Brucella medium

Strains and plasmids are described in Table 1. Burkholderia mallei was cultured at 37°C using Brucella medium (BD) supplemented with 5% (vol/vol) glycerol. When indicated, antibiotics were added at the following concentrations: 7.5 μg/mL Polymyxin B (MP Biomedicals), 5 μg/mL kanamycin (MP Biomedicals), 7.5 μg/mL zeocin (Life Technologies). Burkholderia mallei bacteria used to inoculate mice were cultured on agar plates and suspended in PBS to the indicated concentration, as previously reported [41 (link)]. Aliquots of the bacterial suspension were immediately spread onto agar plates to determine the number of colony forming units (CFU) in the inoculum. Escherichia coli was cultured at 37°C using Low Salt Luria Bertani (LSLB) agar (Teknova) supplemented with 15 μg/mL chloramphenicol, 50 μg/mL kanamycin, 100 μg/mL ampicillin (Sigma-Aldrich), or 50 μg/mL zeocin, where applicable. The cell lines A549 (human type II alveolar epithelium; ATCC CCL85) and J774A.1 (murine macrophages; ATCC TIB-67) were cultured as described elsewhere [14 (link), 38 (link)].

The bacterial strains, plasmids, and primers used in this experiment are listed in Table 1. Attenuated Salmonella and Escherichia coli strains were routinely grown at 37 °C in Luria–Bertani (LB) broth or agar with or without appropriate antibiotics unless otherwise indicated. B. abortus 544 strain was grown in Brucella medium (BD, Sparks, USA) at 37 °C in a 5% CO₂ atmosphere without antibiotics. Appropriate biosafety measures were enforced while handling B. abortus, a biosafety level 3 microorganism.Table 1

Bacterial strains, plasmids, and primers

Strain/Plasmid	Description	Reference
S. Typhimurium
JOL1800	∆lon, ∆cpxR, ∆asd and ∆wpaB mutant of S. Typhimurium	Lab stock
JOL2273	JOL1800 containing pJHL65:: L7/L12 and expressing l7/L12	Lab stock
JOL2080	JOL1800 carrying the empty vector	Lab stock
E. coli
BL21(DE3)pLysS	F−, ompT, hsdSB (rB−, mB−), dcm, gal, λ (DE3), pLysS, Cmr	Progma, USA
JOL1921	BL21(DE3)pLysS harboring pET28a + :: L7/L12	Lab stock
Plasmid
pET28a(+)	IPTG-inducible expression vector; Kanamycin resistant	Novagen, USA
pET28a + :: L7/L12	pET28a + derivative containing L7/L12	Lab stock
pJHL65	asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number	Lab stock
pJHL65:: L7/L12	pJHL65 harboring L7/L12	Lab stock
Primer
L7/L12 EcoRI F	5′-AGAGGAATTCATGGCTGATCTCGCAAAGAT-3′	This study
L7/L12 HindIII R	5′-AGAGAAGCTTTTACTTGAGTTCAACCTTGG-3′	This study

The human Mono Mac-6 (MM6) monocytic cell line was used to study the interaction of Bm with human monocytes. MM6 monocytes were also infected with the genetically-related, non-pathogenic Bt, as a comparison. Mono Mac-6 cells were obtained from the German Culture Collection (DSMZ no.: ACC 124), Braunschweig, Germany and were maintained in 24-well plates in 1 mL of RPMI 1640 containing 25 mM HEPES, supplemented with 10% fetal calf serum, 1% L-Glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 10 μg/mL of recombinant human insulin (Santa Cruz Biotechnology, CAS 11061–68-0) in a humidified 5% CO₂ atmosphere at 37 °C. Cell densities were maintained between 2.5 × 10⁵–1.0 × 10⁶ cells/mL, passaged every 3–4 days, and cultured until approximately passage 35. Bm ATCC23344 was cultured at 37 °C using Brucella medium (BD) supplemented with 5% (vol/vol) glycerol. Bt DW503, an amrAB-oprA efflux pump mutant of the parental strain Bt E264 that is highly sensitive to kanamycin, was cultured at 37 °C using Luria-broth (LB) medium. Bp K96243 was included in the binding assays and was prepared from cultures grown on Trypticase Soy Agar (BD) at 37 °C.