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Supplemental Material
Materials and Methods
Characterization and complementation of S. marcescens
To characterize β
CK2A4
-galactosidase-deficient Mariner transposon mutants, their total genomic DNA was isolated from overnight cultures with a GenElute Bacterial Genomic
DNA Extraction Kit (Sigma-
Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Genomic DNA (5 µg) was digested with RsaI (New England Biolabs,
Ipswich, MA, USA) for four hours at 37

C run on a 0.9% agarose gel in TAE buffer, and the DNA fragments of 1-4 Kb were excised from the gel and purified with the Illustra
GFX

PCR DNA/Gel Band Purification kit (GE Lifesciences, Buckinghamshire, UK).
The purified DNA fragments were incubated with T4 DNA ligase (New England Biolabs) under the conditions that favor intra-molecular ligation at 14

C overnight and resulting
DNA circles were subjected to inverse-
PCR (iPCR) using primers CJK94 5’-
GGCTTGAACGAATTGTTAGGTGGC3’ and CJK95 5’-
CGGCCGCGTAATACGACTCACTA3’ with the following cycle conditions (94 
C, 5 min;
35 cycles of 94

C, 1 min; 53

C, 1 min; 72

C, 2 min; final extension at 72

C, 10 min).
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In an attempt to complement the transposon mutation in CK2A4, the entire malEFG operon was PCR amplified from genomic DNA of S. marcescens PDL100 with primers

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CJK102
5’-GACCCACTATTACCGCGAAACGTC-3’ and CJK103 5’-
TCGATACCCTGATCTACGCCGC3’ and HiFi Platinum Taq polymerase (Invitrogen,
Carlsbad, CA, USA). The 4.8 Kbp PCR product was cloned into pCR2.1, generating plasmid (pCJK16) and subsequently sub-cloned into pBAD18-kan and pBBR1-kan using EcoRI to generate plasmids pCJK17 and pCJK18, which were transformed into chemically competent E. coli DH5 α . These plasmids were moved into CK2A4 through tri-parental mating using pRK600 as a helper plasmid. The complementation vector as well as empty cloning vectors were also electroporated (25 μF, 200 Ω, 2.5 kV, 0.2 cm cuvette, 50 μl cell volume) into each of the individual
E. coli malE, malF or malG mutants using a using a Bio-Rad MicroPulser (Bio-Rad Laboratories, Hercules, CA,
USA). Enzymatic activities in the mutants was tested with Miller assays for each enzyme.
Transductions of individual malE, malF, and malG mutants
Because plasmids containing S. marcescens PDL100 operon did not complement the phenotype of CK2A4, E. coli mutants in malE, malF and malG were constructed.
Individual E. coli
BW25113 mutants were obtained through the Keio Collection (Baba et al., 2006). As
E. coli strain BW25113 is lac -
(Hayashi et al., 2006), each of the mutants
was transduced into strain W3110 ( lac +
) using phage P1 (Lennox, 1955; Ikeda and
Tomizawa, 1965). The resulting frt-kan-frt marker was excised using pCP20 and the
plasmid was cured by incubation
at 37°C (Datsenko and Wanner, 2000). These
mutants were used for the complementation experiments using pCJK17 and pCJK18 plasmids and for the phenotypic assays. Activities of
β
-galactosidase, N-acetyl-
β
-D-

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65 glycopyranosidase, and
α
-D-glucopyranosidase were tested in the wild type and mutant strains of both S. marcescens and E. coli
(Miller, 1972).
Preliminary characterization of inhibitory compounds from Exiguobacterium sp. 33G8
In order to begin to determine the type of inhibitory compounds produced by
Exigobacterium sp. 33G8 in co-culture, cell-free supernatant from the 33G8-PDL100 coincubation experiment was extracted by flash ion exchange chromatography followed by
C18 resin chromatography. At the time of extraction the OD
600
of the 33G8 culture was
~1.5. 500 ml of the culture were pelleted at 14,000 rpm for 10 minutes using an Avanti
J-26 XP centrifuge (Beckman Coulter, Atlanta, GA, USA), the supernatant was transferred to a new centrifuge tube and centrifuged at 14,000 rpm for 10 minutes to remove all of the cells. The cell-free supernatant was applied to an ion exchange resin column (DOWEX-50W, Sigma-Aldrich, St. Louis, MO, USA). The flow-through was then subject to reverse phase Silica-C
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flash chromatography (Alltech Associates, Inc.,
Deerfield, IL, USA) in sequence. For both chromatographies, resin bed volume was 10 ml. DOWEX-50W ion exchange resin was charged with 10 bed volumes of HPLC water prior to the application of the sample. The reverse phase Si-C
18
resin was first washed in reagent ethanol and then equilibrated in 10 bed volumes of HPLC water.
Substances bound to the ion exchange resin were eluted with 3 bed volumes of 2N
NH
4
OH as in (Clark et al., 2011). The reverse phase Si-C
18
column was eluted with 3 bed volumes of 100% isopropanol.

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The NH
4
OH eluent was lyophilized and NH4OH was further sublimated for 48 hrs using a Freezone 18 freeze-drier (Labconco, Kansas City, MO, USA) and extracted in 4 ml of reagent ethanol and the remaining salts were dissolved in 1 ml HPLC water. The isopropanol eluent from the reverse phase Si-C
18
resin was rotary evaporated using a
Collegiate Rotavapor

V-
500 (Büchi Laboratories, Postfach, Switzerland) over water bath at 45

C, the remaining water was lyophilized. The yellowish residue in the boiling
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82 flask was first extracted with isopropanol, then with water:methanol (50:50 vol/vol).
Serial dilutions of these samples were used in bioassays.
References
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M. et al. (2006)
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2 : 2006.0008.
Clark, L.F., Johnson, J.V., and Horenstein, N.A. (2011) Identification of a gene cluster that initiates azasugar biosynthesis in Bacillus amyloliquefaciens . Chembiochem 12 :
2147-2150.
Datsenko, K.A., and Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97 : 6640-6645.
Hayashi, K., Morooka, N., Yamamoto, Y., Fujita, K., Isono, K., Choi, S. et al. (2006)
Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and
W3110. Mol Syst Biol 2 : 2006 0007.
Ikeda, H., and Tomizawa, J.I. (1965) Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J Mol Biol 14 : 85-109.
Lennox, E.S. (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1 : 190-206.
Miller, J.H. (1972) Experiments in Molecular Genetics . Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.