Brickman T J, Armstrong S K
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354, USA.
J Bacteriol. 1996 Jan;178(1):54-60. doi: 10.1128/jb.178.1.54-60.1996.
Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.
通过对与插入标记相关的克隆基因组区域进行限制性图谱分析,发现定义支气管败血波氏杆菌铁载体表型互补III组突变体BRM3和BRM5的染色体插入片段相距约200至300 bp。使用支气管败血波氏杆菌基因组DNA序列对克隆的BRM3插入标记侧翼进行DNA杂交分析,鉴定出同源的百日咳博德特氏菌UT25黏粒,其可互补III组支气管败血波氏杆菌突变体的铁载体生物合成缺陷。亚克隆和互补分析将互补活性定位到一个2.8 kb的百日咳博德特氏菌基因组DNA区域。核苷酸测序确定了一个开放阅读框,预测其编码的多肽在一级氨基酸水平上与几种依赖磷酸吡哆醛的氨基酸脱羧酶具有高度相似性。通过在缺铁培养基中添加腐胺(1,4 - 二氨基丁烷),III组突变体的产碱菌素能力完全恢复,这与产碱菌素铁载体生物合成所需的鸟氨酸脱羧酶活性缺陷一致。同样,BRM3的产碱菌素生物合成缺陷可被编码鸟氨酸脱羧酶活性的异源大肠杆菌speC基因功能性互补。酶活性测定证实,III组支气管败血波氏杆菌铁载体缺陷突变体缺乏产碱菌素生物合成所需的鸟氨酸脱羧酶活性。通过等位基因交换构建的百日咳博德特氏菌类似突变体无法检测到铁载体的产生。我们提议将这些导致产碱菌素铁载体产生缺失的突变所定义的基因命名为odc。腐胺是博德特氏菌属中产碱菌素的必需前体。