MULTIPLE REPLICONS CONSTITUTING THE GENOME OF PSEUDOMONAS-CEPACIA-17616
MULTIPLE REPLICONS CONSTITUTING THE GENOME OF PSEUDOMONAS-CEPACIA-17616
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DOI：
10.1128/jb.176.13.4034-4042.1994
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发表时间：
1994-07-01
影响因子：
3.2
通讯作者：
LESSIE, TG
中科院分区：
文献类型：
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作者：
CHENG, HP;LESSIE, TG
洋葱假单胞菌17616的DNA的宏观限制性片段分析，结合使用含有罕见的限制性内切酶位点的连接片段作为探针的Southern杂交实验，表明该细菌含有3个大的环状复制子3.4，2.5，和0.9兆碱基（Mb）。包含该菌株中存在的170-kb隐蔽质粒给出了7 Mb的基因组大小的总体估计。其他Southern杂交实验表明，这三个大的复制子包含rRNA基因，以及插入序列元件先前在该菌株中确定。测定三个复制子上Swa I、PacI和PmeI位点的分布。携带S，阿威位点的Tn 5 -751衍生物用于在2.5-和3.4-Mb复制子上的测序和定位基因。分离出突变体，其中2.5-和0.9-Mb复制子的大小分别减少到1.8和0.65 Mb。2.5 Mb复制子DNA的丢失与赖氨酸营养缺陷型、β-内酰胺酶缺乏以及不能利用核糖醇和海藻糖作为碳源和能源有关。通过脉冲场凝胶电泳在非限制性DNA中检测到大小与不同复制子的随机线性化形式相对应的DNA片段。这些结果为菌株17616的进一步遗传分析和其他洋葱假单胞菌分离株的基因组复杂性评估提供了框架。
Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a S,waI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.