Baron G S, Nano F E
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Mol Microbiol. 1998 Jul;29(1):247-59. doi: 10.1046/j.1365-2958.1998.00926.x.
Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mgIAB, for Macrophage Growth Locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 or LB (X-p) media. Sequencing on mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgI mutants compared with wild-type F. novicida. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.
新凶手弗朗西斯菌是一种兼性胞内病原体，能够在巨噬细胞中生长。在含有显色磷酸酶底物5-溴-4-氯-3-吲哚基磷酸酯（X-p）的LB培养基上分离出一种在巨噬细胞中生长缺陷的新凶手弗朗西斯菌自发突变体，并将其命名为GB2。采用顺式互补策略，分离出了四株在巨噬细胞中恢复生长的菌株。从其中一个菌株中分离出的一个基因座可使GB2在细胞内生长缺陷和LB（X-p）培养基上的菌落形态得到恢复。该基因座由两个基因组成一个明显的操纵子，命名为mgIAB，即巨噬细胞生长基因座。mglA和mglB转座子插入突变体在细胞内生长方面均有缺陷，且具有与GB2或LB（X-p）培养基相似的表型。对从GB2克隆的mglA进行测序，发现了一个错义突变，这证明mglA和mglB都是新凶手弗朗西斯菌在巨噬细胞内生长所必需的。使用针对重组MglB的抗血清证实了GB2中mglB的表达。细胞分级分离研究揭示了mgI突变体与野生型新凶手弗朗西斯菌相比在蛋白质谱上的几个差异。mglA和mglB推导的氨基酸序列与大肠杆菌和嗜血杆菌属的SspA和SspB蛋白相似。在大肠杆菌中，SspA和/或SspB在营养应激条件下影响多种蛋白质的水平，并且SspA可以与RNA聚合酶全酶结合。综上所述，这些观察结果表明，在弗朗西斯菌中，MglA和MglB可能会影响其产物有助于在巨噬细胞内存活和生长的基因的表达。
