A

Polar Positive

<figure><img src="image_15.png" /></figure>

B

Polar Negative

<figure><img src="image_16.png" /></figure>

C

Nonpolar Positive

<figure><img src="image_17.png" /></figure>

D

Nonpolar Negative

<figure><img src="image_18.png" /></figure>

Membership

CsPs

BtPs

BtCs

BtCsPs

Time (h)

○ 12.5

○ 25

○ 30

○ 35

○ 40

○ 45

1384

1385
A

Polar Positive

<figure><img src="image_16.png" /></figure>

B

Polar Negative

<figure><img src="image_17.png" /></figure>

C

Nonpolar Positive

<figure><img src="image_18.png" /></figure>

D

Nonpolar Negative

<figure><img src="image_19.png" /></figure>

Membership

CsPs

BtPs

BtCs

BtCsPs

Time (h)

○ 12.5

○ 25

○ 30

○ 35

○ 40

○ 45

1384

1385
**Figure 5.** Primary metabolites accumulated in monocultures have altered dynamics in cocultures. A heat map of identified, primary metabolites is shown for C. *subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), *B. thailandensis* monoculture (Bt), *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. These exometabolites were filtered based on their time series accumulation in monocultures (See supplementary methods for details). Data for each sample are the averages from independent time point replicates (n = 3 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.
**Figure 5.** Primary metabolites accumulated in monocultures have altered dynamics in cocultures. A heat map of identified, primary metabolites is shown for C. *subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), *B. thailandensis* monoculture (Bt), *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. These exometabolites were filtered based on their time series accumulation in monocultures (See supplementary methods for details). Data for each sample are the averages from independent time point replicates (n = 3 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.
<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /></figure>
**Figure 6.** *B. thailandensis* upregulates biosynthetic gene clusters (BSGC) in cocultures. Columns represent community membership for *B. thailandensis* cocultures and rows represent BSGCs in *B. thailandensis* that were determined to be upregulated compared to the monoculture control. Genes part of a BSGC were curated from antiSMASH predictions and literature-based evidence. Within each BSGC at each time point, the log2 fold-change (LFC) was calculated by comparing gene counts from a coculture to the monoculture control (n = 3 to 4 LFC calculations/community membership/time point). Log2 fold-changes were then averaged from all biosynthetic genes within the BSGC at each time point. Error bars indicate standard deviations. We defined an upregulated BSGC as a BSGC that had at least two consecutive stationary phase time points with a LFCs > 1 (indicated by the horizontal red dashed line). Note that plots for each BSGC have separate scales for the Y-axis.
**Figure 6.** *B. thailandensis* upregulates biosynthetic gene clusters (BSGC) in cocultures. Columns represent community membership for *B. thailandensis* cocultures and rows represent BSGCs in *B. thailandensis* that were determined to be upregulated compared to the monoculture control. Genes part of a BSGC were curated from antiSMASH predictions and literature-based evidence. Within each BSGC at each time point, the log2 fold-change (LFC) was calculated by comparing gene counts from a coculture to the monoculture control (n = 3 to 4 LFC calculations/community membership/time point). Log2 fold-changes were then averaged from all biosynthetic genes within the BSGC at each time point. Error bars indicate standard deviations. We defined an upregulated BSGC as a BSGC that had at least two consecutive stationary phase time points with a LFCs > 1 (indicated by the horizontal red dashed line). Note that plots for each BSGC have separate scales for the Y-axis.
<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /></figure>
**Figure 7.** Coculture upregulation of BSGCs from *B. thailandensis* translates to temporally accumulated secondary metabolites. Columns represent community membership and rows represent identified secondary metabolites in *B. thailandensis*. Known bioactive secondary metabolites produced by *B. thailandensis* were identified in MZmine 2 through the observation of MS and MS/MS data. The accumulation of each exometabolite was quantified through time (n = 2 to 4 integrated peak areas per time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5× the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.
**Figure 7.** Coculture upregulation of BSGCs from *B. thailandensis* translates to temporally accumulated secondary metabolites. Columns represent community membership and rows represent identified secondary metabolites in *B. thailandensis*. Known bioactive secondary metabolites produced by *B. thailandensis* were identified in MZmine 2 through the observation of MS and MS/MS data. The accumulation of each exometabolite was quantified through time (n = 2 to 4 integrated peak areas per time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5× the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.
Intensity (AU) - relativized to internal reference at each time point

Time (h)

<figure><img src="image_21.png" /></figure>
<figure><img src="image_22.png" /></figure>
**Figure 8.** *B. thailandensis* genes involved in thailandamide production are co-expressed with *C. subtsugae* genes. A network module containing the thailandamide BSGC is shown (A). The network module nodes are color coded by according to the following criteria: thailandamide biosynthetic genes that had interspecies edges (magenta), thailandamide biosynthetic genes that did not have interspecies edges (orange), other *B. thailandensis* genes that were not part of the BSGC (yellow), and genes that were from *C. subtsugae* (blue). The chromosomal organization of the thailandamide BSGC is shown below the network module (B). The same colors are applied to the BSGC operon. The operon also depicts genes that were not detected within the interspecies network, shown in gray. Asterisks indicate core biosynthetic genes in the BSGCs, as predicted from antiSMASH. The table (C) shows upregulated *B. thailandensis* BSGCs (Fig. 6) and whether interspecies edges were detected (check is yes, x is no).
**Figure 8.** *B. thailandensis* genes involved in thailandamide production are co-expressed with *C. subtsugae* genes. A network module containing the thailandamide BSGC is shown (A). The network module nodes are color coded by according to the following criteria: thailandamide biosynthetic genes that had interspecies edges (magenta), thailandamide biosynthetic genes that did not have interspecies edges (orange), other *B. thailandensis* genes that were not part of the BSGC (yellow), and genes that were from *C. subtsugae* (blue). The chromosomal organization of the thailandamide BSGC is shown below the network module (B). The same colors are applied to the BSGC operon. The operon also depicts genes that were not detected within the interspecies network, shown in gray. Asterisks indicate core biosynthetic genes in the BSGCs, as predicted from antiSMASH. The table (C) shows upregulated *B. thailandensis* BSGCs (Fig. 6) and whether interspecies edges were detected (check is yes, x is no).
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

C

<table><thead><tr><th></th><th>B. thailandensis-C. subtsugae interspecies network</th><th>B. thailandensis-P. syringae interspecies network</th></tr></thead><tbody><tr><td>Bactobolin</td><td>✓</td><td>✓</td></tr><tr><td>Beta-lactone</td><td>✓</td><td>×</td></tr><tr><td>Capistruin</td><td>✓</td><td>✓</td></tr><tr><td>Malleilactone</td><td>✓</td><td>✓</td></tr><tr><td>Malleobactin</td><td>✓</td><td>✓</td></tr><tr><td>NRPS-1</td><td>×</td><td>✓</td></tr><tr><td>Pyochelin</td><td>✓</td><td>✓</td></tr><tr><td>Rhamnolipid</td><td>×</td><td>×</td></tr><tr><td>Thailandamide</td><td>✓</td><td>✓</td></tr></tbody></table>
A

<figure><img src="image_23.png" /></figure>

B

<figure><img src="image_24.png" /></figure>

C

<table><thead><tr><th></th><th>B. thailandensis-C. subtsugae<br>interspecies network</th><th>B. thailandensis-P. syringae<br>interspecies network</th></tr></thead><tbody><tr><td>Bactobolin</td><td>✓</td><td>✓</td></tr><tr><td>Beta-lactone</td><td>✓</td><td>×</td></tr><tr><td>Capistruin</td><td>✓</td><td>✓</td></tr><tr><td>Malleilactone</td><td>✓</td><td>✓</td></tr><tr><td>Malleobactin</td><td>✓</td><td>✓</td></tr><tr><td>NRPS-1</td><td>×</td><td>✓</td></tr><tr><td>Pyochelin</td><td>✓</td><td>✓</td></tr><tr><td>Rhamnolipid</td><td>×</td><td>×</td></tr><tr><td>Thailandamide</td><td>✓</td><td>✓</td></tr></tbody></table>
1 Supplemental Materials for

2 Bioactive exometabolites drive maintenance competition in simple bacterial

3 communities.

4 Chodkowski JL and A Shade.

5 Code and data:

6 https://github.com/ShadeLab/Paper_Chodkowski_3member_SynCom_2021

7
1 Supplemental Materials for

2 Bioactive exometabolites drive maintenance competition in simple bacterial

3 communities.

4 Chodkowski JL and A Shade.

5 Code and data:

6 https://github.com/ShadeLab/Paper_Chodkowski_3member_SynCom_2021

7
8

**Supplementary Methods**

9

10

This supplementary information contains expanded Materials and Methods.

11

12

**RNA-seq**

13

*RNA extraction*

14

RNA was extracted using the E.Z.N.A. Bacterial RNA kit (Omega Bio-tek, Inc.). An in-tube DNase I (Ambion, Inc AM2222, 2U) digestion was performed to remove DNA from RNA samples. RNA samples were purified and concentrated using the Qiagen RNAeasy MinElute Clean up Kit (Qiagen, Inc). Ten random samples were chosen to assess RNA integrity (RIN > 7) on an Agilent 2100 Bioanalyzer. Standard operating protocols were performed at the Department of Energy Joint Genome Institute as previously described [1].

15

16

17

18

19

20

21

22

*RNA sample prep, sequencing, QC, read preprocessing, and filtering*

23

Standard operating protocols were performed at the Department of Energy Joint Genome Institute as previously described [1].

24

25

26

*Pseudoalignment and counting*

27

Reads from each library were pseudoaligned to the transcriptome of each member with kallisto [2]. Raw counts from each library were combined into a gene count matrix for each member. The gene count matrix was used for downstream analyses.

28

29

30

31
8

**Supplementary Methods**

9

10

This supplementary information contains expanded Materials and Methods.

11

12

**RNA-seq**

13

*RNA extraction*

14

RNA was extracted using the E.Z.N.A. Bacterial RNA kit (Omega Bio-tek, Inc.). An in-tube DNase I (Ambion, Inc AM2222, 2U) digestion was performed to remove DNA from RNA samples. RNA samples were purified and concentrated using the Qiagen RNAeasy MinElute Clean up Kit (Qiagen, Inc). Ten random samples were chosen to assess RNA integrity (RIN > 7) on an Agilent 2100 Bioanalyzer. Standard operating protocols were performed at the Department of Energy Joint Genome Institute as previously described [1].

15

16

17

18

19

20

21

22

*RNA sample prep, sequencing, QC, read preprocessing, and filtering*

23

Standard operating protocols were performed at the Department of Energy Joint Genome Institute as previously described [1].

24

25

26

*Pseudoalignment and counting*

27

Reads from each library were pseudoaligned to the transcriptome of each member with kallisto [2]. Raw counts from each library were combined into a gene count matrix for each member. The gene count matrix was used for downstream analyses.

28

29

30

31
KEGG pathway analysis 32

Log-2 fold changes (LFC) were extracted from DESeq analysis by comparing
each condition at each time point to the exponential-phase time point (12.5 h) in
monoculture. We then mapped longitudinal LFCs onto KEGG pathways for each strain
using the pathview package in R. First, K numbers were assigned to genes for both C.
*subtsugae* and *P. syringae* using BlastKOALA (version 2.2). K numbers were not
assigned to *B. thailandensis* because KEGG identifiers were available. KEGG identifiers
for *B. thailandensis* and K numbers assigned to *C. subtsugae* and *P. syringae* were
used to map longitudinal LFCs onto KEGG pathways. Pathways of interest were
uploaded to FigShare.

42

Flow cytometry 43

Diluted cultures were stained with the Thermo Scientific LIVE/DEAD BacLight
bacterial viability kit at final concentrations of 1.5 µM Syto9 (live stain) and 2.5 µM
propidium iodide (dead stain). Two hundred microliters of stained cultures were
transferred to a 96-well microtiter U-bottom microplate (Thermo Scientific). Twenty
microliters of sample were analyzed on a BD Accuri C6 flow cytometer (BD
Biosciences) at a fluidics rate of 66 µl/min and a threshold of 500 on an FL2 gate. The
instrument contained the following optical filters: FL1-533, 30 nm; FL2-585, 40 nm; and
FL3, 670-nm longpass. The counting accuracy of the flow cytometer was checked with
green fluorescent protein beads (Thermo Scientific). Data were analyzed using BD
Accuri C6 software version 1.0.264.21 (BD Biosciences).

44

45

46

47

48

49

50

51

52

53

54
KEGG pathway analysis 32

Log-2 fold changes (LFC) were extracted from DESeq analysis by comparing
each condition at each time point to the exponential-phase time point (12.5 h) in
monoculture. We then mapped longitudinal LFCs onto KEGG pathways for each strain
using the pathview package in R. First, K numbers were assigned to genes for both C.
subtsugae and P. syringae using BlastKOALA (version 2.2). K numbers were not
assigned to B. thailandensis because KEGG identifiers were available. KEGG identifiers
for B. thailandensis and K numbers assigned to C. subtsugae and P. syringae were
used to map longitudinal LFCs onto KEGG pathways. Pathways of interest were
uploaded to FigShare.

42

Flow cytometry 43

Diluted cultures were stained with the Thermo Scientific LIVE/DEAD BacLight
bacterial viability kit at final concentrations of 1.5 µM Syto9 (live stain) and 2.5 µM
propidium iodide (dead stain). Two hundred microliters of stained cultures were
transferred to a 96-well microtiter U-bottom microplate (Thermo Scientific). Twenty
microliters of sample were analyzed on a BD Accuri C6 flow cytometer (BD
Biosciences) at a fluidics rate of 66 µl/min and a threshold of 500 on an FL2 gate. The
instrument contained the following optical filters: FL1-533, 30 nm; FL2-585, 40 nm; and
FL3, 670-nm longpass. The counting accuracy of the flow cytometer was checked with
green fluorescent protein beads (Thermo Scientific). Data were analyzed using BD
Accuri C6 software version 1.0.264.21 (BD Biosciences).

44

45

46

47

48

49

50

51

52

53

54
57 Features were normalized by an ITSD reference feature (see Dataset 5
58 at https://github.com/ShadeLab/Paper_Chodkowski_MonocultureExometabolites_2020/tree/master/Datasets) and cube root transformed. Reference features for polar analyses
59 in positive ([¹³C,¹⁵N]proline) and negative ([¹³C,¹⁵N]alanine) modes were determined by
60 the ITSD with the lowest CV value across all samples. The reference feature for
61 nonpolar data sets was the ITSD ABMBA. Heat maps were generated in MetaboAnalyst
62 using Ward's clustering algorithm with Euclidean distances from Z-scored data. Data for
63 each sample are the averages from independent time point replicates (n = 2 to 4). The
64 heatmaps were exported and edited in InkScape for labelling purposes. The normalized
65 and transformed data sets were exported from MetaboAnalyst to generate principal-
66 coordinate analysis (PCoA) plots in R (see main methods).
67

68

69 Selection of Metabolomics Standards Initiative (MSI) level 1 primary metabolites for
70 heatmap analysis

71 DOE-JGI provided a feature table containing MSI level 1 identified primary
72 metabolites from their in-house curated reference database. The metabolites were only
73 identified from polar positive and polar negative analyses. The m/z and retention times
74 (RT) provided by DOE-JGI were used to identify these metabolites from our feature
75 table after MZmine 2 analysis. Internal standards, [¹³C,¹⁵N]proline and [¹³C,¹⁵N]alanine
76 were added to the polar positive feature table and polar negative feature table,
77 respectively. Each feature table, containing all 7 conditions, was uploaded to
57 Features were normalized by an ITSD reference feature (see Dataset 5
58 at https://github.com/ShadeLab/Paper_Chodkowski_MonocultureExometabolites_2020/tree/master/Datasets) and cube root transformed. Reference features for polar analyses
59 in positive ([$^{13}$C, $^{15}$N]proline) and negative ([$^{13}$C, $^{15}$N]alanine) modes were determined by
60 the ITSD with the lowest CV value across all samples. The reference feature for
61 nonpolar data sets was the ITSD ABMBA. Heat maps were generated in MetaboAnalyst
62 using Ward's clustering algorithm with Euclidean distances from Z-scored data. Data for
63 each sample are the averages from independent time point replicates (n = 2 to 4). The
64 heatmaps were exported and edited in InkScape for labelling purposes. The normalized
65 and transformed data sets were exported from MetaboAnalyst to generate principal-
66 coordinate analysis (PCoA) plots in R (see main methods).
67

68

69 Selection of Metabolomics Standards Initiative (MSI) level 1 primary metabolites for
70 heatmap analysis

71 DOE-JGI provided a feature table containing MSI level 1 identified primary
72 metabolites from their in-house curated reference database. The metabolites were only
73 identified from polar positive and polar negative analyses. The m/z and retention times
74 (RT) provided by DOE-JGI were used to identify these metabolites from our feature
75 table after MZmine 2 analysis. Internal standards, [$^{13}$C, $^{15}$N]proline and [$^{13}$C, $^{15}$N]alanine
76 were added to the polar positive feature table and polar negative feature table,
77 respectively. Each feature table, containing all 7 conditions, was uploaded to
78 MetaboAnalyst. Features were normalized by ITSD and cube root transformed. Each
79 feature table was exported and combined. Then, the feature table was split by each
80 member, meaning 3 feature tables were created containing 4 conditions each (1
81 monoculture, 2 pairwise cocultures, and 1, 3-member community). These normalized
82 and log transformed feature tables were re-uploaded to MetaboAnalyst for heatmap
83 analysis.

84 Heat maps were generated in MetaboAnalyst using Ward's clustering algorithm with
85 Euclidean distances from Z-scored data. The dendrograms from each analysis were
86 manually inspected to select clusters of primary metabolites that met the following
87 criteria for each member: 1) accumulated over the time series in monoculture or 2)
88 accumulated substantially in the exponential phase time point in monoculture compared
89 to the stationary phase time points in monoculture. Primary metabolites that met criteria
90 1 were filtered from the normalized and log transformed MSI level 1 feature table and
91 primary metabolites that met criteria 2 were filtered from the normalized and log
92 transformed MSI level 1 feature table, separately. These filtered feature tables,
93 containing all 7 conditions, were re-uploaded to MetaboAnalyst for heatmap analysis.
94 Heat maps were generated in MetaboAnalyst using Ward's clustering algorithm with
95 Euclidean distances from Z-scored data. The heatmaps were exported and edited in
96 InkScape for labeling purposes.

97

98

99

Effects of plate occupancy and resource concentration on gene expression

100

SynCom experiments
78 MetaboAnalyst. Features were normalized by ITSD and cube root transformed. Each
79 feature table was exported and combined. Then, the feature table was split by each
80 member, meaning 3 feature tables were created containing 4 conditions each (1
81 monoculture, 2 pairwise cocultures, and 1, 3-member community). These normalized
82 and log transformed feature tables were re-uploaded to MetaboAnalyst for heatmap
83 analysis.

84 Heat maps were generated in MetaboAnalyst using Ward's clustering algorithm with
85 Euclidean distances from Z-scored data. The dendrograms from each analysis were
86 manually inspected to select clusters of primary metabolites that met the following
87 criteria for each member: 1) accumulated over the time series in monoculture or 2)
88 accumulated substantially in the exponential phase time point in monoculture compared
89 to the stationary phase time points in monoculture. Primary metabolites that met criteria
90 1 were filtered from the normalized and log transformed MSI level 1 feature table and
91 primary metabolites that met criteria 2 were filtered from the normalized and log
92 transformed MSI level 1 feature table, separately. These filtered feature tables,
93 containing all 7 conditions, were re-uploaded to MetaboAnalyst for heatmap analysis.
94 Heat maps were generated in MetaboAnalyst using Ward's clustering algorithm with
95 Euclidean distances from Z-scored data. The heatmaps were exported and edited in
96 InkScape for labeling purposes.

97

98

99

Effects of plate occupancy and resource concentration on gene expression

100 SynCom experiments
We designed an additional experiment to determine whether the observed dynamics in exometabolites and transcription could be attributed solely to population density given resource availability, rather than to interspecies interactions. Additional SynCom experiments (6 conditions, 3 replicates/condition), were prepared as described (see methods section: Bacterial strains and culture conditions and Synthetic Community Experiments). The conditions varied based on plate occupancy (# of wells occupied by each member) and resources (% glucose) in the transwell plate. The conditions were as follows: *B. thailandensis* (31 wells) in M9-0.067% glucose, *B. thailandensis* (62 wells) in M9-0.13% glucose, *B. thailandensis* (93 wells) in M9-0.2% glucose, *B. thailandensis-C. subtsugae* (31 wells/member) in M9-0.13% glucose, *B. thailandensis* *P. syringae* (31 wells/member) in M9-0.13% glucose, and *B. thailandensis-C. subtsugae* *P. syringae* (31 wells/member) in M9-0.2% glucose. Plates were destructed after 45 h incubation and the following procedures were performed: 1) Wells containing spent culture from each member were separately pooled into 15 mL conical tubes, flash frozen in liquid nitrogen, and stored at -80 until further processing. 2) Spent medium (~31 ml) from the shared reservoir was transferred to 50 mL conical tubes, flash-frozen in liquid nitrogen and stored at -80 °C.

RNA extraction, QC, and cDNA synthesis

RNA was extracted using the E.Z.N.A. Bacterial RNA kit (Omega Bio-tek, Inc.). An in-tube DNase I (Ambion, Inc. AM2222, 2U) digestion was performed to remove DNA from RNA samples. RNA samples were purified and concentrated using the Qiagen RNAeasy MinElute Clean up Kit (Qiagen, Inc.). RNA samples were quantified
We designed an additional experiment to determine whether the observed dynamics in exometabolites and transcription could be attributed solely to population density given resource availability, rather than to interspecies interactions. Additional SynCom experiments (6 conditions, 3 replicates/condition), were prepared as described (see methods section: Bacterial strains and culture conditions and Synthetic Community Experiments). The conditions varied based on plate occupancy (# of wells occupied by each member) and resources (% glucose) in the transwell plate. The conditions were as follows: *B. thailandensis* (31 wells) in M9-0.067% glucose, *B. thailandensis* (62 wells) in M9-0.13% glucose, *B. thailandensis* (93 wells) in M9-0.2% glucose, *B. thailandensis-C. subtsugae* (31 wells/member) in M9-0.13% glucose, *B. thailandensis* *P. syringae* (31 wells/member) in M9-0.13% glucose, and *B. thailandensis-C. subtsugae* *P. syringae* (31 wells/member) in M9-0.2% glucose. Plates were destructed after 45 h incubation and the following procedures were performed: 1) Wells containing spent culture from each member were separately pooled into 15 mL conical tubes, flash frozen in liquid nitrogen, and stored at -80 until further processing. 2) Spent medium (~31 ml) from the shared reservoir was transferred to 50 mL conical tubes, flash-frozen in liquid nitrogen and stored at -80 °C.

RNA extraction, QC, and cDNA synthesis

RNA was extracted using the E.Z.N.A. Bacterial RNA kit (Omega Bio-tek, Inc.). An in-tube DNase I (Ambion, Inc. AM2222, 2U) digestion was performed to remove DNA from RNA samples. RNA samples were purified and concentrated using the Qiagen RNAeasy MinElute Clean up Kit (Qiagen, Inc.). RNA samples were quantified
on a Qubit using the RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Inc.).
RNA samples were then sent to the RTSF Genomics Core at Michigan State University
for high sensitivity RNA ScreenTape analysis on an Agilent 4200 TapeStation.
TapeStation analysis confirmed successful digestion of DNA. Total RNA (150
ng/sample) was synthesized to cDNA using the Invitrogen SuperScript III First-Strand
Synthesis kit (Thermo Fisher Scientific, Inc.). cDNA samples were quantified by Qubit in
preparation of target genes for RT-qPCR.

Three genes from the *B. thailandensis* thailandamide operon were targeted for
relative quantification normalized to the *rpoD* reference gene. Primers used for RT-
qPCR are shown in Table S12. We first confirmed amplification of intended targets.
Each of these genes were amplified from *B. thailandensis* gDNA (100 ng) using the
Phusion High-Fidelity DNA Polymerase (New England Biolabs, Inc.) with the following
conditions: 98 °C (30 s), 30 cycles of 98 °C (10 s), 59 °C (10 s), and 72 °C (10 s), and a
final extension at 72 °C (5 min). PCR products were run on gel (100 V for 50 min) and
gel extracted and purified using the Wizard SV Gel and PCR Clean-Up System
(Promega Corporation). PCR amplified and purified products of *rpoD*, *thaF*, *thaK*, and
*thaQ* were sent to the RTSF Genomics Core at Michigan State University for Sanger
sequencing.

RT-qPCR assays were performed using the SsoAdvanced Universal SYBR
Green Supermix (Bio-Rad Laboratories, Inc.). SYBR reactions were placed into Hard-
Shell PCR Plates 96-well, thin wall (Bio-Rad Laboratories, Inc. HSP9601) and analyzed
using a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). First, the
dynamic range of each primer set was determined by making a 10-fold dilution series
on a Qubit using the RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Inc.).
RNA samples were then sent to the RTSF Genomics Core at Michigan State University
for high sensitivity RNA ScreenTape analysis on an Agilent 4200 TapeStation.
TapeStation analysis confirmed successful digestion of DNA. Total RNA (150
ng/sample) was synthesized to cDNA using the Invitrogen SuperScript III First-Strand
Synthesis kit (Thermo Fisher Scientific, Inc.). cDNA samples were quantified by Qubit in
preparation of target genes for RT-qPCR.

Three genes from the *B. thailandensis* thailandamide operon were targeted for
relative quantification normalized to the *rpoD* reference gene. Primers used for RT-
qPCR are shown in Table S12. We first confirmed amplification of intended targets.
Each of these genes were amplified from *B. thailandensis* gDNA (100 ng) using the
Phusion High-Fidelity DNA Polymerase (New England Biolabs, Inc.) with the following
conditions: 98 °C (30 s), 30 cycles of 98 °C (10 s), 59 °C (10 s), and 72 °C (10 s), and a
final extension at 72 °C (5 min). PCR products were run on gel (100 V for 50 min) and
gel extracted and purified using the Wizard SV Gel and PCR Clean-Up System
(Promega Corporation). PCR amplified and purified products of *rpoD*, *thaF*, *thaK*, and
*thaQ* were sent to the RTSF Genomics Core at Michigan State University for Sanger
sequencing.

RT-qPCR assays were performed using the SsoAdvanced Universal SYBR
Green Supermix (Bio-Rad Laboratories, Inc.). SYBR reactions were placed into Hard-
Shell PCR Plates 96-well, thin wall (Bio-Rad Laboratories, Inc. HSP9601) and analyzed
using a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). First, the
dynamic range of each primer set was determined by making a 10-fold dilution series
147 from 10 ng-0.1 pg of cDNA. The following mixture was used for each RT-qPCR assay:
148 10 µL SsoAdvanced universal SYBR Green supermix (2x), 0.5 µL each of forward and
149 reverse primers, 1 µL water, and 8 µL cDNA sample (6 serially diluted samples
150 concentrated between 1.25E-5 and 1.25 ng/uL). The RT-qPCR reaction was run with
151 the following conditions: 95 °C (3 min), 40 cycles of 95 °C (10 s), 59 °C (10 s), and 72
152 °C (10 s). Following the last extension step, the melt curve was run with the following
153 conditions: 95 °C (10 s), then 65 °C to 95 °C in 0.5 °C increments. Each primer set had
154 a 5-fold dynamic range (10 ng-10 pg) with efficiencies between 90-110% (Table S13)
155 The Δslope between the reference gene and each target gene were all ≤0.1, confirming
156 that relative gene expression math models were a viable option for comparing gene
157 expression across conditions.

158 cDNA concentrations across all conditions were diluted to a stock concentration
159 of 0.0125 ng/uL. RT-qPCR reactions and conditions were prepared and run as
160 previously described. Controls for the assay included a gDNA positive control, a no
161 template negative control, and a no amplification (no-RT) negative control. The Livak
162 method (2⁻⁵Cₜ) was used to calculate relative gene expression in each test condition
163 compared to the reference condition (B. thailandensis, 31 wells in M9-0.067% glucose)
164 where target genes were thaF, thaK, and thaQ and the reference gene was rpoD.

165

166 References

167 1. Chodkowski, J. L., & Shade, A. (2020). Exometabolite Dynamics over Stationary Phase Reveal
168 Strain-Specific Responses. MSystems, 5(6), e00493-20.
169 https://doi.org/10.1128/msystems.00493-20

170

171 2. Bray, N. L., Pimentel, H., Melsted, P., & Pachter, L. (2016). Near-optimal probabilistic RNA-seq
172 quantification. Nature Biotechnology, 34, 525–527. https://doi.org/10.1038/nbt.3519

173
147 from 10 ng-0.1 pg of cDNA. The following mixture was used for each RT-qPCR assay:
148 10 µL SsoAdvanced universal SYBR Green supermix (2x), 0.5 µL each of forward and
149 reverse primers, 1 µL water, and 8 µL cDNA sample (6 serially diluted samples
150 concentrated between 1.25E-5 and 1.25 ng/uL). The RT-qPCR reaction was run with
151 the following conditions: 95 °C (3 min), 40 cycles of 95 °C (10 s), 59 °C (10 s), and 72
152 °C (10 s). Following the last extension step, the melt curve was run with the following
153 conditions: 95 °C (10 s), then 65 °C to 95 °C in 0.5 °C increments. Each primer set had
154 a 5-fold dynamic range (10 ng-10 pg) with efficiencies between 90-110% (Table S13)
155 The Δslope between the reference gene and each target gene were all ≤0.1, confirming
156 that relative gene expression math models were a viable option for comparing gene
157 expression across conditions.

158 cDNA concentrations across all conditions were diluted to a stock concentration
159 of 0.0125 ng/uL. RT-qPCR reactions and conditions were prepared and run as
160 previously described. Controls for the assay included a gDNA positive control, a no
161 template negative control, and a no amplification (no-RT) negative control. The Livak
162 method (2⁻⁵Cₜ) was used to calculate relative gene expression in each test condition
163 compared to the reference condition (B. thailandensis, 31 wells in M9-0.067% glucose)
164 where target genes were thaF, thaK, and thaQ and the reference gene was rpoD.

165

166 References

167 1. Chodkowski, J. L., & Shade, A. (2020). Exometabolite Dynamics over Stationary Phase Reveal
168 Strain-Specific Responses. MSystems, 5(6), e00493-20.
169 https://doi.org/10.1128/msystems.00493-20

170

171 2. Bray, N. L., Pimentel, H., Melsted, P., & Pachter, L. (2016). Near-optimal probabilistic RNA-seq
172 quantification. Nature Biotechnology, 34, 525–527. https://doi.org/10.1038/nbt.3519

173
174 3. Funston SJ, Tsaousi K, Rudden M, Smyth TJ, Stevenson PS, Marchant R, Banat IM. (2016).
175 Characterising rhamnolipid production in Burkholderia thailandensis E264, a non-pathogenic
176 producer. Appl Microbiol Biotechnol.,100(18), 7945-56. https://doi.org/10.1007/s00253-016-7564-y
177
178

179

180 Supplementary Files/Datasets

181 **Supplementary File 1:** Pairwise PROTEST analyses comparing the reproducibility of
182 exometabolome profiles across biological replicate time series. Coordinates of the first
183 two PCoA axes were used to perform PROTEST analysis in vegan. File type: .xlsx

184 **Supplementary File 2:** Identification of *B. thailandensis* bioactive exometabolites of
185 interest through observation of mass spectrometry data. File type: .xlsx

186 **Supplementary File 3:** Genes part of the interspecies network, their corresponding
187 modules, and GO enrichment analysis of modules. File type: .xlsx

188 **Supplementary File 4:** GO enrichment analysis on genes with interspecies edges from
189 network analysis. File type: .xlsx

190 **Supplementary File 5:** Gene annotations for *C. subtsugae* and *P. syringae* that
191 contained interspecies edges with *B. thailandensis* thailandamide and malleilactone
192 biosynthetic genes, respectively. File type: .xlsx

193 **Supplementary File 6:** Protein alignment of the DNA starvation/stationary phase
194 protein from *C. subtsugae* and the closest homolog in *B. thailandensis*. File type: .txt

195 **Supplementary File 7:** Protein alignment of the TonB-dependent siderophore receptor
196 family protein from *P. syringae* and the closest homolog in *B. thailandensis*. File type:
197 .txt
174 3. Funston SJ, Tsaousi K, Rudden M, Smyth TJ, Stevenson PS, Marchant R, Banat IM. (2016).
175 Characterising rhamnolipid production in Burkholderia thailandensis E264, a non-pathogenic
176 producer. Appl Microbiol Biotechnol.,100(18), 7945-56. https://doi.org/10.1007/s00253-016-7564-y
177
178

179

180 Supplementary Files/Datasets

181 **Supplementary File 1:** Pairwise PROTEST analyses comparing the reproducibility of
182 exometabolome profiles across biological replicate time series. Coordinates of the first
183 two PCoA axes were used to perform PROTEST analysis in vegan. File type: .xlsx

184 **Supplementary File 2:** Identification of *B. thailandensis* bioactive exometabolites of
185 interest through observation of mass spectrometry data. File type: .xlsx

186 **Supplementary File 3:** Genes part of the interspecies network, their corresponding
187 modules, and GO enrichment analysis of modules. File type: .xlsx

188 **Supplementary File 4:** GO enrichment analysis on genes with interspecies edges from
189 network analysis. File type: .xlsx

190 **Supplementary File 5:** Gene annotations for *C. subtsugae* and *P. syringae* that
191 contained interspecies edges with *B. thailandensis* thailandamide and malleilactone
192 biosynthetic genes, respectively. File type: .xlsx

193 **Supplementary File 6:** Protein alignment of the DNA starvation/stationary phase
194 protein from *C. subtsugae* and the closest homolog in *B. thailandensis*. File type: .txt

195 **Supplementary File 7:** Protein alignment of the TonB-dependent siderophore receptor
196 family protein from *P. syringae* and the closest homolog in *B. thailandensis*. File type:
197 .txt
198

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<figure><img src="image_24.png" /><figcaption>**Figure S1. Cell viability of SynCom members.** Live (green) and dead (blue) flow cytometry cell counts for *B. thailandensis* in monoculture and pairwise cocultures (Top row, panels A-C) and for each member in the 3-member community (Bottom row, panel D; *B. thailandensis*, panel E; *C. subtsugae*, panel F; *P. syringae*). Cells were stained with Syto9- and propdium iodide (n = 4 to 5 wells/time point/community membership/transwell plate and n=4 independent replicates/time point/community membership). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>**Figure S1. Cell viability of SynCom members.** Live (green) and dead (blue) flow cytometry cell counts for *B. thailandensis* in monoculture and pairwise cocultures (Top row, panels A-C) and for each member in the 3-member community (Bottom row, panel D; *B. thailandensis*, panel E; *C. subtsugae*, panel F; *P. syringae*). Cells were stained with Syto9- and propdium iodide (n = 4 to 5 wells/time point/community membership/transwell plate and n=4 independent replicates/time point/community membership). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and</figcaption></figure>
210 smallest value (bottom) was no further away than 1.5× the interquartile range. Points represent outliers

211 that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.

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210 smallest value (bottom) was no further away than 1.5× the interquartile range. Points represent outliers

211 that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.

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214 **Figure S2. Differential gene expression patterns across community memberships.** Venn diagram

215 plots of differentially expressed genes in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*.

216 Differential gene expression was determined using ImpulseDE2 comparing longitudinal gene expression

217 to a monoculture control (FDR-corrected cutoff of 0.01). Bt- *B. thailandensis*, Cs- *C.subtsugae*, and Ps –

218 *P. syringae*.

219
214 **Figure S2. Differential gene expression patterns across community memberships.** Venn diagram

215 plots of differentially expressed genes in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*.

216 Differential gene expression was determined using ImpulseDE2 comparing longitudinal gene expression

217 to a monoculture control (FDR-corrected cutoff of 0.01). Bt- *B. thailandensis*, Cs- *C.subtsugae*, and Ps –

218 *P. syringae*.

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<figure><img src="image_28.png" /><figcaption>**Figure S3.** *B. thailandensis* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>**Figure S3.** *B. thailandensis* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>**Figure S4.** *C. subtsugae* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Figure S4.** *C. subtsugae* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Figure S5.** *P. syringae* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure S5.** *P. syringae* RNA-seq transcript dissimilarity through time. Bray-Curtis dissimilarities were calculated for each coculture condition compared to the monoculture condition at each time point (n= 3-4 replicates/condition/time point). The bottom and top of the box are the first (Q1) and third (Q3) quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) was no further away than 1.5x the interquartile range. Points represent outliers that are less than 1.5x the interquartile range of Q1 or greater than 1.5x the interquartile range of Q3.</figcaption></figure>
<figure><img src="image_31.png" /></figure>
<figure><img src="image_32.png" /></figure>
**Figure S6.** Patterns of transcriptional regulation reveal biological responses of community members to different community memberships. Differentially expressed genes categorized by COG categories in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*. These DEGs were determined by comparing each coculture conditions to the monoculture control. COG categories include: [C] Energy production and conversion, [D] Cell cycle control, cell division, chromosome partitioning, [E] Amino acid transport and metabolism, [F] Nucleotide transport and metabolism, [G] Carbohydrate transport and metabolism, [H] Coenzyme transport and metabolism, [I] Lipid transport and metabolism, [J] Translation, ribosomal structure and biogenesis, [K] Transcription, [L] Replication, recombination and repair, [M] Cell wall/membrane/envelope biogenesis, [N] Cell motility, [O] Post-translational modification, protein turnover, and chaperones, [P] Inorganic ion transport and metabolism, [Q] Secondary metabolites biosynthesis, transport, and catabolism, [S] Function unknown, [T] Signal transduction mechanisms, [U] Intracellular trafficking, secretion, and vesicular transport, and [V] Defense mechanisms. Community memberships are as follows: *B. thailandensis-P. syringae* coculture (BtPs/PsBt), *B. thailandensis-C. subtsugae* coculture (BtCs/CsBt), *C. subtsugae-P. syringae* coculture (CsPs/PsCs), and the 3-member community (BtCsPs/CsPsBt/PsBtCs).

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**Figure S6.** Patterns of transcriptional regulation reveal biological responses of community members to different community memberships. Differentially expressed genes categorized by COG categories in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*. These DEGs were determined by comparing each coculture conditions to the monoculture control. COG categories include: [C] Energy production and conversion, [D] Cell cycle control, cell division, chromosome partitioning, [E] Amino acid transport and metabolism, [F] Nucleotide transport and metabolism, [G] Carbohydrate transport and metabolism, [H] Coenzyme transport and metabolism, [I] Lipid transport and metabolism, [J] Translation, ribosomal structure and biogenesis, [K] Transcription, [L] Replication, recombination and repair, [M] Cell wall/membrane/envelope biogenesis, [N] Cell motility, [O] Post-translational modification, protein turnover, and chaperones, [P] Inorganic ion transport and metabolism, [Q] Secondary metabolites biosynthesis, transport, and catabolism, [S] Function unknown, [T] Signal transduction mechanisms, [U] Intracellular trafficking, secretion, and vesicular transport, and [V] Defense mechanisms. Community memberships are as follows: *B. thailandensis-P. syringae* coculture (BtPs/PsBt), *B. thailandensis-C. subtsugae* coculture (BtCs/CsBt), *C. subtsugae-P. syringae* coculture (CsPs/PsCs), and the 3-member community (BtCsPs/CsPsBt/PsBtCs).

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**Figure S7. Patterns of transcriptional regulation reveal pairwise coculture-specific differences.**

273 Differentially expressed genes categorized by COG categories in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*. These DEGs were determined by comparing gene expression between pairwise cocultures for each member. Analyses were as follows: BtCs-BtPs; *B. thailandensis* coculture with *C. subtsugae* (case) was compared to *B. thailandensis* coculture with *P. syringae* (control), CsBt-CsPs; *C. subtsugae* coculture with *B. thailandensis* (case) was compared to *C. subtsugae* coculture with *P. syringae* (control), and PsBt-PsCs; *P. syringae* coculture with *B. thailandensis* (case) was compared to *P. syringae* coculture with *C. subtsugae* (control). COG categories are labeled in the Figure S6 legend.

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**Figure S7. Patterns of transcriptional regulation reveal pairwise coculture-specific differences.**

273 Differentially expressed genes categorized by COG categories in A) *B. thailandensis* B) *C. subtsugae* and C) *P. syringae*. These DEGs were determined by comparing gene expression between pairwise cocultures for each member. Analyses were as follows: BtCs-BtPs; *B. thailandensis* coculture with *C. subtsugae* (case) was compared to *B. thailandensis* coculture with *P. syringae* (control), CsBt-CsPs; *C. subtsugae* coculture with *B. thailandensis* (case) was compared to *C. subtsugae* coculture with *P. syringae* (control), and PsBt-PsCs; *P. syringae* coculture with *B. thailandensis* (case) was compared to *P. syringae* coculture with *C. subtsugae* (control). COG categories are labeled in the Figure S6 legend.

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<figure><img src="image_35.png" /><figcaption>Figure S8. Exometabolites have membership-specific production and temporal accumulation. A heat map of coculture accumulated exometabolites is shown for polar positive (A), polar negative (B), nonpolar positive (C), and nonpolar negative (D) modes, for *C. subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), *B. thailandensis* monoculture (Bt), *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. Data for each sample are the averages from independent time point replicates (n = 2 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.</figcaption></figure>

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<figure><img src="image_36.png" /><figcaption>Figure S8. Exometabolites have membership-specific production and temporal accumulation. A heat map of coculture accumulated exometabolites is shown for polar positive (A), polar negative (B), nonpolar positive (C), and nonpolar negative (D) modes, for *C. subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), *B. thailandensis* monoculture (Bt), *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. Data for each sample are the averages from independent time point replicates (n = 2 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.</figcaption></figure>

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<figure><img src="image_36.png" /><figcaption>**Figure S9.** Identified exometabolites with exponential phase accumulation. A heat map of exometabolite dynamics is shown for identified metabolites with exponential phase accumulation (time point 12.5 h) determined from *C. subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), and *B. thailandensis* monoculture (Bt), Temporal dynamics for these exometabolites were then plotted for *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. Data for each sample are the averages from independent time point replicates (n = 2 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.</figcaption></figure>

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<caption>**Figure S9.** Identified exometabolites with exponential phase accumulation. A heat map of exometabolite dynamics is shown for identified metabolites with exponential phase accumulation (time point 12.5 h) determined from *C. subtsugae* monoculture (Cs), *P. syringae* monoculture (Ps), and *B. thailandensis* monoculture (Bt), Temporal dynamics for these exometabolites were then plotted for *C. subtsugae*-P. syringae coculture (CsPs), *B. thailandensis*-P. syringae coculture (BtPs), *B. thailandensis*-C. subtsugae coculture (BtCs), and the 3-member community (BtCsPs), where samples are in columns and exometabolites are in rows. Data for each sample are the averages from independent time point replicates (n = 2 to 4). Euclidean distance was calculated from Z-scored mass spectral profiles. Features with similar dynamics were clustered by Ward's method.</caption>
<figure><img src="image_37.png" /><figcaption>**Figure S10.** BSGC downregulated or unaltered in *B. thailandensis*. Biosynthetic genes involved in</figcaption></figure>
<figure><img src="image_38.png" /><figcaption>Figure S10. BSGC downregulated or unaltered in *B. thailandensis*. Biosynthetic genes involved in</figcaption></figure>
315 each BSGC were determined with antiSMASH and evidence from literature. At each time point, the
316 average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC. The
317 horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales for
318 the Y-axis.

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315 each BSGC were determined with antiSMASH and evidence from literature. At each time point, the
316 average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC. The
317 horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales for
318 the Y-axis.

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<figure><img src="image_38.png" /><figcaption>**Figure S11.** Patterns of transcriptional regulation for BSGC in *C. subtsugae*. Biosynthetic genes involved in each BSGC were determined with antiSMASH and evidence from literature. At each time point, the average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC.</figcaption></figure>
<figure><img src="image_39.png" /><figcaption>**Figure S11.** Patterns of transcriptional regulation for BSGC in *C. subtsugae*. Biosynthetic genes involved in each BSGC were determined with antiSMASH and evidence from literature. At each time point, the average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC.</figcaption></figure>
326 The horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales

327 for the Y-axis.

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326 The horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales

327 for the Y-axis.

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<figure><img src="image_39.png" /><figcaption>**Figure S12.** Patterns of transcriptional regulation for BSGC in *P. syringae*. Biosynthetic genes involved in each BSGC were determined with antiSMASH and evidence from literature. At each time point, the average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC. The horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales for the Y-axis.</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**Figure S12.** Patterns of transcriptional regulation for BSGC in *P. syringae*. Biosynthetic genes involved in each BSGC were determined with antiSMASH and evidence from literature. At each time point, the average log2 fold-change (LFC) was determined across all biosynthetic genes for each BSGC. The horizontal line represents a LFC threshold of 1. Note that plots for each BSGC have separate scales for the Y-axis.</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**Figure S13.** Flow diagram for interspecies co-expression network analysis. An interspecies co-expression network was created based on transcript counts from *B. thailandensis*-C. *subtsugae* and *B. thailandensis*-P. *syringae* cocultures. All genes that passed initial quality filtering were included in the analysis to generate networks. Unweighted gene coexpression networks were generated with a Z-score cutoff of 4.5. Intraspecies genes were used to identify network modules. Gene ontology enrichment analysis was performed on nodes with interspecies edges.</figcaption></figure>
<figure><img src="image_41.png" /><figcaption>**Figure S13.** Flow diagram for interspecies co-expression network analysis. An interspecies coexpression network was created based on transcript counts from *B. thailandensis*-C. *subtsugae* and *B. thailandensis*-P. *syringae* cocultures. All genes that passed initial quality filtering were included in the analysis to generate networks. Unweighted gene coexpression networks were generated with a Z-score cutoff of 4.5. Intraspecies genes were used to identify network modules. Gene ontology enrichment analysis was performed on nodes with interspecies edges.</figcaption></figure>
<figure><img src="image_41.png" /><figcaption>**Figure S14.** *B. thailandensis* genes involved in malleilactone production are detected as interspecies edges in the *B. thailandensis-P. syringae* coexpression network and biosynthetic genes organize into network modules. A network module containing the malleilactone BSGC is shown. The network module nodes are color coded by *B. thailandensis* gene type (BSGC or not) and type of connections (interspecies or not): malleilactone biosynthetic genes that had interspecies edges (magenta), malleilactone biosynthetic genes that did not have interspecies edges (orange), or other genes that were not part of the BSGC (yellow); as well as genes that were from *P. syringae* (green). The chromosomal organization of the malleilactone BSGC is shown below the network module. The same colors are applied to the BSGC operons. Asterisks indicate core biosynthetic genes in the BSGCs, as predicted from antiSMASH.</figcaption></figure>

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<figure><img src="image_42.png" /><figcaption>**Figure S14.** *B. thailandensis* genes involved in malleilactone production are detected as interspecies edges in the *B. thailandensis-P. syringae* coexpression network and biosynthetic genes organize into network modules. A network module containing the malleilactone BSGC is shown. The network module nodes are color coded by *B. thailandensis* gene type (BSGC or not) and type of connections (interspecies or not): malleilactone biosynthetic genes that had interspecies edges (magenta), malleilactone biosynthetic genes that did not have interspecies edges (orange), or other genes that were not part of the BSGC (yellow); as well as genes that were from *P. syringae* (green). The chromosomal organization of the malleilactone BSGC is shown below the network module. The same colors are applied to the BSGC operons. Asterisks indicate core biosynthetic genes in the BSGCs, as predicted from antiSMASH.</figcaption></figure>

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<figure><img src="image_42.png" /></figure>

**Figure S15.** The DNA starvation/stationary phase protection gene, *dpsA*, in downregulated in *C. subtsugae* when cocultured with *B. thailandensis* while unaltered in *B. thailandensis*. Transcript abundance trajectories of *dpsA* are plotted for *C. subtsugae* (A) and *B. thailandensis* (B). Time course scatter plots were smooth curve fitted by loess (n=3-4 replicates/condition/time point). Community memberships are as follows: *B. thailandensis* monoculture (Bt; orange), *C. subtsugae* monoculture (Cs; blue), *C. subtsugae-P. syringae* coculture (CsPs; light blue), *B. thailandensis-P. syringae* coculture (BtPs; yellow), *B. thailandensis-C. subtsugae* coculture (BtCs; purple), and the 3-member community (BtCsPs; black).

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<figure><img src="image_43.png" /></figure>

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Figure S15. The DNA starvation/stationary phase protection gene, *dpsA*, in downregulated in *C. subtsugae* when cocultured with *B. thailandensis* while unaltered in *B. thailandensis*. Transcript abundance trajectories of *dpsA* are plotted for *C. subtsugae* (A) and *B. thailandensis* (B). Time course scatter plots were smooth curve fitted by loess (n=3-4 replicates/condition/time point). Community memberships are as follows: *B. thailandensis* monoculture (Bt; orange), *C. subtsugae* monoculture (Cs; blue), *C. subtsugae-P. syringae* coculture (CsPs; light blue), *B. thailandensis-P. syringae* coculture (BtPs; yellow), *B. thailandensis-C. subtsugae* coculture (BtCs; purple), and the 3-member community (BtCsPs; black).

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<figure><img src="image_43.png" /><figcaption>Figure S16. The gene encoding a TonB-dependent siderophore receptor is downregulated in *P. syringae* when cocultured with *B. thailandensis* while upregulated in *B. thailandensis*. Transcript abundance trajectories of *tonB* are plotted for *P. syringae* (A) and *B. thailandensis* (B). Time course scatter plots were smooth curve fitted by loess (n = 3-4 replicates/condition/time point). Community memberships are as follows: *B. thailandensis* monoculture (Bt; orange), *P. syringae* monoculture (Ps; green), *C. subtsugae*-P. syringae coculture (CsPs; light blue), *B. thailandensis*-P. syringae coculture (BtPs; yellow), *B. thailandensis*-C. subtsugae coculture (BtCs; purple), and the 3-member community (BtCsPs; black).</figcaption></figure>

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<figure><img src="image_44.png" /><figcaption>Figure S16. The gene encoding a TonB-dependent siderophore receptor is downregulated in *P. syringae* when cocultured with *B. thailandensis* while upregulated in *B. thailandensis*. Transcript abundance trajectories of *tonB* are plotted for *P. syringae* (A) and *B. thailandensis* (B). Time course scatter plots were smooth curve fitted by loess (n = 3-4 replicates/condition/time point). Community memberships are as follows: *B. thailandensis* monoculture (Bt; orange), *P. syringae* monoculture (Ps; green), *C. subtsugae*-P. syringae coculture (CsPs; light blue), *B. thailandensis*-P. syringae coculture (BtPs; yellow), *B. thailandensis*-C. subtsugae coculture (BtCs; purple), and the 3-member community (BtCsPs; black).</figcaption></figure>
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**Supplementary Table S1.** Percent variation explained on the effect of membership,

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time, and their interaction on transcriptomic profiles.

<table><thead><tr><th></th><th>Membership</th><th>Time</th><th>Membership x Time</th></tr></thead><tbody><tr><td><em>B. thailandensis</em></td><td>46.26</td><td>13.24</td><td>63.11</td></tr><tr><td><em>C. subtsugae</em></td><td>60.60</td><td>3.88</td><td>68.29</td></tr><tr><td><em>P. syringae</em></td><td>77.03</td><td>0.00</td><td>81.40</td></tr></tbody></table>

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**Supplementary Table S1.** Percent variation explained on the effect of membership,

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time, and their interaction on transcriptomic profiles.

<table><thead><tr><th></th><th>Membership</th><th>Time</th><th>Membership x Time</th></tr></thead><tbody><tr><td><i>B. thailandensis</i></td><td>46.26</td><td>13.24</td><td>63.11</td></tr><tr><td><i>C. subtsugae</i></td><td>60.60</td><td>3.88</td><td>68.29</td></tr><tr><td><i>P. syringae</i></td><td>77.03</td><td>0.00</td><td>81.40</td></tr></tbody></table>

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<table><caption>**Supplementary Table S2.** Summary of Protest analyses comparing transcriptional profiles through time across independent replicates. Coordinates of the first two PCA axes were used to perform Protest analyses. Ranges reflect separate Protest analyses performed between all replicates in a community membership. Values in parenthesis represent the median *P* value.</caption><thead><tr><th></th><th>m12</th><th>R</th><th>P</th></tr></thead><tbody><tr><td><strong>B. thailandensis</strong></td><td></td><td></td><td></td></tr><tr><td>Monoculture</td><td>0.048 – 0.820</td><td>0.424 – 0.976</td><td>0.010 – 0.867 (0.200)</td></tr><tr><td><em>P. syringae</em> coculture</td><td>0.018 – 0.049</td><td>0.975 – 0.991</td><td>0.001 – 0.001 (0.001)</td></tr><tr><td><em>C. subtsugae</em> coculture</td><td>0.010 – 0.112</td><td>0.943 – 0.995</td><td>0.001 – 0.003 (0.001)</td></tr><tr><td>3-member</td><td>0.013 – 0.162</td><td>0.916 – 0.994</td><td>0.001 – 0.006 (0.001)</td></tr><tr><td><strong>C. subtsugae</strong></td><td></td><td></td><td></td></tr><tr><td>Monoculture</td><td>0.011 – 0.140</td><td>0.927 – 0.995</td><td>0.004 – 0.067 (0.039)</td></tr><tr><td><em>P. syringae</em> coculture</td><td>0.045 – 0.206</td><td>0.891 – 0.977</td><td>0.003 – 0.042 (0.008)</td></tr><tr><td><em>B. thailandensis</em> coculture</td><td>0.091 – 0.182</td><td>0.905 – 0.954</td><td>0.001 – 0.108 (0.019)</td></tr><tr><td>3-member</td><td>0.190 – 0.543</td><td>0.676 – 0.900</td><td>0.001 – 0.208 (0.013)</td></tr><tr><td><strong>P. syringae</strong></td><td></td><td></td><td></td></tr><tr><td>Monoculture</td><td>0.178 – 0.538</td><td>0.680 – 0.907</td><td>0.008 – 0.136 (0.054)</td></tr><tr><td><em>C. subtsugae</em> coculture</td><td>0.035 – 0.251</td><td>0.865 – 0.982</td><td>0.001 – 0.083 (0.021)</td></tr><tr><td><em>B. thailandensis</em> coculture</td><td>0.021 – 0.290</td><td>0.843 – 0.990</td><td>0.001 – 0.001 (0.001)</td></tr><tr><td>3-member</td><td>0.034 – 0.687</td><td>0.560 – 0.983</td><td>0.007 – 0.317 (0.038)</td></tr></tbody></table>

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<table><caption>**Supplementary Table S2.** Summary of Protest analyses comparing transcriptional profiles through time across independent replicates. Coordinates of the first two PCA axes were used to perform Protest analyses. Ranges reflect separate Protest analyses performed between all replicates in a community membership. Values in parenthesis represent the median *P* value.</caption><thead><tr><th></th><th>m12</th><th>R</th><th>P</th></tr></thead><tbody><tr><td colspan="4"><strong>B. thailandensis</strong></td></tr><tr><td>Monoculture</td><td>0.048 – 0.820</td><td>0.424 – 0.976</td><td>0.010 – 0.867 (0.200)</td></tr><tr><td><em>P. syringae</em> coculture</td><td>0.018 – 0.049</td><td>0.975 – 0.991</td><td>0.001 – 0.001 (0.001)</td></tr><tr><td><em>C. subtsugae</em> coculture</td><td>0.010 – 0.112</td><td>0.943 – 0.995</td><td>0.001 – 0.003 (0.001)</td></tr><tr><td>3-member</td><td>0.013 – 0.162</td><td>0.916 – 0.994</td><td>0.001 – 0.006 (0.001)</td></tr><tr><td colspan="4"><strong>C. subtsugae</strong></td></tr><tr><td>Monoculture</td><td>0.011 – 0.140</td><td>0.927 – 0.995</td><td>0.004 – 0.067 (0.039)</td></tr><tr><td><em>P. syringae</em> coculture</td><td>0.045 – 0.206</td><td>0.891 – 0.977</td><td>0.003 – 0.042 (0.008)</td></tr><tr><td><em>B. thailandensis</em> coculture</td><td>0.091 – 0.182</td><td>0.905 – 0.954</td><td>0.001 – 0.108 (0.019)</td></tr><tr><td>3-member</td><td>0.190 – 0.543</td><td>0.676 – 0.900</td><td>0.001 – 0.208 (0.013)</td></tr><tr><td colspan="4"><strong>P. syringae</strong></td></tr><tr><td>Monoculture</td><td>0.178 – 0.538</td><td>0.680 – 0.907</td><td>0.008 – 0.136 (0.054)</td></tr><tr><td><em>C. subtsugae</em> coculture</td><td>0.035 – 0.251</td><td>0.865 – 0.982</td><td>0.001 – 0.083 (0.021)</td></tr><tr><td><em>B. thailandensis</em> coculture</td><td>0.021 – 0.290</td><td>0.843 – 0.990</td><td>0.001 – 0.001 (0.001)</td></tr><tr><td>3-member</td><td>0.034 – 0.687</td><td>0.560 – 0.983</td><td>0.007 – 0.317 (0.038)</td></tr></tbody></table>

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**Supplementary Table S3.** PERMANOVA results calculated on independently replicated time series within members across all community memberships. PERMANOVA results are presented as *P* values, *R*² values, and pseudo-F statistic results in the first row. Post-hoc pairwise PERMANOVA results are presented below the first row.

<table><thead><tr><th></th><th><i>B. thailandensis</i></th><th><i>C. subtsugae</i></th><th><i>P. syringae</i></th></tr></thead><tbody><tr><td>adonis</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.480,<br><i>F</i> = 27.686</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.619,<br><i>F</i> = 47.15</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.778,<br><i>F</i> = 107.21</td></tr><tr><td>Monoculture vs <i>B. thailandensis</i> coculture</td><td>-</td><td>0.002</td><td>0.001</td></tr><tr><td>Monoculture vs <i>C. subtsugae</i> coculture</td><td>0.001</td><td>-</td><td>0.001</td></tr><tr><td>Monoculture vs <i>P. syringae</i> coculture</td><td>0.001</td><td>0.010</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs <i>C. subtsugae</i> coculture</td><td>-</td><td>-</td><td>0.001</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs <i>P. syringae</i> coculture</td><td>-</td><td>0.002</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>C. subtsugae</i> coculture vs <i>P. syringae</i> coculture</td><td>0.001</td><td>-</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs 3-member <i>C. subtsugae</i> coculture vs 3-member <i>P. syringae</i> coculture</td><td>-</td><td>0.248</td><td>0.068</td></tr><tr><td>Monoculture vs 3-member <i>C. subtsugae</i> coculture vs 3-member <i>P. syringae</i> coculture vs 3-member <i>P. syringae</i> coculture vs 3-member</td><td>0.001</td><td>-</td><td>0.001</td></tr></tbody></table>

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<table><caption>**Supplementary Table S3.** PERMANOVA results calculated on independently replicated time series within members across all community memberships. PERMANOVA results are presented as *P* values, *R*² values, and pseudo-F statistic results in the first row. Post-hoc pairwise PERMANOVA results are presented below the first row.</caption><thead><tr><th></th><th><i>B. thailandensis</i></th><th><i>C. subtsugae</i></th><th><i>P. syringae</i></th></tr></thead><tbody><tr><td>adonis</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.480,<br><i>F</i> = 27.686</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.619,<br><i>F</i> = 47.15</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.778,<br><i>F</i> = 107.21</td></tr><tr><td>Monoculture vs <i>B. thailandensis</i> coculture</td><td>-</td><td>0.002</td><td>0.001</td></tr><tr><td>Monoculture vs <i>C. subtsugae</i> coculture</td><td>0.001</td><td>-</td><td>0.001</td></tr><tr><td>Monoculture vs <i>P. syringae</i> coculture</td><td>0.001</td><td>0.010</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs <i>C. subtsugae</i> coculture</td><td>-</td><td>-</td><td>0.001</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs <i>P. syringae</i> coculture</td><td>-</td><td>0.002</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>C. subtsugae</i> coculture vs <i>P. syringae</i> coculture</td><td>0.001</td><td>-</td><td>-</td></tr><tr><td>Monoculture vs 3-member <i>B. thailandensis</i> coculture vs 3-member <i>C. subtsugae</i> coculture vs 3-member <i>P. syringae</i> coculture</td><td>-</td><td>0.248</td><td>0.068</td></tr><tr><td>Monoculture vs 3-member <i>C. subtsugae</i> coculture vs 3-member <i>P. syringae</i> coculture vs 3-member <i>P. syringae</i> coculture vs 3-member</td><td>0.001</td><td>-</td><td>0.001</td></tr></tbody></table>

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<table><caption>**Supplementary Table S4.** Percent variation explained on the effect of membership, time, and their interaction on exometabolite profiles.</caption><thead><tr><th></th><th>Membership</th><th>Time</th><th>Membership x Time</th></tr></thead><tbody><tr><td>Polar Positive</td><td>45.76</td><td>7.26</td><td>55.89</td></tr><tr><td>Polar Negative</td><td>51.61</td><td>4.12</td><td>58.83</td></tr><tr><td>Nonpolar Positive</td><td>56.92</td><td>9.49</td><td>71.88</td></tr><tr><td>Nonpolar Negative</td><td>64.77</td><td>7.94</td><td>79.38</td></tr></tbody></table>

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<table><caption>**Supplementary Table S4.** Percent variation explained on the effect of membership, time, and their interaction on exometabolite profiles.</caption><thead><tr><th></th><th>Membership</th><th>Time</th><th>Membership x Time</th></tr></thead><tbody><tr><td>Polar Positive</td><td>45.76</td><td>7.26</td><td>55.89</td></tr><tr><td>Polar Negative</td><td>51.61</td><td>4.12</td><td>58.83</td></tr><tr><td>Nonpolar Positive</td><td>56.92</td><td>9.49</td><td>71.88</td></tr><tr><td>Nonpolar Negative</td><td>64.77</td><td>7.94</td><td>79.38</td></tr></tbody></table>

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**Supplementary Table S5.** Summary of Protest analyses comparing exometabolite composition through time across independent replicates. Coordinates of the first two PCoA axes were used to perform Protest analyses. Ranges reflect separate Protest analyses performed for each polarity (polar/nonpolar) and ionization mode (positive/negative).

<table><thead><tr><th></th><th>m12</th><th>R</th><th>P</th></tr></thead><tbody><tr><td>C. subtsugae-P. syringae coculture</td><td>0.022 – 0.906</td><td>0.307 – 0.989</td><td>0.001 – 0.849 (0.025)</td></tr><tr><td>B. thailandensis-P. syringae coculture</td><td>0.015 – 0.592</td><td>0.638 – 0.992</td><td>0.001 – 0.667 (0.050)</td></tr><tr><td>B. thailandensis-C. subtsugae coculture</td><td>0.003 – 0.456</td><td>0.738 – 0.995</td><td>0.001 – 0.250 (0.003)</td></tr><tr><td>3-member community</td><td>0.021 – 0.556</td><td>0.667 – 0.990</td><td>0.001 – 0.133 (0.003)</td></tr></tbody></table>
<table><caption>**Supplementary Table S5.** Summary of Protest analyses comparing exometabolite composition through time across independent replicates. Coordinates of the first two PCoA axes were used to perform Protest analyses. Ranges reflect separate Protest analyses performed for each polarity (polar/nonpolar) and ionization mode (positive/negative).</caption><thead><tr><th></th><th>m12</th><th>R</th><th>P</th></tr></thead><tbody><tr><td>C. subtsugae-P. syringae coculture</td><td>0.022 – 0.906</td><td>0.307 – 0.989</td><td>0.001 – 0.849 (0.025)</td></tr><tr><td>B. thailandensis-P. syringae coculture</td><td>0.015 – 0.592</td><td>0.638 – 0.992</td><td>0.001 – 0.667 (0.050)</td></tr><tr><td>B. thailandensis-C. subtsugae coculture</td><td>0.003 – 0.456</td><td>0.738 – 0.995</td><td>0.001 – 0.250 (0.003)</td></tr><tr><td>3-member community</td><td>0.021 – 0.556</td><td>0.667 – 0.990</td><td>0.001 – 0.133 (0.003)</td></tr></tbody></table>
411 **Supplementary Table 6.** PERMANOVA results calculated on independently replicated time series across coculture community memberships. PERMANOVA results are presented as *P* values, *R*² values, and pseudo-F statistic results in the first row. Post-hoc pairwise PERMANOVA results are presented below the first row.

<table><thead><tr><th></th><th>Polar<br>Positive</th><th>Polar<br>Negative</th><th>Nonpolar<br>Positive</th><th>Nonpolar<br>Negative</th></tr></thead><tbody><tr><td>adonis</td><td>P = 0.001,<br>R<sup>2</sup> = 0.475,<br>F = 27.711</td><td>P = 0.001,<br>R<sup>2</sup> = 0.531,<br>F = 34.773</td><td>P = 0.001<br>R<sup>2</sup> = 0.585,<br>F = 37.549</td><td>P = 0.001,<br>R<sup>2</sup> = 0.662,<br>F = 45.743</td></tr><tr><td>B. thailandensis-C. subtsugae coculture vs<br>C. subtsugae-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>B. thailandensis-C. subtsugae coculture vs<br>B. thailandensis- P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>B. thailandensis- P. syringae coculture vs<br>C. subtsugae-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br>C. subtsugae-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br>B. thailandensis- P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br>B. thailandensis-C. subtsugae coculture</td><td>0.002</td><td>0.008</td><td>0.019</td><td>0.025</td></tr></tbody></table>

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411 **Supplementary Table 6.** PERMANOVA results calculated on independently replicated time series across coculture community memberships. PERMANOVA results are presented as *P* values, *R*² values, and pseudo-F statistic results in the first row. Post-hoc pairwise PERMANOVA results are presented below the first row.

<table><thead><tr><th></th><th>Polar<br>Positive</th><th>Polar<br>Negative</th><th>Nonpolar<br>Positive</th><th>Nonpolar<br>Negative</th></tr></thead><tbody><tr><td>adonis</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.475,<br>F = 27.711</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.531,<br>F = 34.773</td><td><i>P</i> = 0.001<br><i>R</i><sup>2</sup> = 0.585,<br>F = 37.549</td><td><i>P</i> = 0.001,<br><i>R</i><sup>2</sup> = 0.662,<br>F = 45.743</td></tr><tr><td><i>B. thailandensis</i>-C. subtsugae coculture vs<br><i>C. subtsugae</i>-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td><i>B. thailandensis</i>-C. subtsugae coculture vs<br><i>B. thailandensis</i>- P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td><i>B. thailandensis</i>- P. syringae coculture vs<br><i>C. subtsugae</i>-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br><i>C. subtsugae</i>-P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br><i>B. thailandensis</i>- P. syringae coculture</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.001</td></tr><tr><td>3-member community vs<br><i>B. thailandensis</i>-C. subtsugae coculture</td><td>0.002</td><td>0.008</td><td>0.019</td><td>0.025</td></tr></tbody></table>

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**Supplementary Table S7.** Number of predicted biosynthetic gene clusters (BSGCs,

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first row) followed by the quantity of upregulated BSGCs in cocultures.

<table><thead><tr><th></th><th><i>B. thailandensis</i></th><th><i>C. subtsugae</i></th><th><i>P. syringae</i></th></tr></thead><tbody><tr><td>Predicted BSGCs</td><td>28</td><td>14</td><td>10</td></tr><tr><td><i>C. subtsugae</i>-<i>P. syringae</i> coculture</td><td>-</td><td>0</td><td>0</td></tr><tr><td><i>B. thailandensis</i>-<i>P. syringae</i> coculture</td><td>8</td><td>-</td><td>0</td></tr><tr><td><i>B. thailandensis</i>-<i>C. subtsugae</i> coculture</td><td>10</td><td>1</td><td>-</td></tr><tr><td>3-member community</td><td>11</td><td>1</td><td>0</td></tr></tbody></table>

419

420
417

**Supplementary Table S7.** Number of predicted biosynthetic gene clusters (BSGCs,

418

first row) followed by the quantity of upregulated BSGCs in cocultures.

<table><thead><tr><th></th><th><i>B. thailandensis</i></th><th><i>C. subtsugae</i></th><th><i>P. syringae</i></th></tr></thead><tbody><tr><td>Predicted BSGCs</td><td>28</td><td>14</td><td>10</td></tr><tr><td><i>C. subtsugae</i>-<i>P. syringae</i> coculture</td><td>-</td><td>0</td><td>0</td></tr><tr><td><i>B. thailandensis</i>-<i>P. syringae</i> coculture</td><td>8</td><td>-</td><td>0</td></tr><tr><td><i>B. thailandensis</i>-<i>C. subtsugae</i> coculture</td><td>10</td><td>1</td><td>-</td></tr><tr><td>3-member community</td><td>11</td><td>1</td><td>0</td></tr></tbody></table>

419

420
421 **Supplementary Table S8.** One-way ANOVA<sup>a</sup> comparing the quantitation of identified
422 secondary metabolites between community memberships with *B. thailandensis*
423 membership.

<table><thead><tr><th></th><th>Df (between)</th><th>Df (within)</th><th>F value</th><th>p</th></tr></thead><tbody><tr><td>Bactobolin</td><td>6</td><td>160</td><td>392.10</td><td>&lt;2e-16</td></tr><tr><td>Capistruin</td><td>6</td><td>160</td><td>77.83</td><td>&lt;2e-16</td></tr><tr><td>Melleilactone</td><td>6</td><td>121</td><td>150.10</td><td>&lt;2e-16</td></tr><tr><td>Rhamnolipid<sup>b</sup></td><td>6</td><td>136</td><td>39.34</td><td>&lt;2e-16</td></tr><tr><td>Thailandamide</td><td>6</td><td>121</td><td>61.02</td><td>&lt;2e-16</td></tr><tr><td>Pyochelin</td><td>6</td><td>136</td><td>105.20</td><td>&lt;2e-16</td></tr></tbody></table>

424 <sup>a</sup>Formula: aov(formula = log(Value) ~ Membership, data = .)

425 <sup>b</sup>Rhamnolipid congener Rha-Rha-C14-C14

426
421 **Supplementary Table S8.** One-way ANOVA<sup>a</sup> comparing the quantitation of identified
422 secondary metabolites between community memberships with *B. thailandensis*
423 membership.

<table><thead><tr><th></th><th>Df (between)</th><th>Df (within)</th><th>F value</th><th>p</th></tr></thead><tbody><tr><td>Bactobolin</td><td>6</td><td>160</td><td>392.10</td><td>&lt;2e-16</td></tr><tr><td>Capistruin</td><td>6</td><td>160</td><td>77.83</td><td>&lt;2e-16</td></tr><tr><td>Melleilactone</td><td>6</td><td>121</td><td>150.10</td><td>&lt;2e-16</td></tr><tr><td>Rhamnolipid<sup>b</sup></td><td>6</td><td>136</td><td>39.34</td><td>&lt;2e-16</td></tr><tr><td>Thailandamide</td><td>6</td><td>121</td><td>61.02</td><td>&lt;2e-16</td></tr><tr><td>Pyochelin</td><td>6</td><td>136</td><td>105.20</td><td>&lt;2e-16</td></tr></tbody></table>

424 <sup>a</sup>Formula: aov(formula = log(Value) ~ Membership, data = .)

425 <sup>b</sup>Rhamnolipid congener Rha-Rha-C14-C14

426
427

428

429

<table><caption>**Supplementary Table S9.** TukeyHSD post-hoc results comparing quantitation of identified secondary metabolites between community memberships with *B. thailandensis* membership. Values represent the adjusted P-value.</caption><thead><tr><th></th><th>Bactobolin</th><th>Capistruin</th><th>Melleilactone</th><th>Rhamnolipid<sup>a</sup></th><th>Thailandamide</th><th>Pyochelin</th></tr></thead><tbody><tr><td>Monoculture vs <i>P. syringae</i> coculture</td><td>8.41E-01</td><td>8.00E-07</td><td>4.00E-07</td><td>2.26E-01</td><td>1.25E-04</td><td>4.85E-01</td></tr><tr><td>Monoculture vs <i>C. subtsugae</i> coculture</td><td>3.54E-01</td><td>8.45E-02</td><td>&lt; 1.00E-07</td><td>2.53E-02</td><td>&lt; 1.00E-07</td><td>8.55E-01</td></tr><tr><td>Monoculture vs 3-member</td><td>8.27E-01</td><td>5.00E-07</td><td>&lt; 1.00E-07</td><td>1.00E-05</td><td>&lt; 1.00E-07</td><td>7.13E-04</td></tr><tr><td><i>C. subtsugae</i> coculture vs <i>P. syringae</i> coculture</td><td>8.38E-01</td><td>6.48E-03</td><td>5.45E-02</td><td>7.68E-01</td><td>9.27E-03</td><td>9.11E-01</td></tr><tr><td><i>P. syringae</i> coculture vs 3-member</td><td>3.28E-01</td><td>9.99E-01</td><td>1.00E-07</td><td>6.50E-03</td><td>4.32E-03</td><td>4.24E-02</td></tr><tr><td><i>C. subtsugae</i> coculture vs 3-member</td><td>6.08E-02</td><td>4.20E-03</td><td>1.76E-03</td><td>7.76E-02</td><td>9.97E-01</td><td>6.20E-03</td></tr></tbody></table>

430

431

<footnote><sup>a</sup>Rhamnolipid congener Rha-Rha-C14-C14</footnote>
427

428

429

**Supplementary Table S9.** TukeyHSD post-hoc results comparing quantitation of identified secondary metabolites between community memberships with *B. thailandensis* membership. Values represent the adjusted P-value.

<table><thead><tr><th></th><th>Bactobolin</th><th>Capistruin</th><th>Melleilactone</th><th>Rhamnolipid<sup>a</sup></th><th>Thailandamide</th><th>Pyochelin</th></tr></thead><tbody><tr><td>Monoculture vs <i>P. syringae</i> coculture</td><td>8.41E-01</td><td>8.00E-07</td><td>4.00E-07</td><td>2.26E-01</td><td>1.25E-04</td><td>4.85E-01</td></tr><tr><td>Monoculture vs <i>C. subtsugae</i> coculture</td><td>3.54E-01</td><td>8.45E-02</td><td>&lt; 1.00E-07</td><td>2.53E-02</td><td>&lt; 1.00E-07</td><td>8.55E-01</td></tr><tr><td>Monoculture vs 3-member</td><td>8.27E-01</td><td>5.00E-07</td><td>&lt; 1.00E-07</td><td>1.00E-05</td><td>&lt; 1.00E-07</td><td>7.13E-04</td></tr><tr><td><i>C. subtsugae</i> coculture vs <i>P. syringae</i> coculture</td><td>8.38E-01</td><td>6.48E-03</td><td>5.45E-02</td><td>7.68E-01</td><td>9.27E-03</td><td>9.11E-01</td></tr><tr><td><i>P. syringae</i> coculture vs 3-member</td><td>3.28E-01</td><td>9.99E-01</td><td>1.00E-07</td><td>6.50E-03</td><td>4.32E-03</td><td>4.24E-02</td></tr><tr><td><i>C. subtsugae</i> coculture vs 3-member</td><td>6.08E-02</td><td>4.20E-03</td><td>1.76E-03</td><td>7.76E-02</td><td>9.97E-01</td><td>6.20E-03</td></tr></tbody></table>

430

431

<footnote><sup>a</sup>Rhamnolipid congener Rha-Rha-C14-C14</footnote>
**Supplementary Table S10.** Relative gene expression of genes in the thailandamide operon across different SynCom conditions. *B. thailandensis* (31 wells) in M9-0.067% glucose was the control condition and *rpoD* was the reference gene.

<table><thead><tr><th></th><th>thaF</th><th>thaK</th><th>thaQ</th></tr></thead><tbody><tr><td>Bt (62 wells)</td><td>0.523</td><td>0.550</td><td>0.650</td></tr><tr><td>Bt (93 wells)</td><td>0.303</td><td>0.138</td><td>0.188</td></tr><tr><td>Bt-Ps (31 wells/member)</td><td>1.311</td><td>1.675</td><td>2.163</td></tr><tr><td>Bt-Cv (31 wells/member)</td><td>2.375</td><td>2.304</td><td>1.05</td></tr><tr><td>Bt-Cv-Ps (31 wells/member)</td><td>2.048</td><td>2.433</td><td>1.742</td></tr></tbody></table>

435

436
**Supplementary Table S10.** Relative gene expression of genes in the thailandamide operon across different SynCom conditions. *B. thailandensis* (31 wells) in M9-0.067% glucose was the control condition and *rpoD* was the reference gene.

<table><thead><tr><th></th><th>thaF</th><th>thaK</th><th>thaQ</th></tr></thead><tbody><tr><td>Bt (62 wells)</td><td>0.523</td><td>0.550</td><td>0.650</td></tr><tr><td>Bt (93 wells)</td><td>0.303</td><td>0.138</td><td>0.188</td></tr><tr><td>Bt-Ps (31 wells/member)</td><td>1.311</td><td>1.675</td><td>2.163</td></tr><tr><td>Bt-Cv (31 wells/member)</td><td>2.375</td><td>2.304</td><td>1.05</td></tr><tr><td>Bt-Cv-Ps (31 wells/member)</td><td>2.048</td><td>2.433</td><td>1.742</td></tr></tbody></table>

435

436
437 Supplementary Table S11. Network summary results from interspecies coexpression

438 networks.

<table><thead><tr><th rowspan="2">Network</th><th colspan="2">B. thailandensis-C. subtsugae</th><th colspan="2">B. thailandensis-P. syringae</th></tr><tr><th>B. thailandensis</th><th>C. subtsugae</th><th>B. thailandensis</th><th>P. syringae</th></tr></thead><tbody><tr><td><strong>Member</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Total nodes</strong></td><td>2701</td><td>2043</td><td>3254</td><td>3478</td></tr><tr><td><strong>Nodes with only intraspecies edges</strong></td><td>2418</td><td>1814</td><td>2749</td><td>2996</td></tr><tr><td><strong>Nodes with interspecies edges</strong></td><td>283</td><td>229</td><td>505</td><td>482</td></tr><tr><td><strong>Total edges</strong></td><td>9382</td><td>7240</td><td>15801</td><td>23319</td></tr><tr><td><strong>Intraspecies edges</strong></td><td>9074</td><td>6932</td><td>15056</td><td>22574</td></tr><tr><td><strong>Interspecies edges</strong></td><td></td><td>308</td><td></td><td>745</td></tr></tbody></table>

439

440
437 Supplementary Table S11. Network summary results from interspecies coexpression

438 networks.

<table><thead><tr><th rowspan="2">Network</th><th colspan="2">B. thailandensis-C. subtsugae</th><th colspan="2">B. thailandensis-P. syringae</th></tr><tr><th>B. thailandensis</th><th>C. subtsugae</th><th>B. thailandensis</th><th>P. syringae</th></tr></thead><tbody><tr><td><strong>Member</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total nodes</td><td>2701</td><td>2043</td><td>3254</td><td>3478</td></tr><tr><td>Nodes with only intraspecies edges</td><td>2418</td><td>1814</td><td>2749</td><td>2996</td></tr><tr><td>Nodes with interspecies edges</td><td>283</td><td>229</td><td>505</td><td>482</td></tr><tr><td>Total edges</td><td>9382</td><td>7240</td><td>15801</td><td>23319</td></tr><tr><td>Intraspecies edges</td><td>9074</td><td>6932</td><td>15056</td><td>22574</td></tr><tr><td>Interspecies edges</td><td></td><td>308</td><td></td><td>745</td></tr></tbody></table>

439

440
441 **Supplementary Table S12.** Primers used for RT-qPCR analysis of genes in the

442 thailandamide operon.

<table><thead><tr><th>Primer</th><th>Sequence (5' > 3')</th><th>Product size (bps)</th><th>Reference</th></tr></thead><tbody><tr><td>rpoD_F</td><td>ACCGTCGTGGCTACAAATTC</td><td rowspan="2">117</td><td rowspan="2">[3]</td></tr><tr><td>rpoD_R</td><td>TCGTCTCGATCATGTGAACC</td></tr><tr><td>thaF_F</td><td>CATGCACGCGTTTCTGTTTC</td><td rowspan="2">113</td><td rowspan="2">This study</td></tr><tr><td>thaF_F</td><td>TCGTAGCCCAAGATCTCGTT</td></tr><tr><td>thaK_F</td><td>GGTATTGAGGCCATGAACGT</td><td rowspan="2">104</td><td rowspan="2">This study</td></tr><tr><td>thaK_F</td><td>CATCAGCAGATTCGCGAAAC</td></tr><tr><td>thaQ_F</td><td>GAACGCGTCGAAGGATTTTC</td><td rowspan="2">115</td><td rowspan="2">This study</td></tr><tr><td>thaQ_F</td><td>ATTCGTTCGGGTACTTCTGC</td></tr></tbody></table>

443

444
441 **Supplementary Table S12.** Primers used for RT-qPCR analysis of genes in the

442 thailandamide operon.

<table><thead><tr><th>Primer</th><th>Sequence (5' > 3')</th><th>Product size (bps)</th><th>Reference</th></tr></thead><tbody><tr><td>rpoD_F</td><td>ACCGTCGTGGCTACAAATTC</td><td rowspan="2">117</td><td rowspan="2">[3]</td></tr><tr><td>rpoD_R</td><td>TCGTCTCGATCATGTGAACC</td></tr><tr><td>thaF_F</td><td>CATGCACGCGTTTCTGTTTC</td><td rowspan="2">113</td><td rowspan="2">This study</td></tr><tr><td>thaF_F</td><td>TCGTAGCCCAAGATCTCGTT</td></tr><tr><td>thaK_F</td><td>GGTATTGAGGCCATGAACGT</td><td rowspan="2">104</td><td rowspan="2">This study</td></tr><tr><td>thaK_F</td><td>CATCAGCAGATTCGCGAAAC</td></tr><tr><td>thaQ_F</td><td>GAACGCGTCGAAGGATTTTC</td><td rowspan="2">115</td><td rowspan="2">This study</td></tr><tr><td>thaQ_F</td><td>ATTCGTTCGGGTACTTCTGC</td></tr></tbody></table>

443

444
<table><caption>**Supplementary Table S13.** RT-qPCR efficiencies of reference and target genes for relative expression analysis of genes in the thailandamide operon.</caption><thead><tr><td>Gene</td><td>Slope</td><td>R<sup>2</sup></td><td>Efficiency (%)</td></tr></thead><tbody><tr><td>rpoB</td><td>-3.424</td><td>0.999</td><td>102.6</td></tr><tr><td>thaF</td><td>-3.204</td><td>0.996</td><td>105.2</td></tr><tr><td>thaK</td><td>-3.358</td><td>0.995</td><td>98.5</td></tr><tr><td>thaQ</td><td>-3.305</td><td>0.997</td><td>100.7</td></tr></tbody></table>

445

446

447

448

449
445

<caption>**Supplementary Table S13.** RT-qPCR efficiencies of reference and target genes for relative expression analysis of genes in the thailandamide operon.</caption>

446

<table><thead><tr><td>Gene</td><td>Slope</td><td>R<sup>2</sup></td><td>Efficiency (%)</td></tr></thead><tbody><tr><td>rpoB</td><td>-3.424</td><td>0.999</td><td>102.6</td></tr><tr><td>thaF</td><td>-3.204</td><td>0.996</td><td>105.2</td></tr><tr><td>thaK</td><td>-3.358</td><td>0.995</td><td>98.5</td></tr><tr><td>thaQ</td><td>-3.305</td><td>0.997</td><td>100.7</td></tr></tbody></table>

447

448

449