Article Figures & Data
Figures
- Fig. 1.
Schematic diagram of the genetic organization of the B. avium chromosome spanning fhaC-BAV1966, and the sizes (in base pairs) of the DNA regions in between the predicted coding sequences of each gene. Where no size is given, the adjacent coding sequences have overlapping start and stop codons.
- Fig. 2.
RT-PCR analysis of transcription of the B. avium BAV1962-1965 fimbrial locus. RT-PCR products were separated by agarose electrophoresis on a 1.5% gel and stained with ethidium bromide. L, 100-bp DNA ladder. Templates: lane 1, B. avium genomic DNA positive control; lane 2, negative control. No template: RNA from bacteria grown at 22°C (lane 3) or 37°C (lane 4), without reverse transcription, and cDNA reverse transcribed from RNA from bacteria grown at 22°C (lane 5) or 37°C (lane 6).
- Fig. 3.
Transmission electron micrographs of B. avium cells negatively stained with ammonium molybdate. Putative fimbria-like cell surface appendages (highlighted by arrows) were observed on WT bacteria grown at 37°C (A) or 22°C (B). Flagella were observed only on WT bacteria grown at 22°C (C). Immunostaining of bacteria with anti-BAV1965 antiserum and colloidal gold particles revealed immunoreactive material on WT bacteria grown at 37°C (D and E) but not on a BAV1965-1962 deletion mutant (F) or WT bacteria grown at 22°C (data not shown). Due to space constraints, images representative of many are shown.
Tables
- Table 1.
Sequences of primers used in this study
Primer Left sequence Right sequence Fimbrial locus knockout BAV1962-1965 upstream 5′ATCAGGAAGTTCTGAATAGAGCA3′ 5′GCAAGCTTGTAAGGAATCAAAG3′ BAV1962-1965 downstream 5′CCTGGGATTTCTCTCAACG3′ 5′ATGGCCTTCCTCTTTCAAAT3′ RT-PCR tyrB cDNA 5′GCTTCAAAGTCGAAACCTACAC3′ 5′GGTTAGATTGCGTGTTGTAGCTG3′ BAV1965 cDNA 5′GATCACTATTCGCGGTGAAATC3′ 5′GCCAGGCTCAAAATAAATATGG3′ BAV1964 cDNA 5′CTCTCTCGGCCTGGTATTTCTT3′ 5′AAAGGGCACCTCCATTTGAT3′ BAV1963 cDNA 5′TGTCTTATAGCAAGACCCTGGA3′ 5′GAGAGATAGAGGCTGCCGTAAC3′ BAV1962 cDNA 5′GGGAGTTGTTCAGTCATGTCAC3′ 5′GAATCCGTAGTTCTCCAGTTGC3′ BAV1965 expression construct 5′TTCATATGACCATGCATACGATCAAAAAA3′ 5′TTGAATTCTCAGGGGTACATGACAGAGAA3′ - Table 2.
Homologues of the B. avium putative fimbrial proteinsa
Protein Closest homologue(s) Amino acid identity (%) Conserved domain(s) BAV1965 BB3425/FimNBB 48 pfam00419 BB2992/FimABB 47 Fimbrial protein BAV1964 BB2991/FimBBB 52 pfam00345, pfam02753, Gram-negative pilus assembly BAV1963 BB2990/FimCBB 46 pfam00577, fimbrial usher BAV1962 BB2989/FimDBB 25 pfam00419, fimbrial protein BAV1773 BB2354 28 COG3539 BB0338 21 FimA pilin protein BAV1774 BB2990/FimCBB 30 pfam00577, fimbrial usher BAV1775 BB2991/FimBBB 34 pfam00345, Gram-negative pilus assembly BAV1776 STM0200/fimbrial protein—Salmonella typhimurium, LT2 28 pfam00419, fimbrial protein BAV1777 BB3426/FimXBB, BB3425/FimNBB, BB2991/Fim2BB, BB1658/Fim3BB 27 pfam00419, fimbrial protein ↵a B. avium fimbrial biogenesis proteins were subject to BLASTP analysis and conserved domain analysis. BB, B. bronchiseptica.
- Table 3.
Relative quantification of BAV1965 expression at 22°C and 37°C using the 2−ΔΔCT method
Temp CT ΔCTa ΔΔCTb Normalized BAV1965 amtc BAV1965 tyrB 22°C 22.06 14.11 21.88 14.15 22.08 13.93 Avg 22.01 ± 0.11 14.06 ± 0.12 7.94 ± 0.21 0.00 ± 0.21 1.00 (0.87–1.16) 37°C 17.48 15.87 17.92 15.88 17.62 15.90 Avg 17.67 ± 0.23 15.88 ± 0.02 1.79 ± 0.22 −6.15 ± 0.22 71.18 (60.967–83.097) - Table 4.
Adherence of WT and B. aviumΔ1965-1962 to turkey tracheal ring cultures
Strain Fraction of inoculum adhered SD WT 0.0365 0.0251 B. avium Δ1965-1962 0.0083 0.0049a B. avium Δ1965-1962(pBBR1fim) 0.0345 0.0212 B. avium Δ1965-1962(pBBR1) 0.0050 0.0016a ↵a Significantly different (P < 0.05) from values for either WT or the deletion mutant complemented by carriage of BAV1965-1962 on a plasmid (pBBR1fim) as calculated by Student's t test.
