Maltose

For the synthesis of the Glyc•RadiFluor, 1,2-dimethyl-3,5-dinitrobenzene was purchased
from 1ClickChemistry (Kendall Park, NJ, USA) while all other chemicals
(see Scheme S1) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Maltose, maltohexaose, and maltoheptaose standards
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Maltotriose
was purchased from Thermo Scientific (Ward Hill, MA, USA). Maltotetraose
and maltopentaose were acquired from Biosynth Carbosynth (Louisville,
KY, USA). GPC-grade dextran ladder was purchased from Sigma-Aldrich
(Milwaukee, WI, USA). Lacto-N-difucohexaose I (LNDFH
I) and lacto-N-difucohexaose II (LNDFH II) were obtained
from Dextra Laboratories (Reading, UK). Bovine pancreas ribonuclease
B (RNase B) and peptide-N-glycosidase F (PNGase F) were purchased
from New England Biolabs (Ipswich, MA, USA). LC-MS grade ammonium
formate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All
solvents that were used for the purification and analysis of the samples
described herein were of HPLC-grade and purchased from Fisher Scientific
(Nazareth, Pennsylvania, USA). The procedure for glycan release from
RNase B is detailed in the Supporting Information.

First, the growth potential of the LAB strains mentioned above was monitored in WSSM (125 (link)) and in mWSSM at 30°C for 28 h. WSSM contained 0.5 g/L of fructose (Merck), 0.5 g/L of glucose (Merck), 10.0 g/L of maltose (Merck), 2.0 g/L of sucrose (Merck), 12.0 g/L of wheat peptone (Merck), 12.0 g/L of yeast extract (Oxoid), 4.0 g/L of dipotassium phosphate (Merck), 0.2 g/L of magnesium sulfate (Merck), 0.05 g/L of manganese (II) sulfate (Merck), 4.0 g/L of potassium phosphate (Merck), 1.0 mL/L of Tween 80 (Sigma-Aldrich), and 1 mL/L of a vitamin solution (cobalamine, folic acid, nicotinamide, pantothenic acid, pyridoxal-phosphate, and thiamine; 0.2 g/L each). The mWSSM was obtained by the addition of an ester precursor mixture to WSSM. This ester precursor mixture consisted of ethanol (1.0%, vol/vol), acetic acid (0.5%, vol/vol), the organic acids propionic acid, butyric acid, pentanoic acid, hexanoic acid, decanoic acid, and lactic acid (5.0 mg/L each), and the higher alcohols 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and isoamyl alcohol (5.0 mg/L each). Therefore, a single colony of each strain was grown in 5 mL of liquid mMRS-5 medium at 30°C for 24 h. These liquid cultures were used to inoculate (1.0%, vol/vol) 100 mL of WSSM or mWSSM in Scott bottles (VWR International), in duplicate. To draw a growth curve, the optical density at 600 nm was measured (Genesys 20; Sigma-Aldrich) every 2 h. R package GrowthCurver (126 (link)) was used to fit the growth of each strain with the logistic equation
where N_t is the population size at time t, N₀ is the initial population size, K is the maximum population size in the particular environment, and r is the growth rate that would occur if there were no restrictions imposed on the total population size.

Cells were grown in HL5 culture medium without glucose (Formedium) supplemented with 18 g/l maltose (Sigma) and 50 μg/ml ampicillin and 40 μg/ml streptomycin. Cells were grown at 22 °C in polystyrene dishes or shaken in Erlenmeyer flasks at 150 rpm. The parental strain is the axenic Dictyostelium discoideum AX2 strain, which is referred to as wild type. Knockout strains in AX2 background used in this work: iqgC-null [55 (link)], rasG-null [58 (link)], talA-null [14 (link)], paxB-null [21 (link)], myoVII-null (this work). Cell lines rasG-null (DBS0236862), talA-null (DBS0236177) and paxB-null (DBS0236728) are available from the Dicty Stock Center repository, while iqgC-null (DBS0351225) and myoVII-null strains are available upon request from the corresponding author. Cell lines were transfected by electroporation according to the standard protocol. In brief, 1 × 10⁷ cells were washed with electroporation buffer (10 mM potassium phosphate buffer, pH 6.1, 50 mM glucose), and approximately 1 μg of plasmid DNA was electroporated into the cells by pulsing twice at 1000 V with the Xcell gene pulser (Biorad). The cells were incubated with subsequent addition of healing solution (2 mM CaCl₂, 2 mM MgCl₂) and transferred to HL5 medium. After 6–18 h, 10 μg/ml G418 was added and the transfectants were maintained in the presence of 10–20 μg/ml G418. For the comparison of GFP-IqgC dynamics in wild-type and iqgC-null cells, cells were grown on Klebsiella aerogenes lawns on SM agar plates, collected, washed with ice-cold H40 buffer, and transformed using 0.5 µg of pDM1207_IqgC as described [59 (link)]. Wild-type and knockout cells expressing GFP-IqgC were grown in the SorMC buffer in the presence of bacteria and 10 µg/ml of G418.