Fig 1.
K. negevensis strain BB526 produces an exopolysaccharide.
Heat extracts from control K. kingae strains KK01 and KK01pam along with K. negevensis strains BB526 and BB526pam were separated using SDS-PAGE and were then stained with silver. The exopolysaccharide material is evident in the bracketed region of the gel.
Fig 2.
K. negevensis elaborates a polysaccharide capsule composed of N-acetylglucosamine (GlcNAc) and keto-deoxyoctulosonate (Kdo).
(A) Surface acid extracts from control isogenic K. kingae strains Swap-csa, Swap-csb, Swap-csc, Swap-csd, and KK01csaA and K. negevensis strains BB526, Sch1437, and D2292 were separated using SDS-PAGE and were then stained with Alcian blue. The high molecular weight stained material is the capsular polysaccharide. (B) Combined gas chromatography/mass spectrometry (GC/MS) spectrum of the polysaccharide capsule isolated from K. negevensis strain BB526 revealed an abundance of GlcNAc and Kdo.
Table 1.
Wild-type K. negevensis isolates used in this study.
Fig 3.
K. negevensis produce surface fibers.
Strains BB526 (A), BB526pilA (B), Sch1437 (C), Sch1437pilA (D), D2292 (E), and D2292pilA (F) were negatively stained and visualized using TEM. Abundant long surface fibers are present on the surface of the wild type strains BB526, Sch1437, and D2292 but are absent from the surface of each isogenic pilA mutant.
Fig 4.
K. negevensis surface fibers are type IV pili capable of mediating twitching motility.
(A) A multiple sequence alignment of the putative major pilin subunit from K. negevensis strains eburonensis, SW7208426, and Sch538 is shown. (B) Surface fibers were sheared from the bacterial surface, resolved by SDS-PAGE, and stained with Coomassie blue, revealing the major pilin subunit. The major pilin subunit band is evident in wild type strains BB526, D2292, and Sch1437 but is absent from the mutant strains BB526pilA, D2292pilA, and Sch1437pilA. (C) The twitching motility zones were for strains KK01, KK01pilA1, BB526, BB526pilA, D2292, D2292pilA, Sch1437, and Sch1437pilA were determined. The twitching zone diameter for each wild type and isogenic pilA mutant strain were shown to be significantly different using student’s T-test. (**p<0.005, ***p<0.0005).
Fig 5.
K. negevensis produces a Knh homolog.
Outer membrane fractions were isolated from strains KK03, KK03knh, BB526, Sch1437, and D2292, treated with formic acid, and separated using SDS-PAGE. Following transfer to nitrocellulose, samples were probed with GP97, an antiserum targeting the YadA-like head domains of K. kingae Knh. All three K. negevensis strains have a high molecular weight reactive band, indicating production of the Knh homolog, similar to the positive control K. kingae KK03.
Table 2.
K. kingae and K. negevensis mutant strains used in this study.