Sphingomonas wittichiiRW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms
Sphingomonas wittichiiRW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms
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DOI：
10.1111/1758-2229.12276
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发表时间：
2015-06-01
影响因子：
3.3
通讯作者：
van der Meer, Jan R.
中科院分区：
文献类型：
--
作者：
Coronado, Edith;Valtat, Annabelle;van der Meer, Jan R.
为了更好地了解细菌进入污染物质的命运和活性，以提高生物降解率，我们构建了带有询问二苯并呋喃代谢活性的基因报告者的鞘氨单胞菌irw1变异。从二苯并呋喃代谢网络中选择三个潜在的启动子并融合到增强绿色荧光蛋白（EGFP）基因上。对RW1遗传结构的稳定性进行了检测，发现基于广泛宿主范围的载体pME6012的质粒是最可靠的。其中一个启动子位于2-羟基-2,4-戊二酸水合酶基因Swit_4925的上游，可以在二苯并呋喃上诱导生长。配有Swit_4925启动子egfp融合的威氏鞘单胞菌（Sphingomonas wittichiiRW1）在被二苯并呋喃和前气化场所的物质污染的各种非无菌沙质微生物中生长。该菌株在没有添加双苯并呋喃的情况下也能在微环境中生长，但生长程度非常有限，EGFP表达表明形成了一致的小细胞亚群，这些细胞亚群具有活跃的推断双苯并呋喃代谢网络。有证据表明，气化现场物质中常驻细菌对二苯并呋喃代谢物的竞争导致RW1种群的下降速度更快。本研究结果表明，低接种密度对观察引入菌群发育的重要性，并进一步说明了独特碳基质的有限可用性可能是影响生长的最重要因素。
In order to better understand the fate and activity of bacteria introduced into contaminated material for the purpose of enhancing biodegradation rates, we constructed Sphingomonas wittichiiRW1 variants with gene reporters interrogating dibenzofuran metabolic activity. Three potential promoters from the dibenzofuran metabolic network were selected and fused to the gene for enhanced green fluorescent protein (EGFP). The stability of the resulting genetic constructions in RW1 was examined, with plasmids based on the broad-host range vector pME6012 being the most reliable. One of the selected promoters, upstream of the gene Swit_4925 for a putative 2-hydroxy-2,4-pentadienoate hydratase, was inducible by growth on dibenzofuran. Sphingomonas wittichiiRW1 equipped with the Swit_4925 promoter egfp fusion grew in a variety of non-sterile sandy microcosms contaminated with dibenzofuran and material from a former gasification site. The strain also grew in microcosms without added dibenzofuran but to a very limited extent, and EGFP expression indicated the formation of consistent small subpopulations of cells with an active inferred dibenzofuran metabolic network. Evidence was obtained for competition for dibenzofuran metabolites scavenged by resident bacteria in the gasification site material, which resulted in a more rapid decline of the RW1 population. Our results show the importance of low inoculation densities in order to observe the population development of the introduced bacteria and further illustrate that the limited availability of unique carbon substrate may be the most important factor impinging growth.