Supporting Information Welander and Summons 10.1073/pnas.1208255109 SI Text SI Materials and Methods. Physiological studies. For all physiological

studies, 10 mL starter cultures of Methylococcus capsulatus strains were grown for 3 d in nitrate minimal salts (NMS). A 2% inoculum from this starter culture was then used to inoculate 50 mL of NMS in triplicate. For growth curves, absorbance was measured by transferring a 100 μL aliquot to a 96-well plate and measuring the optical density at 600 nm on a Synergy 2 Microplate Reader (BioTek). Methane in the headspace was measured by removing 100 μL of the headspace in a gas tight Hamilton syringe and injecting into a GC-2014 Gas Chromatogram (Shimadzu) equipped with a flame ionization detector and a Carboxen1000 capillary column. For survival assay, aliquots of each culture were serially diluted and colony forming units (cfu) were determined using the drop plate method (1). For transmission electron microscopy, samples were fixed on day 2 and day 14 of growth by adding an equal amount of a standard morphology fixative (2% gluteraldehyde, 3% paraformaldehyde, 5% sucrose in 0.1 M sodium cacadylate buffer) to the culture. Fixed cells were submitted to the W.M. Keck Biological Imaging Facility (Whitehead Institute) for processing and imaging. Construction of M. capsulatus hpnR mutant. The deletion plasmid construct pPVW87 was constructed by cloning 1 kb of the hpnR upstream and downstream regions into the suicide vector pJQ200SK (2) as previously described (3). To delete hpnR, an overnight culture (5 mL) of Escherichia coli BW29427, a dap auxotroph requiring diaminopimelic acid (DAP) supplementation 1. Herigstad B, Hamilton M, Heersink J (2001) How to optimize the drop plate method for enumerating bacteria. J Microbiol Methods 44:121–129. 2. Quandt J, Hynes MF (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127:15–21.

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for growth, carrying pPVW87 was pelleted, rinsed three times with NMS, and resuspended in 0.5 mL of NMS. M. capsulatus was grown for 3 d in 50 mL NMS plus methane and the entire culture was pelleted and resuspended in 0.5 mL of NMS. The two cultures were mixed, pelleted, and resuspended in 0.2 mL of NMS. The mixed cultures were spotted (50 μL∕spot) on NMS plates containing 600 μM DAP and incubated overnight in sealed jars at 37 °C. The spots were scraped off the agar with sterile sticks, resuspended in 1 mL of NMS and plated on NMS plus gentamicin. Plates were incubated for 10 d at 37 °C. One gentamicin-resistant colony was grown in NMS alone for 3 d and dilutions were plated on NMS plus 5% sucrose. Sucrose-resistant colonies were screened for deletion of hpnR with primers MCA11 and MCA14. One mutant was detected and genomic DNA was isolated from this strain and further verified by PCR (Fig. S1). Complementation of ΔhpnR. Primers (MCA33 and MCA34) were designed to PCR amplify hpnR with the addition of an NheI site at the 3′ end and fusing an NdeI site with the start codon at the 5′ end. The gene was ligated into NdeI/NheI cut pSRK-Km to generate pPVW100 and was transferred into the M. capsulatus hpnR mutant. For lipid analysis and physiological studies, the mutant strain carrying the complementation plasmid was grown at 37 °C for 3 d in 50 mL of NMS supplemented with kanamycin and 1 mM IPTG to induce expression of the complementing gene. 3. Welander PV, et al. (2009) Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. J Bacteriol 191:6145–6156.

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A upstream region

Deletion plasmid

gentR

sacB

downstream region gene of interest

wild type

A

select for gentamicin resistance

B

merodiploid gent R sucS sacB

gentR

sacB

removal of plasmid and deletion of gene of interest

gentR

removal of plasmid and regeneration of wild type

select for sucrose resistance

deletion mutant

wild type

B

1 WT

2 WT

3 WT

4 WT

3000 bp 2000 bp 1500 bp 1000 bp

500 bp

Fig. S1. (A) Strategy for constructing unmarked gene deletions in M. capsulatus. Two possible scenarios are shown. In both cases, homologous recombination results in insertion of the entire deletion plasmid next to the gene of interest. The resulting merodiploid is gentamicin resistant and sucrose sensitive. Sucrose sensitivity results from the acquisition of sacB. Growth of the merodiploid on nonselective medium promotes a second recombination event that can excise the plasmid with or without the gene of interest. In scenario A, the second recombination event results in generating a deletion mutant whereas in scenario B the wild type strain is regenerated. Loss of the plasmid is selected for by sucrose resistance. Screening of sucrose resistant colonies by PCR is required to identify deletion mutants. (B) PCR verification of ΔhpnR. WT, wild type; Δ, ΔhpnR. Expected band size for each PCR reaction: 1: WT: 535 bp, Δ: 0 bp; 2: WT: 4,147 bp, Δ: 2,599 bp; 3: WT: 3,090 bp, Δ: 1,542 bp; 4: WT: 3,354 bp, Δ: 1,806 bp. Primers used for each reaction are MCA27 and MCA28 (PCR 1), MCA 31 and MCA32 (PCR 2), MCA29 and MCA32 (PCR 3), and MCA30 and MCA31 (PCR 4) and listed in Table S1.

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411 – 2H

397 – 2H

OAc OAc

OAc OAc

OAc OAc NHAc

OAc OAc NHAc

aminotetrol

H3 C

aminopentol

3-methylaminotetrol

772.5356

Full MS

786.5513

600 650 700 750 800 850 900

3-methylaminopentol

830.5424

600 650 700 750 800 850 900

844.5576

600 650 700 750 800 850 900 191

191

from 772

from 786

205

395

652

409

300

450

600

750

m/z

395

300

450

600

750

150

300

m/z

450

m/z

544

710

726

150

485 409

666

712

150

from 844

471 530

546

532

600 650 700 750 800 850 900

205

from 830

487 473

OAc OAc NHAc

OAc OAc NHAc

726

712 H3C

MS-MS

OAc OAc OAc

OAc OAc OAc

205

191

600

750

724

150

300

450

600

750

m/z

Fig. S2. Mass spectra of the acetylated methyl and desmethyl aminobacteriohopanepolyols produced by M. capsulatus. The top row shows the protonated molecular ions, obtained under atmospheric pressure chemical ionization (APCI) conditions, of the four hopanoid structures from Fig. 2. Accurate mass measurements of these ions are, as follows: 772.5356 (calculated 772.5364), 786.5513 (calculated 786.5520), 830.5424 (calculated 830.5418), and 844.5576 (calculated 844.5575) consistent with the elemental compositions of C45 H74 NO9 , C46 H76 NO9 , C47 H76 NO11 , and C48 H78 NO11 , respectively. The bottom row shows the MS-MS fragmentation patterns of the molecular ion for each acetylated bacteriohopanepolyol (BHP). The spectra of corresponding methyl and desmethyl compounds are closely similar apart from the 14 Da shift of MH þ and those fragments (e.g., 191 plus 395 and 205 plus 409 Da) derived from the triterpane ring system. In each compound, high mass fragment ions correspond to sequential losses of acetic acid (HOAc) and these are also shifted 14 Da from each desmethyl BHP to its 3-methyl counterpart.

0.1

HpnR Sequences e-value < e-100 Fig. S3. Unrooted maximum likelihood tree of 192 radical S-adenosylmethionine (AdoMet) sequences retrieved through a translated Basic Local Assignment Search Tool (TBLASTN) query against the M. capsulatus HpnR sequence. Amino acid sequences were aligned via the Multiple Sequence Comparison by LogExpectation (MUSCLE) program, the tree was constructed through phylogenetic estimation using maximum likelihood (PhyML), and edited on the Interactive Tree of Life (iTOL). This tree demonstrates that sequences with an e-value lower than e −100 , shown in red bold, cluster together and most likely represent true HpnR homologues.

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M

A

l

Growth d Meth Consumption 2

0.14 0.12

1.5

y

0. 08 1 0.06

Growth

0.04

0.5

Methane

0.02 0

0 0

B

CH (mM)

0.1

2.5

5

7.5

10

12.5

15

0.14 0.12 0.1 0.08 0.06

wild type

0.04 0.02 0 0

2.5

5

7.5

10

12.5

15

Fig. S4. (A) Growth and methane consumption by wild type M. capsulatus and (B) growth of M. capsulatus and ΔhpnR in NMS medium at 37 °C as monitored by optical density over 2 wk. Each time point represents the average of three separate growth curves. The error bars are standard deviations and may not be visible beneath the point markers.

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Table S1. Strains, plasmids, and primers used in this study Strains E. coli DH10B BW29427 M. capsulatus strain Bath M. capsulatus ΔhpnR Plasmids

Genotype/Description

Source

Strain used for constructing plasmids F − endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC)λ − DAP auxotroph used for conjugation with M. capsulatusthrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341∷[erm pir (wt)]; wild type

D.K. Newman (California Institute of Technology, Pasadena, CA) D.K. Newman (California Institute of Technology, Pasadena, CA) M.G. Klotz (University of North Carolina, Charlotte, NC) This study

Open reading frame MCA0738 deleted Description

Primers

Mobilizable suicide vector; sacB Gm r Broad host range self-replicating plasmid; Km r lacZ hpnR upstream region (1 kb) PCR amplified with primers MCA11 and MCA12 and cloned into pJQ200SK at NotI/SpeI hpnR downstream region (1 kb) PCR amplified with primers MCA13 and MCA14 and cloned into pJQ200SK at SpeI/PstI hpnR downstream region subcloned from pPVW86 and ligated into pPVW85 at SpeI/PstI hpnR was PCR amplified with primers MCA33 and MCA34 and cloned into pSRK-Gm at NdeI/NheI for complementation of M. capsulatus ΔhpnR Sequence*

MCA11 MCA12 MCA13 MCA14 MCA27 MCA28 MCA29 MCA30 MCA31 MCA32 MCA33 MCA34

gaattcgcggccgcCAACCAGAGCGCCAATCT gaattcactagtGGGATCTCGGCTCCTTCCTT gaattcactagtCGACGCACGCCCGGAGATAT gaattcctgcagCGCCAGCTTCAGTATCTCGT CCGCAGCTATCGTGTAATGA GATGTCGAACAGGCGGTAGT TCCGTTTATCTGGTCGAAGC GGTGCCGACTTGAAAACTTC GAACGCCATTTCAACCAACT GGACGATCCACCTTGAATTG gaattccatatgAAGGTCTTCTGCGTACA gaattcgctagcTCAACCGGTCGCCGCTCCCA

pJQ200SK pSRK-Km pPVW85 pPVW86 pPVW87 pPVW100

(1) (2) This study This study This study This study Restriction sites EcoRI/NotI EcoRI/SpeI EcoRI/SpeI EcoRI/PstI

EcoRI/NdeI EcoRI/NheI

1 Quandt J, Hynes MF (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127:15–21. 2 Khan SR, Gaines J, Roop RM, Farrand SK (2008) Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 74:5053–5062.

*Restriction sites shown in lower case letters

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