Abstract：
Over 300 essential genes are predicted using transposon sequencing in the genome of Pseudomonas aeruginosa. However, methods for reverse genetic analysis of essential genes are scarce. To address this issue, we developed a three-step protocol consisting of integration of deletion plasmid, introduction of temperature-sensitive rescue plasmid, and excision of integrated-deletion plasmid to construct the plasmid-based temperature-sensitive allele of essential genes. Using PA0006 as an example, we showed that PA0006(Ts) exhibited wild-type cell morphology at permissive temperature but filamentous form at restrictive temperatures. We further showed that the glycerol-mannoheptose-bisphosphate phosphatase GmhB in Escherichia coli shared 32.4% identity with that of PA0006p and functionally complemented the defect of PA0006(Ts) at 42°C. SDS-PAGE and Western blotting indicated the presence and absence of the complete core lipopolysaccharide (LPS) and B-band O-antigen in PA0006(Ts) at 30 and 42°C, respectively. An isolated suppressor sup displayed wild-type-like cell morphology but no complete core LPS or O-antigen. Genome resequencing together with comparative transcriptomic profiling identified a candidate suppressor fructose-bisphosphate phosphatase in which the promoter harbored a SNP and the transcription level was not downregulated at 42°C compared to 30°C in sup. It was further validated that fbp overexpression suppressed the lethality of PA0006(Ts) at 42°C. Taken together, our results demonstrate that PA0006 plays a role in regulation of cell morphology and biosynthesis of core LPS. This three-step protocol for construction of conditional lethal allele in P. aeruginosa should be widely applicable for genetic analysis of other essential genes of interest, including analysis of bypass suppressibility. IMPORTANCE Microbial essential genes encode nondispensable function for cell growth and therefore are ideal targets for the development of new drugs. Essential genes are readily identified using transposon-sequencing technology at the genome scale. However, genetic analysis of essential genes of interest was hampered by limited methodologies. To address this issue, we developed a three-step protocol for construction of conditional allele of essential genes in the opportunistic pathogen Pseudomonas aeruginosa. Using PA0006 as an example, we demonstrated that the plasmid-based PA0006(Ts) mutant exhibited defects in regulation of cell morphology, formation of intact core LPS, and attachment of the O-antigen at restrictive temperatures but not at permissive temperatures. A suppressor of PA0006(Ts) was isolated through spontaneous mutations and showed restored cell morphology but not core oligosaccharide or O-antigen. This method should be widely applicable for phenotype and suppressibility analyses of other essential genes of interest in P. aeruginosa.
摘要：
使用铜绿假单胞菌基因组中的转座子测序预测超过300个必需基因。然而,对必需基因进行反向遗传分析的方法很少。为了解决这个问题，我们开发了一个由缺失质粒整合组成的三步方案，引入温度敏感的拯救质粒，并切除整合缺失质粒，构建基于质粒的必需基因温度敏感等位基因。以PA0006为例，我们显示PA0006（Ts）在允许温度下表现出野生型细胞形态，但在限制性温度下表现出丝状形式。我们进一步表明，大肠杆菌中的甘油-甘露庚糖-二磷酸磷酸酶GmhB与PA0006p具有32.4％的同一性，并且在42°C时在功能上补充了PA0006（Ts）的缺陷。SDS-PAGE和Western印迹表明在30和42°C下PA0006（Ts）中存在和不存在完整的核心脂多糖（LPS）和B带O抗原，分别。分离的抑制子表现出野生型样细胞形态，但没有完整的核心LPS或O抗原。基因组重新测序与比较转录组分析一起鉴定了候选抑制因子果糖-二磷酸磷酸酶，其中启动子具有SNP，并且与sup中的30°C相比，转录水平在42°C未下调。进一步验证了fbp过表达在42°C下抑制PA0006(Ts)的致死性。一起来看,我们的结果表明，PA0006在调节细胞形态和核心LPS的生物合成中起作用。在铜绿假单胞菌中构建条件致死等位基因的三步方案应广泛适用于其他感兴趣的必需基因的遗传分析，包括旁路抑制性分析。重要性微生物必需基因编码细胞生长的非必需功能，因此是开发新药物的理想目标。使用转座子测序技术在基因组规模上容易地鉴定必需基因。然而,有限的方法阻碍了对重要基因的遗传分析。为了解决这个问题，我们开发了一个三步方案，用于在机会病原体铜绿假单胞菌中构建必需基因的条件等位基因。以PA0006为例，我们证明了基于质粒的PA0006（Ts）突变体在细胞形态调节方面表现出缺陷，完整核心LPS的形成，在限制性温度下而不是在允许的温度下附着O-抗原。通过自发突变分离出PA0006（Ts）的抑制子，并显示恢复的细胞形态，但未显示核心寡糖或O抗原。该方法应广泛适用于铜绿假单胞菌中其他感兴趣的必需基因的表型和抑制分析。
