄M,⁄et⁄ 667⁄

al.⁄The⁄RIN:⁄an⁄RNA⁄integrity⁄number⁄for⁄assigning⁄integrity⁄values⁄to⁄RNA⁄

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measurements.⁄BMC⁄Mol⁄Biol⁄2006;7:3.⁄doi:10.1186/1471-2199-7-3.⁄

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672⁄

2010;48:1358 65.⁄doi:10.1128/JCM.02123-09.⁄

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671⁄

al.⁄InhA1,⁄NprA,⁄and⁄HlyII⁄as⁄candidates⁄for⁄markers⁄to⁄differentiate⁄ pathogenic⁄from⁄nonpathogenic⁄Bacillus⁄cereus⁄strains.⁄J⁄Clin⁄Microbiol⁄

670⁄

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675⁄

in⁄bacteria⁄of⁄the⁄Bacillus⁄cereus⁄group.⁄Mol⁄Microbiol⁄2011;82:619 33.⁄

676⁄

doi:10.1111/j.1365-2958.2011.07839.x.⁄

EP

674⁄

677⁄ [29]⁄ Hy⁄A,⁄Briandet⁄R,⁄Aymerich⁄S,⁄Gohar⁄M.⁄Involvement⁄of⁄motility⁄and⁄ flagella⁄in⁄Bacillus⁄cereus⁄biofilm⁄formation.⁄Microbiology⁄2010;156:1009

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18.⁄doi:10.1099/mic.0.034827-0.⁄

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678⁄

680⁄ [30]⁄ Vlamakis⁄H,⁄Aguilar⁄C,⁄Losick⁄R,⁄Kolter⁄R.⁄Control⁄of⁄cell⁄fate⁄by⁄the⁄ 681⁄

formation⁄of⁄an⁄architecturally⁄complex⁄bacterial⁄community.⁄Genes⁄Dev⁄

682⁄

2008;22:945 53.⁄doi:10.1101/gad.1645008.⁄

683⁄ [31]⁄ Verplaetse⁄E,⁄Slamti⁄L,⁄Gohar⁄M,⁄Lereclus⁄D.⁄Cell⁄Differentiation⁄in⁄a⁄ 684⁄

Bacillus⁄thuringiensis⁄Population⁄during⁄Planktonic⁄Growth,⁄Biofilm⁄

685⁄

Formation,⁄and⁄Host⁄Infection.⁄MBio⁄2015;6:e00138 15.⁄

686⁄

doi:10.1128/mBio.00138-15.⁄

ACCEPTED MANUSCRIPT 687⁄ [32]⁄ Sheppard⁄AE,⁄Poehlein⁄A,⁄Rosenstiel⁄P,⁄Liesegang⁄H,⁄Schulenburg⁄H.⁄ 688⁄

Complete⁄Genome⁄Sequence⁄of⁄Bacillus⁄thuringiensis⁄Strain⁄407⁄Cry-.⁄Genome⁄

689⁄

Announc⁄2013;1.⁄doi:10.1128/genomeA.00158-12.⁄

690⁄ [33]⁄ Medema⁄MH,⁄Blin⁄K,⁄Cimermancic⁄P,⁄de⁄Jager⁄V,⁄Zakrzewski⁄P,⁄Fischbach⁄MA,⁄

et⁄al.⁄antiSMASH:⁄rapid⁄identification,⁄annotation⁄and⁄analysis⁄of⁄

692⁄

secondary⁄metabolite⁄biosynthesis⁄gene⁄clusters⁄in⁄bacterial⁄and⁄fungal⁄

693⁄

genome⁄sequences.⁄Nucleic⁄Acids⁄Res⁄2011;39:⁠339 46.⁄

694⁄

doi:10.1093/nar/gkr466.⁄

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691⁄

695⁄ [34]⁄ ⁠eber⁄T,⁄Blin⁄K,⁄Duddela⁄S,⁄Krug⁄D,⁄Kim⁄HU,⁄Bruccoleri⁄R,⁄et⁄al.⁄antiSMASH⁄ 3.0--a⁄comprehensive⁄resource⁄for⁄the⁄genome⁄mining⁄of⁄biosynthetic⁄gene⁄

697⁄

clusters.⁄Nucleic⁄Acids⁄Res⁄2015:gkv437⁄ ⁄.⁄doi:10.1093/nar/gkv437.⁄

SC

696⁄

698⁄ [35]⁄ Altschul⁄SF,⁄Madden⁄TL,⁄Schäffer⁄AA,⁄Zhang⁄J,⁄Zhang⁄Z,⁄Miller⁄⁠,⁄et⁄al.⁄ Gapped⁄BLAST⁄and⁄PSI-BLAST:⁄a⁄new⁄generation⁄of⁄protein⁄database⁄search⁄

700⁄

programs.⁄Nucleic⁄Acids⁄Res⁄1997;25:3389 402.⁄

M AN U

699⁄

701⁄ [36]⁄ Sievers⁄F,⁄⁠ilm⁄A,⁄Dineen⁄D,⁄Gibson⁄TJ,⁄Karplus⁄K,⁄Li⁄⁠,⁄et⁄al.⁄Fast,⁄ 702⁄

scalable⁄generation⁄of⁄high-quality⁄protein⁄multiple⁄sequence⁄alignments⁄

703⁄

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705⁄

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708⁄

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EP

707⁄

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711⁄

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710⁄

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714⁄

doi:10.1371/journal.pone.0009490.⁄

715⁄ [41]⁄ ⁠aterhouse⁄AM,⁄Procter⁄JB,⁄Martin⁄DMA,⁄Clamp⁄M,⁄Barton⁄GJ.⁄Jalview⁄Version⁄ 716⁄

2--a⁄multiple⁄sequence⁄alignment⁄editor⁄and⁄analysis⁄workbench.⁄

717⁄

Bioinformatics⁄2009;25:1189 91.⁄doi:10.1093/bioinformatics/btp033.⁄

718⁄ [42]⁄ Van⁄Ert⁄MN,⁄Easterday⁄⁠R,⁄Huynh⁄LY,⁄⁠kinaka⁄RT,⁄Hugh-Jones⁄ME,⁄Ravel⁄J,⁄et⁄ 719⁄

al.⁄Global⁄Genetic⁄Population⁄Structure⁄of⁄Bacillus⁄anthracis.⁄PLoS⁄⁠ne⁄

720⁄

2007;2:e461.⁄doi:10.1371/journal.pone.0000461.⁄

ACCEPTED MANUSCRIPT 721⁄ [43]⁄ Huang⁄L,⁄Cheng⁄T,⁄Xu⁄P,⁄Cheng⁄D,⁄Fang⁄T,⁄Xia⁄Q.⁄A⁄genome-wide⁄survey⁄for⁄ 722⁄

host⁄response⁄of⁄silkworm,⁄Bombyx⁄mori⁄during⁄pathogen⁄Bacillus⁄

723⁄

bombyseptieus⁄infection.⁄PLoS⁄⁠ne⁄2009;4:e8098.⁄

724⁄

doi:10.1371/journal.pone.0008098.⁄

725⁄ [44]⁄ Guinebretière⁄M-H,⁄Thompson⁄FL,⁄Sorokin⁄A,⁄Normand⁄P,⁄Dawyndt⁄P,⁄EhlingSchulz⁄M,⁄et⁄al.⁄Ecological⁄diversification⁄in⁄the⁄Bacillus⁄cereus⁄Group.⁄

727⁄

Environ⁄Microbiol⁄2008;10:851 65.⁄doi:10.1111/j.1462-2920.2007.01495.x.⁄

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726⁄

728⁄ [45]⁄ Tourasse⁄NJ,⁄⁠kstad⁄⁠A,⁄Kolstø⁄A-B.⁄HyperCAT:⁄an⁄extension⁄of⁄the⁄SuperCAT⁄ database⁄for⁄global⁄multi-scheme⁄and⁄multi-datatype⁄phylogenetic⁄analysis⁄

730⁄

of⁄the⁄Bacillus⁄cereus⁄group⁄population.⁄Database⁄(⁠xford)⁄

731⁄

2010;2010:baq017.⁄doi:10.1093/database/baq017.⁄

SC

729⁄

732⁄ [46]⁄ Moran⁄CP,⁄Lang⁄N,⁄LeGrice⁄SF,⁄Lee⁄G,⁄Stephens⁄M,⁄Sonenshein⁄AL,⁄et⁄al.⁄ Nucleotide⁄sequences⁄that⁄signal⁄the⁄initiation⁄of⁄transcription⁄and⁄

734⁄

translation⁄in⁄Bacillus⁄subtilis.⁄Mol⁄Gen⁄Genet⁄1982;186:339 46.⁄

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733⁄

735⁄ [47]⁄ Chu⁄F,⁄Kearns⁄DB,⁄Branda⁄SS,⁄Kolter⁄R,⁄Losick⁄R.⁄Targets⁄of⁄the⁄master⁄ 736⁄

regulator⁄of⁄biofilm⁄formation⁄in⁄Bacillus⁄subtilis.⁄Mol⁄Microbiol⁄

737⁄

2006;59:1216 28.⁄doi:10.1111/j.1365-2958.2005.05019.x.⁄

738⁄ [48]⁄ Spiegelman⁄GB,⁄Bird⁄TH,⁄Voon⁄Y.⁄Transcription⁄regulation⁄by⁄the⁄Bacillus⁄

subtilis⁄response⁄regulator⁄Spo0A.⁄In:⁄Hoch⁄JA,⁄Silhavy⁄TJ,⁄editors.⁄Two⁄

740⁄

Compon.⁄signal⁄Transduct.⁄American⁄S,⁄⁠ashington⁄D.C.:⁄1995,⁄p.⁄159 79.⁄

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739⁄

741⁄ [49]⁄ Finking⁄R,⁄Marahiel⁄MA.⁄Biosynthesis⁄of⁄nonribosomal⁄peptides.⁄Annu⁄Rev⁄ Microbiol⁄2004;58:453 88.⁄doi:10.1146/annurev.micro.58.030603.123615.⁄

EP

742⁄

743⁄ [50]⁄ Lereclus⁄D,⁄Agaisse⁄H,⁄Gominet⁄M,⁄Chaufaux⁄J.⁄⁠verproduction⁄of⁄ Encapsulated⁄Insecticidal⁄Crystal⁄Proteins⁄in⁄a⁄Bacillus⁄thuringiensis⁄

745⁄

spo0A⁄Mutant.⁄Bio/Technology⁄1995;13:67 71.⁄doi:10.1038/nbt0195-67.⁄

AC C

744⁄

746⁄ [51]⁄ Agaisse⁄H,⁄Lereclus⁄D.⁄Structural⁄and⁄functional⁄analysis⁄of⁄the⁄promoter⁄ 747⁄

region⁄involved⁄in⁄full⁄expression⁄of⁄the⁄cryIIIA⁄toxin⁄gene⁄of⁄Bacillus⁄

748⁄

thuringiensis.⁄Mol⁄Microbiol⁄1994;13:97 107.⁄

749⁄ 750⁄ 751⁄ 752⁄

[52]⁄ Lauderdale⁄KJ,⁄Malone⁄CL,⁄Boles⁄BR,⁄Morcuende⁄J,⁄Horswill⁄AR.⁄Biofilm⁄ dispersal⁄of⁄⁄ community-associated⁄methicillin-resistant⁄Staphylococcus⁄ aureus⁄on⁄orthopedic⁄implant⁄⁄material.⁄J⁄⁠rthop⁄Res⁄2010;28:55 61.⁄ doi:10.1002/jor.20943.

ACCEPTED MANUSCRIPT 753⁄ Legends⁄to⁄Fig. Legends⁄to⁄Fig.s Fig.s⁄ 754⁄ Fig.⁄1.⁄Krs+⁄strains⁄mainly⁄belong⁄to⁄cluster⁄II.⁄ 755⁄ The⁄ sequences⁄ of⁄ the⁄ products⁄ of⁄ the⁄ six⁄ krs⁄ genes⁄ were⁄ concatenated⁄ for⁄ each⁄ 756⁄ strain⁄and⁄aligned⁄with⁄Clustal

.⁄This⁄global⁄alignment⁄was⁄used⁄by⁄FastTree⁄to⁄

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757⁄ build⁄ the⁄ approximately-maximum-likelihood⁄ tree⁄ of⁄ all⁄ Krs+⁄ loci.⁄ Branch⁄

759⁄ belong⁄to⁄clade⁄III.⁄ 760⁄ ⁄

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761⁄ Fig.⁄2.⁄RT-PCR⁄analysis⁄of⁄the⁄krs⁄locus.⁄ ⁄

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758⁄ supports⁄ are⁄ indicated.⁄ Strains⁄ in⁄ black⁄ belong⁄ to⁄ clade⁄ II;⁄ strains⁄ in⁄ gray⁄

762⁄ A.⁄ A. Schematic⁄ representation⁄ to⁄ scale⁄ of⁄ the⁄ krs⁄ locus⁄ of⁄ B.⁄thuringiensis⁄ 407.⁄ 763⁄ E:⁄ krsE,⁄ A:⁄ krsA,⁄ B:⁄ krsB,⁄ C:⁄ krsC,⁄ s:⁄ sfp,⁄ D:⁄ krsD.⁄ Note⁄ the⁄ scale⁄ break⁄ in⁄ 764⁄ krsC.⁄ The⁄ stem-loop⁄ structure⁄ in⁄ 3

⁄ represents⁄ a⁄ putative⁄ transcription⁄

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765⁄ terminator.⁄Below⁄the⁄locus⁄are⁄represented⁄the⁄primers⁄pairs⁄used⁄in⁄the⁄RT-PCR⁄ 766⁄ (see⁄experimental⁄procedures).⁄ B.⁄Photograph⁄after⁄electrophoretic⁄migration⁄of⁄ B.

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767⁄ the⁄ RT-PCR⁄ products⁄ in⁄ ⁄ 0.8⁠⁄ agarose⁄ gel⁄ containing⁄ ethidium⁄ bromide.⁄ Each⁄ 768⁄ primer⁄pair⁄was⁄used⁄in⁄a⁄reaction⁄with⁄or⁄without⁄reverse⁄transcriptase⁄(RT+⁄or⁄

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769⁄ RT-,⁄ respectively)⁄ using⁄ cDNA⁄ synthesized⁄ from⁄ total⁄ RNA⁄ extracted⁄ from⁄ 770⁄ B.⁄thuringiensis⁄ 407⁄ as⁄ a⁄ template,⁄ and⁄ a⁄ reaction⁄ using⁄ B.⁄thuringiensis⁄ 407⁄ 771⁄ genomic⁄DNA⁄as⁄a⁄template⁄(G).⁄ 772⁄ ⁄ 773⁄ Fig.⁄3.⁄Mapping⁄of⁄the⁄krs⁄promoter⁄region.⁄ ⁄ 774⁄ A.⁄ A. Total⁄ RNA⁄ was⁄ subjected⁄ to⁄ primer⁄ extension⁄ analysis⁄ using⁄ oligonucleotide⁄ 775⁄ tss_bc2450⁄ complementary⁄ to⁄ the⁄ 5

⁄ end⁄ of⁄ the⁄ krsE⁄ gene.⁄ The⁄ same⁄

ACCEPTED MANUSCRIPT 776⁄ oligonucleotide⁄ was⁄ used⁄ to⁄ prime⁄ dideoxy⁄ sequencing⁄ reactions⁄ from⁄ the⁄ 777⁄ B.⁄thuringiensis⁄ 407⁄ chromosome⁄ (lanes⁄ T,⁄ G,⁄ C,⁄ A).⁄ Position⁄ of⁄ the⁄ 778⁄ transcription⁄start⁄site⁄is⁄given⁄with⁄reference⁄to⁄the⁄translation⁄start⁄codon⁄ 779⁄ of⁄ krsE.⁄B. B.⁄Sequence⁄of⁄the⁄intergenic⁄space⁄upstream⁄of⁄ krsE.⁄The⁄two⁄putative⁄ B.

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780⁄ SinR⁄ binding⁄ sites⁄ are⁄ framed⁄ (dashes).⁄ The⁄ putative⁄ Spo0A⁄ binding⁄ site⁄ is⁄ 781⁄ framed⁄ (plain).⁄ The⁄ palindrome⁄ is⁄ highlighted⁄ in⁄ gray.⁄ The⁄ TSS⁄ is⁄ marked⁄ by⁄ a⁄

SC

782⁄ star.⁄The⁄putative⁄-35⁄and⁄-10⁄boxes⁄are⁄underlined⁄and⁄the⁄putative⁄ATG⁄of⁄krsE⁄ 783⁄ is⁄ in⁄ bold⁄ characters.⁄ C.⁄ C. Schematic⁄ representation⁄ of⁄ the⁄ different⁄ fragments⁄

785⁄ pHT304-18Z⁄ plasmid⁄ D.⁄ D. Relative⁄

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784⁄ that⁄ were⁄ used⁄ in⁄ transcriptional⁄ fusions⁄ with⁄ the⁄ lacZ⁄ reporter⁄ gene⁄ on⁄ the⁄ -galactosidase⁄ specific⁄ activity⁄ generated⁄ by⁄

786⁄ the⁄different⁄constructions⁄described⁄in⁄B,⁄at⁄t3⁄in⁄HCT⁄at⁄30°C,⁄normalized⁄to⁄ 787⁄ the⁄activity⁄of⁄the⁄FL⁄reference⁄construct.⁄NS:⁄not⁄significant,⁄**:⁄ p⁄<⁄0.005.⁄

789⁄ the⁄

-galactosidase⁄specific⁄activity⁄generated⁄by⁄the⁄FL⁄construct⁄in⁄

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788⁄ E.⁄Relative⁄ E.

spo0A⁄strain⁄in⁄HCT⁄at⁄30°C,⁄normalized⁄to⁄its⁄activity⁄in⁄the⁄⁠T⁄strain.⁄

791⁄ ⁄

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790⁄ Measures⁄were⁄taken⁄hourly⁄starting⁄at⁄t2.⁄NS:⁄not⁄significant,⁄*:⁄p⁄<⁄0.05.⁄

793⁄ A.⁄ A.

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792⁄ Fig.⁄4.⁄krs⁄transcription⁄is⁄sensitive⁄to⁄growth⁄conditions.⁄ ⁄ -galactosidase⁄ specific⁄ activity⁄ of⁄ the⁄ krsC::lacZ⁄ chromosomal⁄ fusion⁄ in⁄

794⁄ shaken⁄ LB,⁄ BHI,⁄ LBP⁄ and⁄ HCT⁄ at⁄ 30°C.⁄ Time⁄ 0⁄ marks⁄ the⁄ beginning⁄ of⁄ the⁄ 795⁄ transition⁄ between⁄ exponential⁄ and⁄ stationary⁄ phase.⁄ Error⁄ bars⁄ were⁄ plotted⁄ 796⁄ asymmetrically⁄ for⁄ the⁄ sake⁄ of⁄ readability.⁄ B.⁄ B. Flow⁄ cytometry⁄ analysis⁄ of⁄ the⁄ 797⁄ fluorescence⁄ distribution⁄ in⁄ cells⁄ harboring⁄ the⁄ pHT304

Pkrs(FL)

-sgfp⁄ plasmid⁄

798⁄ in⁄shaken⁄LB⁄(upper⁄panel)⁄and⁄a⁄colony⁄grown⁄on⁄LB⁄agar⁄(lower⁄panel).⁄Samples⁄

ACCEPTED MANUSCRIPT 799⁄ were⁄taken⁄20⁄h⁄after⁄the⁄end⁄of⁄stationary⁄phase⁄in⁄shaken⁄LB,⁄and⁄72⁄h⁄after⁄ 800⁄ colony⁄ inoculation⁄ on⁄ LB⁄ agar.⁄ Autofluorescence⁄ of⁄ a⁄ reporter-less⁄ strain⁄ is⁄ 801⁄ represented⁄by⁄the⁄gray⁄area;⁄fluorescence⁄of⁄the⁄bacteria⁄harboring⁄the⁄plasmid⁄ 802⁄ is⁄represented⁄in⁄black⁄outline.⁄This⁄experiment⁄is⁄representative⁄of⁄at⁄least⁄4⁄

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803⁄ independent⁄replicates.⁄ 804⁄ ⁄

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805⁄ Fig.⁄5.⁄Detection⁄of⁄kurstakin⁄in⁄the⁄B.⁄thuringiensis⁄407⁄strain.⁄ ⁄

806⁄ A.⁄⁠hole-cell⁄⁄mass⁄spectrum⁄of⁄ B.⁄thuringiensis⁄407;⁄ m/z⁄peak⁄of⁄906,⁄928⁄and⁄ A.

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807⁄ 944⁄represents⁄H+,⁄Na+⁄and⁄K+⁄adducts⁄of⁄C13⁄kurstakin⁄isoform,⁄respectively.⁄B. B.⁄ B. 808⁄ ⁠hole-cell⁄ mass⁄ spectrum⁄ of⁄ B.⁄thuringiensis⁄ 407⁄

krsC⁄ mutant.⁄ The⁄ peaks⁄

809⁄ corresponding⁄ to⁄ kurstakin⁄ could⁄ not⁄ be⁄ observed⁄ on⁄ mass⁄ spectra⁄ obtained⁄ from⁄ 810⁄ the⁄

krsC⁄strain.⁄

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811⁄ ⁄ 812⁄ Fig.⁄6.⁄Kurstakin⁄is⁄required⁄for⁄proper⁄biofilm⁄structuration.⁄ ⁄

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813⁄ A.⁄Biofilm⁄density⁄in⁄glass⁄tubes⁄after⁄48⁄h⁄incubation⁄and⁄PVC⁄plates⁄after⁄a⁄ A. 814⁄ 24⁄h⁄⁄incubation⁄in⁄HCT⁄at⁄30°C.⁄B. B.⁄Air-liquid⁄pellicles⁄of⁄biofilms⁄formed⁄by⁄ B.

krsC⁄strains⁄in⁄PVC-plate⁄wells,⁄observed⁄from⁄above⁄with⁄a⁄stereo⁄

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815⁄ the⁄⁠T⁄and⁄

816⁄ microscope⁄ C.⁄ C. 3D⁄ reconstructions⁄ of⁄ confocal⁄ laser⁄ scanning⁄ spectrum⁄ 817⁄ micrographs.⁄ ⁄

ACCEPTED MANUSCRIPT 818⁄ Tables Tables⁄ bles

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819⁄ Table⁄1.⁄krs⁄is⁄not⁄evenly⁄distributed⁄within⁄the⁄B.⁄cereus⁄group⁄ ⁄

ACCEPTED MANUSCRIPT 820⁄ Supplementary⁄material⁄for⁄onSupplementary⁄material⁄for⁄on-line⁄submission⁄ line⁄submission 821⁄ Table⁄S1.⁄B.⁄thuringiensis⁄407⁄derived⁄strains⁄used⁄in⁄this⁄study.

⁄

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822⁄ ⁄

ACCEPTED MANUSCRIPT 823⁄ Table⁄S2.⁄Plasmids⁄used⁄in⁄this⁄study.⁄

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⁄

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824⁄

ACCEPTED MANUSCRIPT 825⁄ Table⁄S3.⁄Primers⁄used⁄in⁄this⁄study.⁄

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826⁄ ⁄

ACCEPTED MANUSCRIPT

827⁄ Table⁄S4.⁄Krs+⁄genomes.⁄

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828⁄ ⁄

ACCEPTED MANUSCRIPT 829⁄ Fig.⁄S1.⁄The⁄krs⁄locus⁄in⁄the⁄B.⁄thuringiensis⁄407⁄reference⁄genome.⁄ 830⁄ ⁄ 831⁄ The⁄ top⁄ schematic⁄ represents⁄ our⁄ annotation⁄ of⁄ the⁄ krs⁄ locus;⁄ the⁄ bottom⁄ one⁄

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832⁄ represents⁄the⁄annotation⁄on⁄the⁄B.⁄thuringiensis⁄407⁄reference⁄genome⁄[32]

.⁄

ACCEPTED MANUSCRIPT 833⁄ Fig.⁄S2.⁄Alignment⁄of⁄17⁄krs⁄upstream⁄sequences.⁄ 834⁄ ⁄ 835⁄ Conservation⁄ at⁄ each⁄ position⁄ is⁄ indicated⁄ by⁄ shades⁄ of⁄ blue:⁄ the⁄ darker,⁄ the⁄

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836⁄ more⁄conserved.⁄

ACCEPTED MANUSCRIPT -lacZ⁄ fusion⁄ has⁄ ⁄ higher⁄ activity⁄ than⁄ the⁄ chromosomal⁄

837⁄ Fig.⁄ S3.⁄ Plasmidic⁄ Pkrs

838⁄ krsC::lacZ⁄fusion,⁄but⁄has⁄a⁄comparable⁄pattern.⁄ 839⁄ ⁄ 840⁄

-galactosidase⁄ specific⁄ activity⁄ generated⁄ by⁄ strains⁄ harboring⁄ chromosomal⁄

842⁄ (pHT304-18

Pkrs(FL)

-lacZ)⁄strain)⁄Pkrs

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841⁄ (B.⁄thuringiensis⁄ 407⁄ krsC::lacZ⁄ strain)⁄ or⁄ plasmidic⁄ (B.⁄thuringiensis⁄ 407⁄ -lacZ⁄fusions⁄cultured⁄in⁄HCT⁄medium⁄at⁄

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843⁄ 30°C⁄ was⁄ quantified⁄ hly⁄ from⁄ t0⁄ to⁄ t5.⁄ Two⁄ independent⁄ experiments⁄ are⁄

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844⁄ presented.⁄

ACCEPTED MANUSCRIPT 845⁄ Fig.⁄S4.⁄SinR⁄is⁄not⁄involved⁄in⁄⁄⁄regulation⁄of⁄krs⁄transcription.⁄ 846⁄ ⁄ 847⁄ Relative⁄

sinI-sinR⁄strain⁄in⁄HCT⁄at⁄30°C,⁄normalized⁄to⁄its⁄activity⁄in⁄the⁄⁠T⁄strain.⁄

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849⁄ Measures⁄were⁄taken⁄hly⁄starting⁄at⁄t2.⁄

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848⁄

-galactosidase⁄specific⁄activity⁄generated⁄by⁄the⁄FL⁄construct⁄in⁄the⁄

ACCEPTED MANUSCRIPT 850⁄ Fig.⁄S5.⁄The⁄krs⁄operon⁄is⁄not⁄essential⁄for⁄necrotrophism.⁄ 851⁄ ⁄ 852⁄ CFUs⁄ were⁄ counted⁄ daily⁄ from⁄ day⁄ 0⁄ (infection)⁄ to⁄ day⁄ 4;⁄ 3⁄ and⁄ 2⁄ independent⁄ 853⁄ infections⁄ with⁄ the⁄ ⁠T⁄ and⁄

krsC⁄ strains,⁄ respectively,⁄ are⁄ plotted⁄ together⁄

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854⁄ with⁄their⁄respective⁄means.⁄

ACCEPTED MANUSCRIPT 855⁄ Fig.⁄S6.⁄Biofilms⁄formed⁄by⁄ B.⁄thuringiensis⁄407⁄

krsC⁄are⁄thinner⁄and⁄contain⁄

856⁄ fewer⁄cells⁄than⁄biofilms⁄formed⁄by⁄the⁄⁠T⁄strain.⁄ 857⁄ ⁄ 858⁄ A.⁄ A. Representative⁄ macroscopic⁄ picture⁄ of⁄ biofilms⁄ formed⁄ by⁄ the⁄ ⁠T⁄ and⁄

krsC⁄

860⁄ from⁄ confocal⁄ microscopy⁄ of⁄ all⁄ three⁄ ⁠T⁄ and⁄

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859⁄ strains⁄ in⁄ the⁄ conditions⁄ used⁄ for⁄ confocal⁄ microscopy.⁄ B.⁄ B. 3D⁄ reconstructions⁄

krsC⁄ biofilms⁄ (representative⁄

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861⁄ fields).⁄ Note⁄ that⁄ the⁄ second⁄ ⁠T⁄ biofilm⁄ fell⁄ off⁄ its⁄ filter⁄ during⁄ sample⁄ 862⁄ preparation,⁄before⁄observation.⁄

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863⁄ ⁄

ACCEPTED MANUSCRIPT 864⁄ Movie⁄ S1.⁄ Pellicles⁄ formed⁄ by⁄ B.⁄thuringiensis⁄ 407⁄ ⁠T⁄ and⁄ B.⁄thuringiensis⁄ 407⁄

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krsC⁄react⁄differently⁄to⁄pressure⁄applied⁄from⁄above.⁄

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865⁄

ACCEPTED MANUSCRIPT Species of the B. cereus group

Frequency of Krs+ genomes

Bacillus anthracis

0/31

Bacillus cereus

3/26

Bacillus cytotoxicus

0/1

Bacillus mycoides

1/2 12/23 (10/21)*

Bacillus toyonensis

0/1

Bacillus weihenstephanensis

1/2

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Bacillus thuringiensis

* The frequency was corrected from 12/23 to 10/21 to take into account redundancy of three

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B. thuringiensis genomes (strains Bt407, CT-43 and IS5056).

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ACCEPTED MANUSCRIPT

AC C

EP

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ACCEPTED MANUSCRIPT

A

B

SC

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ACCEPTED MANUSCRIPT

D

E

AC C

EP

TE D

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C

ACCEPTED MANUSCRIPT

B

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Colony

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Planktonic

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A

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

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A

WT

ΔkrsC

AC C

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C

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B

WT

ΔkrsC