Genetic engineering of bee gut microbiome bacteria with a toolkit for

Publication Date (Web): April 2, 2018 ... a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF101...
0 downloads 4 Views 2MB Size
Subscriber access provided by UNIV OF DURHAM

Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids Sean P. Leonard, Jiri Perutka, J Elijah Powell, Peng Geng, Darby Richhart, Michelle Byrom, Shaunak Kar, Bryan W. Davies, Andrew D. Ellington, Nancy Moran, and Jeffrey E Barrick ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00399 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids Sean P. Leonard, Jiri Perutka, J. Elijah Powell, Peng Geng, Darby D. Richhart, Michelle Byrom, Shaunak Kar, Bryan W. Davies, Andrew D. Ellington, Nancy A. Moran, Jeffrey E. Barrick

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Genetic engineering of bee gut microbiome bacteria with a

2

toolkit for modular assembly of broad-host-range plasmids

3 4

Sean P. Leonard1, Jiri Perutka2, J. Elijah Powell4, Peng Geng2, Darby D. Richhart4,

5

Michelle Byrom3, Shaunak Kar1, Bryan W. Davies1,2,3, Andrew D. Ellington1,2,3,

6

Nancy A. Moran1,2,4*, Jeffrey E. Barrick1,2,3*

7 8

*corresponding authors: [email protected], [email protected]

9 10

1

Institute for Cellular and Molecular Biology, 2Center for Systems and Synthetic Biology,

11

3

12

University of Texas at Austin, Austin, Texas 78712, United States.

Department of Molecular Biosciences, 4Department of Integrative Biology, The

1

ACS Paragon Plus Environment

Page 2 of 50

Page 3 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

13

Abstract

14

Engineering the bacteria present in animal microbiomes promises to lead to

15

breakthroughs in medicine and agriculture, but progress is hampered by a dearth of

16

tools for genetically modifying the diverse species that comprise these communities.

17

Here we present a toolkit of genetic parts for the modular construction of broad-host-

18

range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in

19

this toolkit can be used to rapidly test various antibiotic resistance markers, promoters,

20

fluorescent reporters and other coding sequences in newly isolated bacteria. We

21

demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native

22

to the gut microbiomes of honey bees (Apis mellifera) and bumble bees (Bombus sp.).

23

Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella

24

apis, and Serratia strains enables us to visualize how these bacteria colonize the bee

25

gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated

26

knockout of an S. alvi adhesion gene to show that it is important for colonization of the

27

gut. Beyond characterizing how the gut microbiome influences the health of these

28

prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering

29

bacteria found in other natural microbial communities.

30 31

Keywords

32

host-associated microbiome, probiotics, symbiotic bacteria, colony collapse disorder

2

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33 34

Symbiotic communities of microorganisms live in close association with many animals

35

and plants. Bacteria in these communities influence the development, metabolism, and

36

health of their hosts.1,2 Genetically engineering bacteria in these microbiomes to

37

manipulate these interactions and add novel functions has enormous practical potential,

38

including such diverse applications as treating human disease,3-5 deploying

39

environmental biosensors,6-9 controlling crop pests,10 and mitigating the spread of

40

disease by insect vectors.11,12 However, these communities are largely composed of

41

bacteria that have not yet been extensively characterized in the laboratory, which

42

presents a major obstacle to achieving these goals.

43

Decades of study and recent advances in synthetic biology have generated fully

44

featured toolsets for genetically manipulating model microbial species such as

45

Escherichia coli13,14 and Saccharomyces cerevisiae.15 For these organisms, complex

46

synthetic assemblies of heterologous genes can be designed in silico,16 built from sets

47

of well characterized genetic parts, and then tested. One approach for genetically

48

manipulating a microbiome is to add engineered versions of these platform organisms,

49

but model bacteria that function robustly under laboratory conditions often die or fail to

50

proliferate and persist when introduced into natural microbial communities.

51

A promising alternative for therapeutic applications in animal microbiomes is

52

engineering the bacteria that naturally live in these environments. This is difficult,

53

however, because technologies for genetically manipulating recently isolated bacteria

54

are limited. While some genetic tools exist for human gut-associated bacteria, notably

55

Bacteroides thetaiotaomicron,9,17-19 few bacteria from other gut microbiomes have

3

ACS Paragon Plus Environment

Page 4 of 50

Page 5 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

56

received such attention. Extensive trial and error is required to validate transformation

57

methods, antibiotic markers, plasmid backbones, promoters, and ribosome binding sites

58

(RBS) in a new bacterial species before it can be genetically engineered.20

59

One natural community worth engineering is the gut microbiome of bees. Bee

60

pollination of crops is a multibillion dollar industry that is crucial to agriculture, and

61

honey bee colonies are currently suffering high mortality rates for reasons that are not

62

yet fully understood.21 In Western honey bees (Apis mellifera), the hindgut community is

63

dominated by eight core bacterial species that can all be cultivated in the laboratory,

64

and most of these also occur in guts of bumble bees (Bombus sp.).22-24 The bee gut

65

microbiome (BGM) contributes to host nutrition and growth25 and to protection against

66

pathogens.26 Like the human gut microbiome, the BGM is socially acquired and

67

transmitted27 and has a history of antibiotic exposure.22,26,28,29 Major members of the

68

core BGM — Snodgrassella alvi, Gilliamella apicola, and Bartonella apis — have been

69

the subject of detailed genomic analyses.30-32 But aside from random transposon

70

mutagenesis in S. alvi,33 no genetic tools have been reported for these species.

71

Here, we describe a plasmid toolkit for combining a broad-host-range (BHR) replicon

72

with a set of modular genetic parts and its application to bacteria from the honey bee

73

and bumble bee gut microbiomes. We show that plasmids constructed using this bee

74

microbiome toolkit (BTK) function reliably in multiple species of Proteobacteria found in

75

the BGM. The BTK can be used to express heterologous genes or to repress or disrupt

76

genes in the bacterial chromosome. We generate fluorescent reporter strains of S. alvi,

77

B. apis, G. apicola and a pathogenic Serratia marcescens34 isolate to visualize how they

78

colonize honey bee guts. Finally, we use the BTK to validate the importance of a

4

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

79

specific adhesion gene (staA) for S. alvi colonization of the gut. The broad-host-range

80

replicon used in the BTK promises to make it suitable for use in other newly isolated or

81

poorly characterized bacterial species found in animal and plant microbiomes.

82 83

Results and Discussion

84 85

Design of the bee microbiome toolkit (BTK)

86

We performed a preliminary screen with a variety of broad-host-range plasmids with

87

different replication origins (RP4, pBBR1, RSF1010) and antibiotic resistance markers

88

(kanamycin, ampicillin, chloramphenicol, spectinomycin) for their ability to be transferred

89

by conjugation and stably maintained in two bacterial species, S. alvi and G. apicola,

90

which are both abundant in the honey bee gut (Supplementary Table 1). Plasmid

91

pMMB67EH, a synthetic plasmid containing an RSF1010 origin,35 was the most

92

versatile: it replicated in both species. Plasmids containing an RSF1010 origin are

93

known to be extremely broad-host-range (BHR) because they encode multiple ORFs

94

that make them less dependent on the presence of specific proteins in a host cell for

95

replication.36,37 Additionally, they contain a promiscuous origin of transfer (oriT) that

96

enables one-way transfer of the plasmid to a recipient cell from a donor cell encoding a

97

conjugation apparatus in trans on the chromosome, such as E. coli MFDpir.38

98 99

Because of these characteristics of pMMB67EH, we decided to create a toolkit of genetic parts for hierarchical and combinatorial assembly into its RSF1010-derived

100

backbone for testing in additional species (Figure 1A). These BTK parts are compatible

101

with the Golden Gate cloning scheme used by the Yeast Toolkit (YTK)15, and connector

5

ACS Paragon Plus Environment

Page 6 of 50

Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

102

parts from the YTK are required for BTK assembly. BTK parts are classified into eight

103

types defined by the specific flanking overhangs generated by type IIS restriction

104

enzyme cleavage. Entry vectors containing any complete set of parts labeled 1-8 can be

105

combined via one BsaI Golden Gate assembly reaction into a complete plasmid (Figure

106

1B). This assembly creates a Stage 1 plasmid comprising parts 2-4 flanked by

107

assembly connector parts 1 and 5 with vector backbone components in parts 6-8.

108

Transcriptional units from multiple Stage 1 plasmids that have matching sets of

109

connector parts can be further composed into one vector by Stage 2 assembly using

110

BsmBI (Figure 1C).

111

To create BTK vector backbone parts, we replaced the high-copy number bacterial

112

ColE1 origin of YTK part 8 plasmids (AmpR, KanR, SpecR) with the RSF1010 origin from

113

pMMB67EH. These backbones retain the oriT for delivery into recipient cells via

114

conjugation, which is useful for genetically modifying bacterial species and strains

115

lacking established chemical or electrical transformation techniques. In the original YTK,

116

Type 6 and 7 parts encode a yeast marker and a yeast origin, respectively. We

117

repurposed Type 6 part overhangs for flanking a DNA sequence encoding an optional

118

additional CDS (such as a repressor) in the reverse orientation relative to the Type 2

119

part CDS, and Type 7 part overhangs for incorporating an optional reverse promoter for

120

driving expression of the part 6 CDS. A combined Type 6-7 linker (pBTK301) can be

121

used in lieu of these parts to create constructs lacking this extra reverse gene.

122

We used these vectors to construct a variety of plasmids containing a single

123

fluorescent protein driven by a broad-host-range promoter. To build more complex

124

assemblies, such as those with T7 RNA polymerase driving inducible expression of

6

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

125

GFP, we combined multiple vectors from BsaI Stage 1 assembly in a Stage 2 BsmBI

126

assembly. Notable components of the BTK that expand on the set of genetic parts

127

available in the YTK for compatible assembly include:

128 129

1) 3 BHR plasmids with different antibiotic resistance cassettes and oriT as Type 8 origin parts for Stage 1 assembly

130

2) 2 BHR plasmids ready for Stage 2 assembly (SpecR, KanR)

131

3) 11 bacterial promoter/RBS combinations as Type 2 parts

132

4) 8 new CDSs including E2-Crimson39 and Nanoluc40 for in vivo visualization as

133

Type 3 parts

134

5) 3 bacterial terminators as Type 4 parts

135

6) 1 transcriptional repressor (LacI) as a Type 6 part

136

7) 2 R6K-origin plasmid backbones to assemble suicide plasmids for gene

137 138 139

disruption or chromosomal modification 8) Pre-assembled plasmids with BHR promoters for immediate testing in new bacterial strains

140 141

Dataset S1 summarizes BTK parts, assembled BTK plasmids, and their validation. In

142

the next sections, we describe and evaluate the functions of plasmids for control of

143

gene expression and disruption of chromosomal genes in non-model bacteria from the

144

honey bee (A. mellifera) and bumble bee (Bombus sp.) gut microbiomes. Component

145

and assembled BTK plasmids have been deposited with Addgene (accession number

146

pending acceptance) for distribution to other researchers.

147

7

ACS Paragon Plus Environment

Page 8 of 50

Page 9 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

148

BTK plasmids function in diverse bacterial species found in the bee gut

149

We next sought to explore the host range of the RSF1010 origin used as a basis for the

150

BTK in the context of a larger set of bee-associated bacterial strains. Simultaneously,

151

we needed to identify antibiotic resistance genes able to function in each bacterial

152

strain. To do so, we constructed three BTK plasmids, each with a different antibiotic

153

resistance marker and encoding GFP driven by the PA1 promoter: pBTK501 (AmpR),

154

pBTK519 (KanR), pBTK520 (SpecR). We performed biparental matings between E. coli

155

MFDpir donors containing each plasmid and bee gut-associated strains (see Methods).

156

Stable transconjugants were obtained for all of the Gram-negative strains we tested with

157

at least one of these three plasmids, as verified by further passaging on antibiotic-

158

containing media, PCR amplification of plasmid sequences, and GFP expression

159

(Figure 2A). Successfully transformed bacterial species include Alpha-, Beta-, and

160

Gammaproteobacteria and strains isolated from different bee species (A. mellifera,

161

Bombus terrestris, Bombus impatiens, and Bombus pensylvanicus). Several of the

162

bacterial species (S. alvi, G. apicola, B. apis, and Parasaccharibacter apium) are

163

phylogenetically distant from any established model organisms and have no previously

164

reported genetic tools. Transfer of the BTK plasmids was efficient, with >10–3

165

transconjugants per CFU for four diverse bacterial species (Figure 2B).

166 167

Identifying functional promoters in BGM species

168

While some sequence features of transcriptional promoters are conserved across

169

bacterial species, there is no guarantee that promoters designed to function in model

170

organisms will function effectively in new bacterial isolates from a natural community of

8

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

171

interest.19 The BTK includes BHR promoters and RBS combinations as Type 2 parts

172

that can be used to build plasmids to identify functional sequences for driving protein

173

expression in new bacterial hosts. We compared the function of the BHR promoters

174

PA1 (pBTK501), PA2 (pBTK509), PA3 (pBTK510), and CP25 (pBTK503) in S. alvi

175

wkB2, G. apicola wkB7, B. apis PEB0150, and S. marcescens N10A28, all isolated from

176

honey bee gut communities. Promoters PA1, PA2, PA3 are strong early promoters from

177

bacteriophage T7.41 The synthetic CP25 promoter was originally designed to function in

178

Lactococcus strains,42 and the BTK includes other promoters from this series.

179

Using flow cytometry, we characterized GFP expression from each of these

180

promoters when they were combined with the same RBS sequence (Figure 3A). As

181

expected, all of the promoters showed the highest activity in S. marcescens, which is

182

most closely related to E. coli. Expression was generally weaker in the other BGM

183

strains, but there was a clear signal well above background for a least two of the four

184

promoters in all strains. In addition to the overall level of gene expression that a

185

promoter affords, the amount of variation in its activity among different cells in a

186

population is an important property to characterize for many synthetic biology

187

applications. We found that the amount of fluorescence per cell was broadly distributed

188

or noticeably bimodal in many cases, including in E. coli. This variation could be due to

189

intrinsic properties of these promoter-RBS combinations leading to stochastic variation

190

in gene expression or due to the rapid accumulation of broken plasmids with mutations

191

in the promoter-RBS that lower burdensome GFP expression43, or both of these

192

processes. In any case, our measurements show which promoters can be used to

193

achieve useful levels of gene expression in each BGM strain. For example, PA3

9

ACS Paragon Plus Environment

Page 10 of 50

Page 11 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

194

expression was strong in S. alvi, and we used this observation to design a constitutive

195

E2-Crimson-expressing plasmid (pBTK570) to demonstrate expression in vivo, as

196

described in later sections. Validation of additional parts (E2-Crimson, Nanoluc, and

197

other CP-series promoters) is available in Supplemental Figures S1-S3.

198 199

Inducible gene expression in BGM species

200

Induction systems are required for the temporal control of gene expression, and are

201

useful for testing the functional roles of microbes in gut environments.18 We tested two

202

lacI induction systems: one simple system composed of a modified CP25 promoter with

203

lacO sites and a more complex system that uses T7 RNA polymerase (T7 RNAP). We

204

tested IPTG-induction of these systems in E. coli MFDpir, S. alvi wkB2, G. apicola

205

wkB7, B. apis PEB0150, and S. marcescens N10A28. The simple system (pBTK552)

206

showed robust induction of GFP in all strains tested (Figure 3B). Interestingly, G.

207

apicola GFP expression with this system surpassed that of E. coli and S. marcescens.

208

For the T7 RNAP system, we built two transcriptional unit plasmids (pBTK549d,

209

pBTK541), one bearing lacI driven by the CP25 promoter and T7 RNAP under control of

210

the inducible lac promoter and the other with GFP expressed from a T7 promoter with

211

lacO sites, and combined them into a composite plasmid (pBTK550d). (Figure 3C).

212

Expression was strong in S. marcescens N10A28 and E. coli MFDpir, with maximal

213

GFP expression after induction surpassing the simpler system in which lacI directly

214

regulates GFP expression. However, in G. apicola wkB7, S. alvi wkB2, and B. apis

215

PEB0150, we saw weaker induction of GFP compared to the simpler system. The

216

cause of this weak expression is unknown. It may be due to poor transcription from the

10

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

217

lac promoter driving T7 RNAP or to an intrinsic incompatibility between T7 RNAP and

218

the intracellular environment in the BGM species tested. In all strains, the inducible T7

219

RNAP construct showed appreciable background expression when not induced.

220 221

CRISPRi repression of chromosomal gene expression in Bartonella apis

222

We next used the BTK to suppress gene activity in a BGM bacterium. Catalytic mutants

223

of Cas9 (dCas9) have been used to reduce transcription of target genes, an approach

224

termed CRISPR interference (CRISPRi), in diverse mammalian and bacterial systems.44

225

To expand this approach to new non-model bacterial species, we established a

226

modified dCas9 system in which targeting is achieved by a BTK part encoding a small

227

guide RNA (sgRNA) (Figure 4A).45 To test the system, we targeted the sgRNA to a

228

PA1-driven GFP gene in B. apis PEB0150, which we inserted into the chromosome

229

using Tn7-based integration.46 GFP expression was significantly reduced in the

230

presence of a sgRNA targeted to the GFP sequence (Figure 4B). Coupled with the

231

induction system, this ability to repress a target gene enables functional studies of

232

essential genes that cannot be disrupted entirely.

233 234

Cas9-assisted gene disruption in the BGM

235

Gene disruption is an important tool for establishing gene function and for studying

236

interactions between genes. After identifying functional antibiotic cassettes in our earlier

237

plasmid-replication screen, we attempted to use homologous recombination to disrupt

238

chromosomal genes in our BGM strains. To improve the efficacy of targeted gene

239

disruption, we also implemented a two-step approach based on using Cas9 cleavage

11

ACS Paragon Plus Environment

Page 12 of 50

Page 13 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

240

for chromosomal modifications44 (see Methods). In step one, Cas9 is introduced into a

241

cell on the BTK backbone (pBTK601) without any targeting sgRNA. In step two, a

242

second round of conjugation is used to deliver a suicide plasmid with the replacement

243

cassette (~1000 bp homology flanking a functional antibiotic resistance gene) and the

244

sgRNA targeting the desired chromosomal location. The suicide plasmid is made with

245

Golden Gate assembly using repurposed Type 2-4 overhangs and an R6K origin of

246

replication (Figure 5A-B). The sgRNA can be retargeted using MEGAWHOP cloning47

247

(see Methods). A detailed description of suicide plasmid assembly and validation of

248

mutants is shown in Supplemental Figure S4. We expected that Cas9 cleavage might

249

facilitate recombination into the chromosome and that it would also select against

250

single-crossover integrations, in which the suicide plasmid backbone is incorporated

251

into the chromosome, because they preserve the cleavage site, whereas double-

252

crossover integrations result in replacement of the targeted gene sequence with just the

253

antibiotic resistance cassette and delete the cleavage site. The Cas9 plasmid has a low

254

to medium copy number and lacks a partitioning system, so cells can be readily cured of

255

this plasmid after the gene disruption procedure is complete.37

256

To test the utility of this scheme, we attempted to generate gene disruptions in three

257

BGM species. In S. alvi wkB2 we targeted staA (SALWKB2_RS11470), an adhesion

258

gene previously implicated in a genome-wide screen as important for gut colonization

259

(Supplemental Figure S5).33 In G. apicola wkB7 we targeted acetate kinase ackA

260

(A9G17_RS12535) (Supplemental Figure S6), and in B. apis PEB0150 we targeted

261

nitrate reductase narG (PEB0150_RS00755) (Supplemental Figure S7). We designed

262

our homology regions to be internal to each coding sequence, so that even single-

12

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

263

crossover events would disrupt gene function. For S. alvi wkB2, our multi-step system

264

showed higher efficiency compared to basic homologous recombination not using Cas9.

265

In the presence of Cas9, wkB2 mutants were obtained more frequently and were more

266

often double-crossover mutants (Figure 5D). In contrast, B. apis PEB0150 showed

267

relatively high gene disruption efficiency even in the absence of Cas9, and the Cas9

268

system had little effect on improving the number of double-crossover mutants (Figure

269

5C). In G. apicola wkB7 the Cas9 was also not helpful, and we obtained no double-

270

crossover mutants (Figure 5E). The G. apicola wkB7 mutants isolated showed irregular

271

PCR amplification at the expected junctions (Supplemental Figure S6), indicating we

272

could not effectively disrupt ackA, perhaps because it is an essential gene in this

273

species. Unfortunately, the limited sample size in these trials makes it difficult to draw

274

conclusions from these experiments. This Cas9 approach may be useful to increase

275

double-crossover mutants in S. alvi, but not in other species. While we validated this

276

general approach to gene disruption in two BGM species, it will be necessary to repeat

277

this procedure on more target genes and test more clones to gain a broader

278

understanding of the effectiveness of Cas9-assisted gene disruption in these species.

279 280

Engineered strains colonize bees and can be directly visualized in the ileum

281

We next tested the ability of engineered BGM strains to colonize newly emerged worker

282

bees removed from the hive before they acquire a normal microbiota. Previously, S. alvi

283

and G. apicola have been visualized in bees using fluorescent in situ hybridization.48

284

However, this technique can only be used at one time point because it requires

285

sacrificing the bee. In contrast, fluorescent reporter strains can be used to non-

13

ACS Paragon Plus Environment

Page 14 of 50

Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

286

destructively estimate bacterial abundance and observe how bacterial community

287

structure changes over time in live bees. Previous studies have examined how S. alvi

288

colonizes the honey bee gut,31,49 but colonization by G. apicola, B. apis, and S.

289

marcescens has not been investigated.26,34

290

We inoculated newly emerged workers with ~104 CFU per bee of either S.

291

marcescens N10A28 or S. alvi wkB2, each carrying a constitutively expressed E2-

292

Crimson fluorescent protein (pBTK570). After 5 days, we dissected bees from each

293

group and examined their guts (Figure 6). Fluorescent bacteria were successfully

294

imaged directly in guts without preparation or fixation, preserving natural community

295

structure. S. marcescens N10A28 shows robust colonization in all gut compartments,

296

while other species show spatially restricted colonization. As previously reported, S. alvi

297

wkB2 robustly colonizes the ileum, with little colonization in the midgut and rectum.

298

Additionally, we performed co-inoculations with S. alvi wkB2 and either B. apis

299

PEB0150 or G. apicola wkB7 engineered to express GFP (pBTK520). We again

300

dissected the guts of colonized bees and were able to fluorescently image in vivo co-

301

colonization of these defined communities (Figure 7A-B). While both B. apis and G.

302

apicola are found in the ileum co-located with S. alvi, they also colonize the rectum, in

303

contrast to S. alvi.

304 305

Snodgrassella staA contributes to gut colonization in vivo

306

Finally, we sought to validate the usefulness of the BTK for disrupting specific genes in

307

BGM species in order to investigate their function. StaA belongs to a family of YadA-like

308

adhesion proteins important for colonization and pathogenicity in multiple host-

14

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

309

associated species.50 These trimeric autotransporter proteins localize to the bacterial

310

membrane and form “lollipop” structures that allow bacteria to adhere to epithelial

311

cells.51,52 Orthologs of these genes are found in multiple S. alvi genomes, including

312

those from honey bee- and bumble bee-associated strains.31 Our previous work

313

screening a transposon mutant library identified staA (SALWKB2_RS11470) as

314

necessary for the fitness of S. alvi during gut colonization.33 However, we were unable

315

to isolate a mutant from our library with a transposon disrupting staA, and thus we could

316

not fully characterize and validate the role of this gene.

317

Using the BTK, we generated a ∆staA mutant in S. alvi wkB2 (as described

318

above). We then labeled the ∆staA mutant and a wild-type control with a BTK plasmid

319

expressing E2-Crimson (pBTK570) to assess the effects of disrupting this gene in the

320

context of the bee gut. The wkB2 ∆staA mutant shows reduced colonization efficacy

321

compared to a wild-type control, as measured by qPCR of S. alvi 16S rRNA gene

322

copies (Figure 7C). The colonization pattern of this mutant in terms of its localization

323

within the gut (Figure 7D) is distinct from that of wild-type S. alvi (Figure 6E). After 6

324

days, the mutant does not form the contiguous, robust colonization of the ileum wall

325

seen for the wild type strain. Instead, colonization is apparently restricted to small

326

patches, while the majority of the ileum remains uncolonized.

327 328

Summary

329

We built the BTK, the first Golden Gate toolkit designed specifically for the combinatorial

330

assembly of broad-host-range plasmids, with the aim of expanding synthetic biology into

331

diverse bacteria native to non-laboratory environments. In this study, we applied the

15

ACS Paragon Plus Environment

Page 16 of 50

Page 17 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

332

BTK to modify bacteria found in the honey bee gut microbiome. These species are

333

typical of many other bacteria in natural microbial communities of interest: they have

334

only been cultured recently, are phylogenetically diverse, and have few or no

335

established genetic tools. We validated fundamental BTK components needed for

336

genetic modification, including antibiotic selection markers, conjugation procedures, and

337

promoters to express proteins under constitutive or inducible control. BGM strains

338

engineered with the BTK colonize the guts of newly emerged bees, and fluorescent in

339

vivo imaging revealed a characteristic spatial distribution of each species in the gut.

340

The species we engineered are within the Proteobacteria, a diverse Gram-

341

negative phylum that is a common component of animal- and plant-associated

342

communities. Although we have not yet tested it more broadly, BTK components should

343

also be useful for genetically modifying other bacteria native to other natural

344

communities. The core of the BTK is the RSF1010 plasmid origin, which is known to

345

replicate in diverse bacterial lineages including Cyanobacteria, Agrobacterium, and

346

others.36,37,53,54 The BTK also includes promoters that have previously been shown to

347

function in both Gram-negative and Gram-positive bacteria.42 The E2-Crimson reporter

348

gene fluoresces at far-red excitation wavelengths, which is ideal for in vivo imaging of

349

bacteria through tissue in host-associated systems.39 While broad-host-range plasmids

350

have already been extensively used to study newly isolated bacteria in the past,54 the

351

combinatorial nature of this new toolkit makes it possible to test multiple antibiotic

352

resistance markers and promoters, which are more difficult to replace in plasmids that

353

rely on classical cloning approaches.

16

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

354

Standard part definitions enable researchers to customize a toolkit by adding

355

new capabilities for their own applications, as we did with re-using parts from the yeast

356

toolkit (YTK).15 Several aspects of the BTK could be improved and fleshed out in future

357

work. Separating antibiotic resistance cassettes and replication origins into different

358

subparts and adding to the library of choices available for each function would allow

359

more combinations of antibiotics and origins to be tested when first working with a new

360

species. Gram-positive origins, such as pAMβ1,54 would be especially useful for the

361

manipulation of other common host-associated phyla such as Firmicutes and

362

Actinobacteria.55 Further validation of some BTK parts, such as the dCas9 and Cas9

363

systems, is needed to conclude that they will function reliably across diverse species.

364

Other established broad-host-range tools—such as Tn7-transposon integration46, Group

365

II intron-based gene disruption57, and emerging CRISPR methods for targeted

366

mutagenesis58—could also be incorporated into the BTK-compatible Golden Gate

367

framework in the future.

368

Application of the BTK to engineering bee gut bacteria enables new approaches

369

to microbiome research in these insect species that are important pollinators and model

370

systems for studying social behavior and learning. For example, gene disruption

371

combined with fluorescent visualization of bacterial cells in living bees can be used to

372

improve our understanding of the molecular basis of host-microbe and microbe-microbe

373

interactions and their relevance to host health. The BTK can also be used to implement

374

and test biotechnological approaches for mitigating threats to bee health. For example,

375

it could be used to engineer commensal gut bacteria to degrade pesticides or suppress

17

ACS Paragon Plus Environment

Page 18 of 50

Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

376

pathogen populations (i.e., paratransgenesis)59. These efforts could one day profoundly

377

affect the health of the bee colonies that sustain modern agriculture.

378 379

Methods

380

Bacterial Culture

381

A complete list of bacterial strains used in this work and their sources is available as

382

Table S2. Unless otherwise specified, bacterial strains S. alvi wkB2, G. apicola wkB7,

383

Parasaccharibacter apium wkB6, B. apis PEB0150, G. apicola PEB0183, B. apis

384

PEB0149, and S. alvi PEB0171, S. marcescens N10A28 were grown on Columbia agar

385

supplemented with 5% sterile sheep’s blood (B-COL) and incubated at 35°C in a 5%

386

CO2 atmosphere as static cultures. E. coli were cultured at 37°C with orbital shaking at

387

225 rpm over a 1-inch diameter. E. coli MFDpir was grown in LB supplemented with 0.3

388

mM diaminopimelic acid (DAP). E. coli EC100D and E. coli DH5α were grown in LB.

389

For antibiotic selection, the following concentrations were used: ampicillin (100

390

µg/mL E. coli, 30 µg/mL S. alvi, 30 µg/mL G. apicola, 30 µg/mL B. apis, 300 µg/mL S.

391

marcescens), kanamycin (50 µg/mL E. coli, 20 µg/mL S. alvi, 20 µg/mL G. apicola, 20

392

µg/mL B. apis), spectinomycin (60 µg/mL E. coli, 30 µg/mL for S. alvi, 30 µg/mL G.

393

apicola, 30 µg/mL B. apis, 30 µg/mL P. apium, 180 µg/mL S. marcescens).

394

Biparental Conjugation

395

MFDpir with mobilizable plasmid (“donor strain”) was grown overnight, shaking in LB

396

with appropriate selective antibiotics and DAP (0.3 mM) supplementation. Recipient

397

strains (wkB2, wkB7, PEB0150, PEB0183, PEB0171, N10A28, wkB6, wkB12, Snod 2-

398

15, Pens 2-2-5) were grown overnight on solid media. Recipient and donor strains were

18

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

399

washed in 1 mL PBS, spun down (1006 × g for 5 minutes), and resuspended with 1 mL

400

of PBS. These two suspensions were mixed in a 9:1 OD ratio of recipient:donor and

401

spotted (without filter) onto a B-COL plate supplemented with 0.3 mM DAP.

402

Conjugations proceeded overnight (~12-14 hours) and were scraped from the plate into

403

PBS the next morning. Conjugation mixtures were again gently spun down (1006 × g)

404

and washed twice in PBS to remove residual DAP. Approximately 100 µL of this mixture

405

(and 1:10, 1:100 dilutions) was plated onto selective antibiotic plates and incubated 2-3

406

days to obtain transconjugant colonies. Transconjugants were passaged again on

407

selective media and confirmed by PCR amplification of a plasmid sequence and visible

408

fluorescence, when appropriate. For the initial broad-host-range plasmid screen,

409

transconjugants were further verified by plasmid re-isolation and electroporation into E.

410

coli DH5α cells. To determine conjugation efficiency, mating mixtures were serially

411

diluted and plated on selective and non-selective plates. Conjugation frequency was

412

calculated as the number of fluorescent transconjugant CFUs on selective plates per

413

total CFUs on non-selective plates.

414

BTK construction

415

Construction of the BTK backbone was carried out with Gibson assembly60 following

416

established protocols. New part plasmids were constructed using a previously published

417

BsmBI assembly protocol for the yeast toolkit (YTK)15 with inserts synthesized as

418

double-stranded DNA gBlocks (IDT). New parts were cloned into the pYTK001 entry

419

vector. The BTK kit uses the entry vector plasmid, connector parts (Type 1 and Type 5),

420

and part sequence overhangs of the YTK. In contrast to the YTK, Type 3 parts of the

421

BTK include a stop codon, as the Type 4 terminators do not include a stop codon. The

19

ACS Paragon Plus Environment

Page 20 of 50

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

422

entire list of BTK parts is available in Dataset S1. A complete list of non-BTK plasmids

423

used to generate data for this work is available in Table S3.

424

Measuring BGM GFP in vitro

425

To measure fluorescence, 50 µL of ~0.2 OD bacterial cultures were pooled on B-COL

426

agar plates and incubated for 48 hours. Cells were scraped into PBS and then loaded

427

into wells of a 96 well plate to measure fluorescent excitation using a Tecan Spark 10M

428

multimode microplate reader at excitation/emission wavelengths of 485/535.

429

Fluorescent readings were corrected with blank values, and then normalized by OD.

430

Gain was set manually and consistent throughout experiments.

431

Flow cytometry analysis of GFP expression

432

As with plate reader measurements, we pooled 50 µL of ~0.2 OD bacterial culture onto

433

B-COL agar plates and incubated for 48 hours. Bacteria were scraped into PBS,

434

washed, and then gently spun down (1006 × g for 5 minutes). Cells were resuspended

435

vigorously to disrupt any biofilm, and then diluted to ~0.1 OD in HPLC-grade water. We

436

counterstained cells with SYTO 17 red nucleic acid stain (Thermo-Fisher), and then ran

437

samples on a BD LSRFortessa SORP Flow Cytometer at the UT Austin flow cytometry

438

core. Data were acquired with FACSDiva v6.1.3, and then analyzed with FlowJo

439

v10.4.2. All samples were run under identical conditions. GFP-A voltage was consistent

440

throughout experiments. Non-fluorescent controls were used to determine forward-

441

scatter, side-scatter, and APC-A (counterstain) gates that were then set individually for

442

each species.

443

Tn7-transposition in B. apis

20

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

444

For the chromosomal insertion of gfp into B. apis, a tri-parental mating was performed

445

with B. apis, E. coli MFDpir with pTNS2, and E. coli MFDpir with pTN7-PA1-gfp-kan in

446

an 8:1:1 ratio. Conjugation proceeded for 12 hours, and transformed B. apis was

447

selected with kanamycin as in biparental conjugation.

448

CRISPRi gene repression

449

Broad-host-range dCas9 plasmids are created by BsmBI assembly of 3 parts plasmids

450

containing: (1) the sgRNA transcriptional unit (pBTK615), (2) the dCas9 transcriptional

451

unit (pBTK614), and (3) the broad-host-range backbone with ConLE and ConRE

452

connector sequences (pBTK527a). To repress gfp expression in B. apis, we targeted

453

the gfp non-template strand by using the N20 sequence: 5′–

454

CGTCTAATTCCACGAGGATT. The sgRNA plasmid can be retargeted using

455

MEGAWHOP cloning47. Briefly, in MEGAWHOP cloning a double-stranded PCR

456

product containing the sequence change to be introduced, but otherwise identical to a

457

portion of the plasmid, is used as a “megaprimer” to re-amplify the whole plasmid in a

458

second PCR reaction. Because the sgRNA targeting sequence is short, it is possible to

459

include a new target sequence flanked by 20 bp of homology to the plasmid on either

460

side in one of the primers used in the initial PCR reaction to generate the megaprimer.

461

The fully assembled CRISPRi plasmid (pBTK618) was conjugated into B. apis with

462

chromosomally integrated PA1-gfp, and GFP fluorescence was measured as above.

463

Chromosomal disruption using Cas9 and homologous recombination

464

Plasmid pBTK601 contains Cas9 driven by the kanamycin resistance gene promoter on

465

the broad-host-range backbone. This plasmid was conjugated into S. alvi wkB2, G.

466

apicola wkB7, and B. apis PEB0150 and maintained with spectinomycin. The CP25-

21

ACS Paragon Plus Environment

Page 22 of 50

Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

467

driven sgRNA is on plasmid pBTK615 and can be retargeted using MEGAWHOP

468

cloning.47 A full description of homology donor plasmid assembly is available in

469

Supplemental Figure S4. Briefly, a genomic homology segment upstream of the gene

470

of interest to disrupt or replace is amplified with Type 2 part overhangs, and a

471

downstream genomic homology segment is amplified as a Type 4 part. Upstream

472

homology, antibiotic resistance cassette (Type 3), and downstream homology are

473

combined in a single BsaI reaction with ConLE and ConRE to form a Stage 1 assembly

474

of the replacement cassette. The final BsmBI assembly includes: (1) the sgRNA

475

plasmid, (2) the replacement cassette plasmid, and (3) pBTK599 (R6K suicide plasmid

476

backbone). This final assembly must be transformed into pir+ strains, such as EC100D

477

or MFDpir.

478

Efficiency of chromosomal disruption with and without Cas9

479

Recipient BGM strains (wkB2, wkB2::pBTK601, wkB7, wkB7::pBTK601, PEB0150,

480

PEB0150::pBTK601) were grown on B-COL plates for 48 hours prior to conjugation.

481

Donor E. coli strains were grown in liquid culture overnight prior to conjugation. Donor

482

and recipients were washed in PBS and mixed in a 1:9 ratio (by OD), and 100 µL was

483

plated on B-COL + 0.3 mM DAP media for overnight conjugation. After 14 hours, the

484

entire conjugation mixtures were scraped into PBS and washed twice to remove

485

residual DAP, and dilutions were plated on selective agar plates (B-COL + Kanamycin

486

20 µg/mL) and non-selective agar plates (B-COL). Efficiency of gene disruption was

487

calculated as (# of transconjugant cells) / (# of total cells). To identify single-crossover

488

and double-crossover mutants, a series of PCR reactions were conducted as described

489

in Supplemental figure S4. Briefly, transconjugants were screened for the appropriate

22

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

490

upstream and downstream junctions with colony PCR. Potential double-crossover

491

mutants were then further screened for the size of the disrupted region, and loss of the

492

suicide plasmid backbone.

493

Laboratory care of honey bees

494

Microbiota-free bees were obtained and raised using methods described previously.31

495

Briefly, pupae were pulled under sterile conditions from brood combs obtained from

496

outdoor hives. These pupae emerged in a sterile incubator (becoming newly emerged

497

adult workers) and were then sorted into individual cup cages for further development in

498

the laboratory. Prior to inoculation, newly emerged workers were allowed to feed on

499

sterile irradiated pollen (Betterbee) and 50% sucrose solution ad libitum. For any

500

individual experiment, all pupae were obtained from the same hive. When raised in this

501

manner, Apis mellifera workers remain uncolonized by core BGM bacteria species and

502

show very low levels of environmental bacteria in their guts.27 It is critical to pull the

503

pupae from frames at an early stage, before the mouthparts have hardened, as later

504

pupal stages will begin to ingest hive material and may be colonized.

505

Mono- and Co-inoculation of engineered BGM into honey bees

506

After obtaining newly emerged workers, bees were chilled at 4°C for 30 minutes and

507

then coated in sugar syrup containing resuspended bacterial inoculum, transferred to

508

cup cages, and allowed to groom each other. The inoculum generally contained 200 µL

509

of OD ~0.1 bacterial suspension combined with 800 µL of 1:1 sucrose:water solution.

510

Approximately 30 µL of this solution per bee was used for inoculations (corresponding

511

to 104 bacteria per bee to ensure robust inoculation). Plate counts of the inoculum were

512

used to confirm concentrations.

23

ACS Paragon Plus Environment

Page 24 of 50

Page 25 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

513

In vivo imaging of bacterial burden using E2-Crimson

514

To visualize in vivo expression of E2-Crimson in living bees, we used a Syngene G:Box

515

Chemi XX6 gel doc system at the UT ICMB Microscopy Core. Bees were chilled on ice

516

for 30 min to minimize movement, then imaged using manufacturers recommended

517

instructions for far-red fluorescent probe visualization: “Red LED” light source and “Filter

518

705M” emission filter. All bees were imaged under identical conditions: 5 minutes

519

exposure time for whole bee and 30 seconds for bees with dissected guts. Images were

520

saved as TIFF files for further analysis in FIJI.61 In FIJI, fluorescence intensity was

521

mapped to the “mpl-magma” scale. A representative bee for each condition is shown.

522

No further image manipulation was performed. Different scales are used for comparing

523

fluorescent S. marcescens and fluorescent BGM species due to the increased

524

fluorescent protein production and titer of S. marcescens.

525

Confocal fluorescence microscopy

526

Fluorescent images were obtained at the UT ICMB Microscopy core on a Zeiss 710

527

Laser Scanning Confocal microscope. Bees were chilled and then dissected to expose

528

rectum, ileum, and midgut. Without puncturing the gut, the entire gut compartment was

529

transferred to an Ibidi µ-Dish 35 mm (CAT #81156) and then placed on the microscope.

530

Images were taken with a 20× objective and tiled using Zeiss software. Z-stack 2-

531

channel fluorescent images were taken and combined using Imaris software. Intensity

532

on individual channels was false colored to correspond to species-specific coloring.

533

Display intensity of individual channels was scaled linearly to aid in visualization of

534

different species, but no further transformations or background reduction was used.

535

qPCR to assess colonization of staA mutant

24

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

536

Absolute quantification of 16S rRNA gene copies specific to S. alvi was performed as

537

described previously33. Cohorts of newly emerged bees were hand-fed with equal

538

amounts (~104 CFU/bee) of either wild-type S. alvi or the staA mutant. Control bees

539

were maintained identically but remained uninoculated. After five days, five bees from

540

each group were dissected and DNA was isolated from individual bee guts using the

541

cetyltrimethylammonium bromide (CTAB) extraction method outlined previously27. After

542

extraction, S. alvi-specific primers were used for quantitative PCR and absolute

543

quantification based on 10-fold dilution of the target sequence in a pGEM-T plasmid

544

vector. Reactions were run in triplicate.

545

Quantification and Statistical Analysis

546

All data processing and statistical analyses were done in R. Kruskal-Wallis rank sum

547

tests were used to assess significance in the dCas9 gene repression experiment and

548

the Cas9-assisted genome modification experiments.

549 550

Competing Interests

551

N.A.M, J.E.B., and S.P.L. have filed a provisional patent application (62/529,754) for

552

engineering bee gut bacteria with BTK components to improve bee health.

25

ACS Paragon Plus Environment

Page 26 of 50

Page 27 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

553

Supporting Information

554

Supplementary Tables and Figures

555

Supplementary Table S1. Broad-host-range plasmid screen in bee gut bacteria

556

Supplementary Table S2. Bacterial strains

557

Supplementary Table S3. Non-BTK plasmids

558

Supplementary Table S4. Oligonucleotides

559

Supplementary Table S5. Assembled BTK plasmids

560

Supplementary Figure S1. Expression of Nanoluc in BGM strains

561

Supplementary Figure S2. Expression of E2-Crimson in BGM strains

562

Supplementary Figure S3. Validation of additional BTK promoters in E. coli

563

Supplementary Figure S4. Construction of replacement cassette plasmids

564

Supplementary Figure S5. Schematic and validation of staA disruption in S. alvi

565

Supplementary Figure S6. Schematic and validation of ackA disruption in G.

566

apicola

567

Supplementary Figure S7. Schematic and validation of narG disruption in B. apis

568

Supplementary References

569

Supplementary Dataset S1. BTK parts

570

Supplementary Dataset S2. Complete sequences for BTK plasmids

571 572

Abbreviations

573

BTK – Bee microbiome toolkit

574

BHR – Broad-host-range

575

BGM – Bee gut microbiome

26

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

576 577

Author Contributions

578

All authors conceived the study. JP designed the BTK assembly scheme. SPL, JP, JEP,

579

MB, SK, PG, DR conducted experiments. SPL, JEB, and NAM wrote the manuscript.

580

SPL and JEP performed data analysis and imaging. All authors participated in

581

manuscript revision.

582 583

Acknowledgements

584

We thank members of the Moran, Barrick, Davies, and Ellington laboratories for

585

discussion and ideas. Special thanks to Kim Hammond for care and maintenance of

586

bee hives and laboratory equipment. Thanks to Dan Deatherage for assistance with the

587

Flow Cytometry experiment. Thanks to Vidya Pandarinath and Kate Elston for help

588

organizing BTK parts for submission to Addgene. Thanks to Waldan Kwong, Phillip

589

Engel and Hauke Koch for bacterial strains used in this study. Thanks to Julie Hayes

590

and Anna Webb of the UT Microscopy and Imaging Facility for confocal microscopy

591

assistance and instruction.

592 593

Funding

594

This work was supported by DARPA (HR0011-15-C0095 to NAM, ADE, BWD, JEB) and

595

the NIH (1R01GM108477-01 to NAM).

596 597 598

References

27

ACS Paragon Plus Environment

Page 28 of 50

Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

599

(1) Sommer, F., and Bäckhed, F. (2013) The gut microbiota — masters of host

600

development and physiology. Nat. Rev. Microbiol. 11, 227–238.

601

(2) Shin, S. C., Kim, S.-H., You, H., Kim, B., Kim, A. C., Lee, K.-A., Yoon, J.-H., Ryu, J.-

602

H., and Lee, W.-J. (2011) Drosophila microbiome modulates host developmental and

603

metabolic homeostasis via insulin signaling. Science 334, 670–674.

604

(3) Shen, T.-C. D., Albenberg, L., Bittinger, K., Chehoud, C., Chen, Y.-Y., Judge, C. A.,

605

Chau, L., Ni, J., Sheng, M., Lin, A., Wilkins, B. J., Buza, E. L., Lewis, J. D., Daikhin, Y.,

606

Nissim, I., Yudkoff, M., Bushman, F. D., and Wu, G. D. (2015) Engineering the gut

607

microbiota to treat hyperammonemia. J. Clin. Invest. 125, 2841–2850.

608

(4) Palmer, J. D., Piattelli, E., McCormick, B. A., Silby, M. W., Brigham, C. J., and Bucci,

609

V. (2017) Engineered probiotic for the inhibition of Salmonella via tetrathionate-Induced

610

production of microcin H47. ACS Infect. Dis. 4, 39–45

611

(5) Mimee, M., Citorik, R. J., and Lu, T. K. (2016) Microbiome therapeutics — advances

612

and challenges. Adv. Drug Delivery Rev. 105, 44–54.

613

(6) Daeffler, K. N.-M., Galley, J. D., Sheth, R. U., Ortiz-Velez, L. C., Bibb, C. O.,

614

Shroyer, N. F., Britton, R. A., and Tabor, J. J. (2017) Engineering bacterial thiosulfate

615

and tetrathionate sensors for detecting gut inflammation. Mol. Syst. Biol. 13, 923.

616

(7) Riglar, D. T., Giessen, T. W., Baym, M., Kerns, S. J., Niederhuber, M. J., Bronson,

617

R. T., Kotula, J. W., Gerber, G. K., Way, J. C., and Silver, P. A. (2017) Engineered

618

bacteria can function in the mammalian gut long-term as live diagnostics of

619

inflammation. Nat. Biotechnol. 105, 1–8.

620

(8) Kotula, J. W., Kerns, S. J., Shaket, L. A., Siraj, L., Collins, J. J., Way, J. C., and

621

Silver, P. A. (2014) Programmable bacteria detect and record an environmental signal

28

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

622

in the mammalian gut. Proc. Natl. Acad. Sci. U. S. A. 111, 4838–4843.

623

(9) Mimee, M., Tucker, A. C., Voigt, C. A., and Lu, T. K. (2015) Programming a human

624

commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in

625

the murine gut microbiota. Cell Syst. 1, 62–71.

626

(10) Abrieux, A., and Chiu, J. C. (2016) Oral delivery of dsRNA by microbes: Beyond

627

pest control. Commun. Integr. Biol. 9, e1236163.

628

(11) Wang, S., Dos-Santos, A. L. A., Huang, W., Liu, K. C., Oshaghi, M. A., Wei, G.,

629

Agre, P., and Jacobs-Lorena, M. (2017) Driving mosquito refractoriness to Plasmodium

630

falciparum with engineered symbiotic bacteria. Science 357, 1399–1402.

631

(12) Wang, S., Ghosh, A. K., Bongio, N., Stebbings, K. A., Lampe, D. J., and Jacobs-

632

Lorena, M. (2012) Fighting malaria with engineered symbiotic bacteria from vector

633

mosquitoes. Proc. Natl. Acad. Sci. U. S. A. 109, 12734–12739.

634

(13) Iverson, S. V., Haddock, T. L., Beal, J., and Densmore, D. M. (2015) CIDAR

635

MoClo: improved MoClo assembly standard and new E. coli part library enable rapid

636

combinatorial design for synthetic and traditional biology. ACS Synth. Biol. 5, 99–103.

637

(14) Moore, S. J., Lai, H.-E., Kelwick, R. J. R., Chee, S. M., Bell, D. J., Polizzi, K. M.,

638

and Freemont, P. S. (2016) EcoFlex: a multifunctional MoClo kit for E. coli synthetic

639

biology. ACS Synth. Biol. 10, 1059-1069

640

(15) Lee, M. E., DeLoache, W. C., Cervantes, B., and Dueber, J. E. (2015) A highly

641

characterized yeast toolkit for modular, multipart assembly. ACS Synth. Biol. 4, 975–

642

986.

643

(16) Nielsen, A. A. K., Der, B. S., Shin, J., Vaidyanathan, P., Paralanov, V., Strychalski,

644

E. A., Ross, D., Densmore, D., and Voigt, C. A. (2016) Genetic circuit design

29

ACS Paragon Plus Environment

Page 30 of 50

Page 31 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

645

automation. Science 352, aac7341–aac7341.

646

(17) Salyers, A. A., Shoemaker, N. B., and Guthrie, E. P. (1987) Recent advances in

647

Bacteroides genetics. Crit. Rev. Microbiol. 14, 49–71.

648

(18) Lim, B., Zimmermann, M., Barry, N. A., and Goodman, A. L. (2017) Engineered

649

regulatory systems modulate gene expression of human commensals in the gut. Cell

650

169, 547–558.

651

(19) Whitaker, W. R., Shepherd, E. S., and Sonnenburg, J. L. (2017) Tunable

652

expression tools enable single-cell strain distinction in the gut microbiome. Cell 169,

653

538–538.e12.

654

(20) Salyers, A. A., Bonheyo, G., and Shoemaker, N. B. (2000) Starting a new genetic

655

system: lessons from Bacteroides. Methods 20, 35–46.

656

(21) Cox-Foster, D. L., Conlan, S., Holmes, E. C., Palacios, G., Evans, J. D., Moran, N.

657

A., Quan, P.-L., Briese, T., Hornig, M., Geiser, D. M., Martinson, V., vanEngelsdorp, D.,

658

Kalkstein, A. L., Drysdale, A., Hui, J., Zhai, J., Cui, L., Hutchison, S. K., Simons, J. F.,

659

Egholm, M., Pettis, J. S., and Lipkin, W. I. (2007) A metagenomic survey of microbes in

660

honey bee colony collapse disorder. Science 318, 283–287.

661

(22) Kwong, W. K., and Moran, N. A. (2016) Gut microbial communities of social bees.

662

Nat. Rev. Microbiol. 14, 374–384.

663

(23) Kwong, W. K., and Moran, N. A. (2013) Cultivation and characterization of the gut

664

symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov.,

665

sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and

666

Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord.

30

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

667

nov., a sister taxon to the order “Enterobacteriales” of the Gammaproteobacteria. Int. J.

668

Syst. Evol. Microbiol. 63, 2008–2018.

669

(24) Moran, N. A., Hansen, A. K., Powell, J. E., and Sabree, Z. L. (2012) Distinctive gut

670

microbiota of honey bees assessed using deep sampling from individual worker bees.

671

PLoS One 7, e36393.

672

(25) Zheng, H., Powell, J. E., Steele, M. I., Dietrich, C., and Moran, N. A. (2017)

673

Honeybee gut microbiota promotes host weight gain via bacterial metabolism and

674

hormonal signaling. Proc. Natl. Acad. Sci. U. S. A. 114, 4775–4780.

675

(26) Raymann, K., Shaffer, Z., and Moran, N. A. (2017) Antibiotic exposure perturbs the

676

gut microbiota and elevates mortality in honeybees. PLoS Biol. 15, e2001861.

677

(27) Powell, J. E., Martinson, V. G., Urban-Mead, K., and Moran, N. A. (2014) Routes of

678

acquisition of the gut microbiota of the honey bee Apis mellifera. Appl. Environ.

679

Microbiol. 80, 7378–7387.

680

(28) Ludvigsen, J., Porcellato, D., L'Abée-Lund, T. M., Amdam, G. V., and Rudi, K.

681

(2017) Geographically widespread honeybee-gut symbiont subgroups show locally

682

distinct antibiotic-resistant patterns. Mol. Ecol. 26, 6590–6607.

683

(29) Tian, B., Fadhil, N. H., Powell, J. E., Kwong, W. K., and Moran, N. A. (2012) Long-

684

term exposure to antibiotics has caused accumulation of resistance determinants in the

685

gut microbiota of honeybees. mBio 3, e00377–12.

686

(30) Zheng, H., Nishida, A., Kwong, W. K., Koch, H., Engel, P., Steele, M. I., and Moran,

687

N. A. (2016) Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella

688

apicola. mBio 7, e01326–16.

31

ACS Paragon Plus Environment

Page 32 of 50

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

689

(31) Kwong, W. K., Engel, P., Koch, H., and Moran, N. A. (2014) Genomics and host

690

specialization of honey bee and bumble bee gut symbionts. Proc. Natl. Acad. Sci. U. S.

691

A.111, 11509–11514.

692

(32) Segers, F. H., Kešnerová, L., Kosoy, M., and Engel, P. (2017) Genomic changes

693

associated with the evolutionary transition of an insect gut symbiont into a blood-borne

694

pathogen. ISME J. 60, 810.

695

(33) Powell, J. E., Leonard, S. P., Kwong, W. K., Engel, P., and Moran, N. A. (2016)

696

Genome-wide screen identifies host colonization determinants in a bacterial gut

697

symbiont. Proc. Natl. Acad. Sci. U. S. A. 113, 13887–13892.

698

(34) Burritt, N. L., Foss, N. J., Neeno-Eckwall, E. C., and Church, J. O. (2016) Sepsis

699

and hemocyte loss in honey bees (Apis mellifera) infected with Serratia marcescens

700

strain Sicaria. PLoS One. 11, e0167752.

701

(35) Fürste, J. P., Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M.,

702

and Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-

703

host-range tacP expression vector. Gene 48, 119–131.

704

(36) Jain, A., and Srivastava, P. (2013) Broad host range plasmids. FEMS Microbiol.

705

Lett. 348, 87–96.

706

(37) Meyer, R. (2009) Replication and conjugative mobilization of broad host-range

707

IncQ plasmids. Plasmid 62, 57–70.

708

(38) Ferrières, L., Hémery, G., Nham, T., Guérout, A.-M., Mazel, D., Beloin, C., and

709

Ghigo, J.-M. (2010) Silent mischief: bacteriophage Mu insertions contaminate products

710

of Escherichia coli random mutagenesis performed using suicidal transposon delivery

32

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

711

plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192,

712

6418–6427.

713

(39) Strack, R. L., Hein, B., Bhattacharyya, D., Hell, S. W., Keenan, R. J., and Glick, B.

714

S. (2009) A rapidly maturing far-red derivative of dsRed-Express2 for whole-cell

715

labeling. Biochemistry 48, 8279–8281.

716

(40) Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G.,

717

Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A.,

718

Eggers, C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P.,

719

and Wood, K. V. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing

720

a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857.

721

(41) Siebenlist, U. (1979) Nucleotide sequence of the three major early promoters of

722

bacteriophage T7. Nucleic Acids Res. 6, 1895–1907.

723

(42) Jensen, P. R., and Hammer, K. (1998) The sequence of spacers between the

724

consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ.

725

Microbiol. 64, 82–87.

726

(43) Sleight, S. C., Bartley, B. A., Lieviant, J. A., and Sauro, H. M. (2010) Designing and

727

engineering evolutionary robust genetic circuits. J. Biol. Eng. 4, 12.

728

(44) Barrangou, R., and Horvath, P. (2017) A decade of discovery: CRISPR functions

729

and applications. Nat. Microbiol. 2, 1–9.

730

(45) Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., and Marraffini, L. A.

731

(2013) Programmable repression and activation of bacterial gene expression using an

732

engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437.

33

ACS Paragon Plus Environment

Page 34 of 50

Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

733

(46) Choi, K.-H., Gaynor, J. B., White, K. G., Lopez, C., Bosio, C. M., Karkhoff-

734

Schweizer, R. R., and Schweizer, H. P. (2005) A Tn7-based broad-range bacterial

735

cloning and expression system. Nat. Meth. 2, 443–448.

736

(47) Miyazaki, K. (2011) MEGAWHOP cloning: a method of creating random

737

mutagenesis libraries via megaprimer PCR of whole plasmids. Meth. Enz. 498, 399–

738

406.

739

(48) Martinson, V. G., Moy, J., and Moran, N. A. (2012) Establishment of characteristic

740

gut bacteria during development of the honeybee worker. Appl. Environ. Microbiol. 78,

741

2830–2840.

742

(49) Engel, P., James, R. R., Koga, R., Kwong, W. K., McFrederick, Q. S., and Moran,

743

N. A. (2013) Standard methods for research on Apis mellifera gut symbionts. J. of Api.

744

Res. 52, 1–24.

745

(50) Linke, D., Riess, T., Autenrieth, I. B., Lupas, A., and Kempf, V. A. J. (2006) Trimeric

746

autotransporter adhesins: variable structure, common function. Trends Microbiol. 14,

747

264–270.

748

(51) Ribet, D., and Cossart, P. (2015) How bacterial pathogens colonize their hosts and

749

invade deeper tissues. Microbes Infect. 17, 173–183.

750

(52) Tahir, El, Y., and Skurnik, M. (2001) YadA, the multifaceted Yersinia adhesin. Int. J.

751

Med. Microbiol. 291, 209–218.

752

(53) Taton, A., Unglaub, F., Wright, N. E., Zeng, W. Y., Paz-Yepez, J., Brahamsha, B.,

753

Palenik, B., Peterson, T. C., Haerizadeh, F., Golden, S. S., and Golden, J. W. (2014)

754

Broad-host-range vector system for synthetic biology and biotechnology in

755

cyanobacteria. Nucleic Acids Res. 42, gku673–e136.

34

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

756

(54) pAMB1 citeClewell, D. B., Yagi, Y., Dunny, G. M., Schultz, S. K. (1974)

757

Characterization of three plasmid deoxyribonucleic acid molecules in a strain of

758

Streptococcus faecaelis: identification of a plasmid determining erythromycin resistance.

759

J. Bacteriol. 117, 283–289.

760

(55) Robinson, C. J., Bohannan, J. M., Young, V. B. (2010) From structure to function:

761

the ecology of host-associated microbial communities. Microbiol. Mol. Biol. Rev. 74,

762

453–476.

763

(56) Martínez-García, E., Aparicio, T., Goñi-Moreno, A., Fraile, S., and de Lorenzo, V.

764

(2015) SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-

765

construction of bacterial functionalities. Nucleic Acids Res. 43, D1183–D1189.

766

(57) Enyeart, P. J., Chirieleison, S. M., Dao, M. N., Perutka, J., Quandt, E. M., Yao, J.,

767

Whitt, J. T., Keatinge-Clay, A. T., Lambowitz, A. M., and Ellington, A. D. (2013)

768

Generalized bacterial genome editing using mobile group II introns and Cre‐lox. Mol.

769

Syst. Biol. 9, 685–685.

770

(58) Banno, S., Nishida, K., Arazoe, T., Mitsunobu, H., and Kondo, A. (2018)

771

Deaminase-mediated multiplex genome editing in Escherichia coli. Nat. Microbiol. 339,

772

819.

773

(59) Rangberg, A., Diep, D. B., Rudi, K., and Amdam, G. V. (2012) Paratransgenesis:

774

an approach to improve colony health and molecular insight in honey bees (Apis

775

mellifera)? Integr. Comp. Biol. 52, 89–99.

776

(60) Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and

777

Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred

778

kilobases. Nat. Meth. 6, 343–345.

35

ACS Paragon Plus Environment

Page 36 of 50

Page 37 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

779

(61) Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T.,

780

Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J.,

781

Hartenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A. (2012) Fiji: an open-source

782

platform for biological-image analysis. Nat. Meth. 9, 676–682.

783

(62) Galdzicki, M., Clancy, K. P., Oberortner, E., Pocock, M., Quinn, J. Y., Rodriguez, C.

784

A., Roehner, N., Wilson, M. L., Adam, L., Anderson, J. C., Bartley, B. A., Beal, J.,

785

Chandran, D., Chen, J., Densmore, D., Endy, D., Grünberg, R., Hallinan, J., Hillson, N.

786

J., Johnson, J. D., Kuchinsky, A., Lux, M., Misirli, G., Peccoud, J., Plahar, H. A., Sirin,

787

E., Stan, G.-B., Villalobos, A., Wipat, A., Gennari, J. H., Myers, C. J., and Sauro, H. M.

788

(2014) The Synthetic Biology Open Language (SBOL) provides a community standard

789

for communicating designs in synthetic biology. Nat. Biotechnol. 32, 545–550.

790

36

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

791

Tables and Figures

792 793

Figure 1. Design of the bee microbiome toolkit (BTK) and schematic assembly. (A) The

794

BTK was designed for Golden Gate assembly according to a scheme with eight part

795

types compatible with the yeast toolkit (YTK).15 Parts of each type generated in this

796

study are shown in the top panel. Type 1-5 and Type 8 parts are defined as in the YTK

797

except that Type 3 open-reading frames include the stop codon. Type 6 and 7 parts are

798

either replaced with a linker part or used to incorporate a reverse reading frame

799

encoding a transcriptional regulator for inducible expression of the main Type 3 open-

800

reading frame and its promoter, respectively, during Stage 1 assembly, so that costly or

801

toxic genes can be repressed while they are assembled into transcriptional units. (B)

37

ACS Paragon Plus Environment

Page 38 of 50

Page 39 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

802

Schematic of Stage 1 (BsaI) assembly. Plasmid parts are shown, but PCR products with

803

appropriate overhangs can be substituted. (C) Schematic Stage 2 (BsmBI) assembly.

804

Compatible Stage 2 connectors are described in the YTK documentation.

38

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

805

806 807

Figure 2. The bee microbiome toolkit (BTK) functions in diverse bee-associated

808

bacteria. (A) Replication of the BTK backbone and the function of three antibiotic

809

resistance cassettes were tested in eight honey bee-associated bacterial strains as

810

described in the Methods. At least one antibiotic resistance cassette functioned in each

811

strain, and the kanamycin cassette functioned in all eight strains. (B) Replication of the

39

ACS Paragon Plus Environment

Page 40 of 50

Page 41 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

812

BTK backbone and the function of three antibiotic resistance cassettes were tested in

813

three bumble bee-associated bacterial strains. Again, the plasmid with kanamycin

814

resistance was maintained in all three bacteria. (C) Conjugation frequency in four bee

815

gut-associated strains. Black bars are the geometric mean and each point is an

816

independent conjugation. Conjugation in B. apis is the most efficient, with conjugation

817

efficiency approximately 10%.

40

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

818 819

Figure 3. Constitutive and inducible control of in vitro gene expression in bee gut

820

bacteria. (A) Flow cytometry results of GFP fluorescence from four broad-host-range

821

promoters in each of four honey bee-associated bacterial strains and an E. coli control.

822

One representative fluorescence distribution for each promoter is shown, with the

823

medians from three biological replicates plotted as open circles. Spotted grey line

824

indicates maximum detected fluorescence in wild-type cells. Median fluorescent values

825

were calculated from cells more fluorescent than wild-type. (B) GFP fluorescence from a

826

designed CP25 (lacO) promoter at different levels of IPTG-induction, measured in four

827

BGM strains and an E. coli control. All tested species are responsive to IPTG induction,

828

and G. apicola shows the highest expression across all strains. (C) GFP fluorescence

829

from a T7 (lacO) promoter at different levels of IPTG-induction of T7 RNAP expression

830

in the same four BGM strains. Schematics in (A)—(C) show the design of tested 41

ACS Paragon Plus Environment

Page 42 of 50

Page 43 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

831

constructs using Synthetic Biology Open Language (SBOL) standard glyphs.62 Error

832

bars are standard deviations (n = 3).

42

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

833 834

Figure 4. dCas9 gene silencing in Bartonella apis. (A) Schematic assembly of dCas9

835

plasmids for gene suppression. (B) Fluorescence from chromosomally integrated GFP

836

in PEB0150 in the presence and absence of dCas9 and sgRNA targeting GFP.

837

Background fluorescence of wild-type PEB0150 was subtracted. GFP fluorescence

838

decreased in presence of dCas9 targeting GFP (p = 0.004, Kruskal-Wallis rank sum

839

test). Error bars are 95% confidence intervals (n = 4).

43

ACS Paragon Plus Environment

Page 44 of 50

Page 45 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

840 841

Figure 5. Cas9 assisted gene disruption in species from the bee gut microbiota. (A)

842

Schematic assembly of R6K-based suicide plasmids. Assembly strategy and validation

843

primers are described in Supplementary Figure S4. (B) The two tested approaches for

844

gene disruption. The suicide plasmids were introduced into either wild-type bacteria or

845

bacteria possessing the constitutively active Cas9 (pBTK601). (C) Transconjugation

846

frequency and percent of desired mutants in B. apis, in the presence and absence of

847

Cas9. The Cas9 plasmid did not increase the efficiency of genome modification.

848

Numbers above each bar indicate the number of clones evaluated. (D)

849

Transconjugation frequency and proportion of desired mutants in S. alvi. S. alvi wkB2

850

showed increased efficiency of genome modification in the presence of the Cas9

851

plasmid (p = 0.0007, Kruskal-Wallis rank sum test). (E) Transconjugation frequency and

44

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

852

proportion of desired mutants in G. apicola. Each point in C-E is from an independent

853

conjugation experiment. Bars in C-E represent the geometric mean of estimated

854

transconjugation efficiencies.

45

ACS Paragon Plus Environment

Page 46 of 50

Page 47 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

855 856

Figure 6. Visualization of engineered bacteria in the honey bee gut. (A) Intact honey

857

bee worker and dissection of honey bee gut showing brightfield microscopy of midgut,

858

ileum, and rectum. (B) Fluorescent imaging of whole bee (left) and dissected bee (right)

859

5 days after inoculation with S. marcescens N10A28 expressing E2-Crimson (plasmid

860

pBTK570). Control bee is uninoculated. Color corresponds to pixel fluorescence

861

intensity. Engineered S. marcescens N10A28 is present in the midgut, ileum, and

862

rectum. (C) Similar to (B), with S. alvi wkB2 expressing E2-Crimson as inoculum.

863

Control bee is identical to (B), but different fluorescent intensity scales are used for

864

comparison between bees inoculated with S. alvi and S. marcescens. Engineered S.

865

alvi wkB2 is visibly fluorescent in the midgut and ileum. (D) Confocal imaging of partial

46

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

866

ileum and rectum in bees inoculated with S. marcescens N10A28 expressing E2-

867

Crimson (red). As in (B), S. marcescens can be seen robustly colonizing throughout the

868

ileum and rectum. (E) Similar to (D), with S. alvi wkB2 expressing E2-Crimson (green).

869

Snodgrassella alvi wkB2 colonizes the ileum, but not the rectum. Scale bars in (D) and

870

(E) are 100 µm. Images are representative of multiple bees inspected (n = 3-5 per

871

condition) for (B–E). White and black arrows correspond to the ileum-rectum junction

872

across images (A–E).

873

47

ACS Paragon Plus Environment

Page 48 of 50

Page 49 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

874 875

Figure 7. Visible co-inoculation of the bee gut with species from the bee gut microbiota

876

and the role of staA in colonization. (A) The ileum-rectum junction imaged by confocal

877

fluorescence microscopy 5 days after co-inoculating B. apis PEB0150 (purple) and S.

878

alvi wkB2 (green). When co-inoculated, B. apis and S. alvi are co-located in the ileum,

879

but only B. apis colonizes the rectum. (B) Similar to (A), with images taken 5 days after

880

co-inoculation of G. apicola wkB7 (blue) and S. alvi wkB2 (green). As in (A), S. alvi

881

remains restricted to the ileum, while G. apicola is present in both ileum and rectum.

882

Scale bars are 100 µm. Images are representative of multiple bees inspected (n = 3 per

883

condition). (C) The number of S. alvi 16S ribosomal DNA copies 5 days after inoculating

48

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

884

newly emerged worker bees with the S. alvi WT or ∆staA mutant, based on quantitative

885

PCR. Horizontal bars represent means per condition (n = 5). The ∆staA has a significant

886

colonization defect compared to WT (p = 2.7 × 10-6, Kruskall-Wallis rank sum test). (D)

887

The ileums of bees inoculated with S. alvi wkB2∆staA expressing E2-Crimson

888

(pBTK570) were imaged 5 days after colonization. Mutants achieved lower colonization

889

levels than did S. alvi WT (see Fig. 6E). Localized colonization was typical of multiple

890

ileums inspected (n = 3). Scale bar is 100 µm.

49

ACS Paragon Plus Environment

Page 50 of 50