Rozsa F W, Marrs C F
Department of Epidemiology, University of Michigan, Ann Arbor 48109.
J Bacteriol. 1991 Jul;173(13):4000-6. doi: 10.1128/jb.173.13.4000-4006.1991.
The bacterium Moraxella lacunata is a causative agent of human conjunctivitis and keratitis. We have previously reported construction of plasmid pMxL1, which includes a 5.9-kb fragment on which the pilin gene inversion region of M. lacunata resides. The inversion region of pMxL1 was shown to invert when pMxL1 was in an Escherichia coli host cell. In this report, we present Western immunoblot analysis using Moraxella bovis Epp63 anti-I and anti-Q pilin sera which demonstrate that pMxL1 makes pilin only when in orientation 1. The sequence of the pMxL1 plasmid containing the invertible region contains a perfect tandem repeat of 19 bp in the orientation 1 nonexpressed pilin gene at the middle of the recombination junction site. This 19-bp insert causes a frameshift and disrupts the pilin gene. The predicted amino acid sequence of this nonfunctional pilin gene (with the 19-bp repeat subtracted) bears closest resemblance to M. bovis Epp63 Q pilin sequence, although the other (functional) M. lacunata pilin encoded by pMxL1 shows slightly higher homology to Q pilin. Comparison of the pMxL1 sequence with that of the M. bovis Epp63 sequence shows two other particularly interesting differences. One is a 15-bp sequence addition found in pMxL1 at the 60-bp region previously reported as a possible M. bovis recombinational enhancer. The second is an AT deletion in pMxL1 compared with Epp63 within an open reading frame (tfpB) which results in the pMxL1 tfpB open reading frame being one-third shorter than in Epp63. The DNA sequences in these three altered regions from the M. lacunata strain from which pMxL1 was derived were amplified by polymerase chain reaction and sequenced. The parent strain was found to contain the differences seen in pMxL1. Comparison of the M.bovis and M. lacunata pilin gene amino acid sequences is also presented.
腔隙莫拉菌是人类结膜炎和角膜炎的病原体。我们之前报道了质粒pMxL1的构建，该质粒包含一个5.9 kb的片段，腔隙莫拉菌的菌毛蛋白基因倒位区域位于其上。当pMxL1处于大肠杆菌宿主细胞中时，pMxL1的倒位区域会发生倒位。在本报告中，我们展示了使用牛莫拉菌Epp63抗I和抗Q菌毛蛋白血清进行的蛋白质免疫印迹分析，结果表明pMxL1仅在方向1时产生菌毛蛋白。包含可倒位区域的pMxL1质粒序列在重组连接位点中间的方向1非表达菌毛蛋白基因中含有一个19 bp的完美串联重复序列。这个19 bp的插入导致移码并破坏了菌毛蛋白基因。这个无功能菌毛蛋白基因（减去19 bp重复序列）的预测氨基酸序列与牛莫拉菌Epp63 Q菌毛蛋白序列最为相似，尽管pMxL1编码的另一个（功能性）腔隙莫拉菌菌毛蛋白与Q菌毛蛋白的同源性略高。将pMxL1序列与牛莫拉菌Epp63序列进行比较，发现了另外两个特别有趣的差异。一个是在pMxL1中，在先前报道的可能作为牛莫拉菌重组增强子的60 bp区域发现了一个15 bp的序列添加。第二个是与Epp63相比，pMxL1在一个开放阅读框（tfpB）内有一个AT缺失，这导致pMxL1的tfpB开放阅读框比Epp63中的短三分之一。通过聚合酶链反应扩增并测序了来自构建pMxL1的腔隙莫拉菌菌株的这三个改变区域的DNA序列。发现亲本菌株包含pMxL1中所见的差异。还展示了牛莫拉菌和腔隙莫拉菌菌毛蛋白基因氨基酸序列的比较。
