Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U,S, Food and Drug Administration, Laurel, MD 20708, USA.
BMC Microbiol. 2013 May 1;13:94. doi: 10.1186/1471-2180-13-94.
The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C.
Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations.
Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway.
通过对大肠杆菌 K-12 中 aga/gam 调控子的生物信息学分析，以及对大肠杆菌 C 的研究，提出了 N-乙酰-D-半乳糖胺（Aga）和 D-半乳糖胺（Gam）在大肠杆菌中的分解代谢途径。在这个基因簇的十三个基因中，推测 agaA、agai 和 agaS 分别编码 Aga-6-P-脱乙酰酶、Gam-6-P 脱氨酶/异构酶和酮糖-醛缩酶同工酶的作用尚未通过实验测试。在这里，我们研究了它们在大肠杆菌 O157:H7 和大肠杆菌 C 中利用 Aga 和 Gam 的作用。
构建了 agaA、agai 和 agaS 的敲除突变体，以测试它们在利用 Aga 和 Gam 中的作用。还构建了 N-乙酰葡萄糖胺（GlcNAc）途径基因 nagA 和 nagB 的敲除突变体，分别编码 GlcNAc-6-P 脱乙酰酶和葡萄糖胺-6-P 脱氨酶/异构酶，以及双敲除突变体ΔagaAΔnagA 和ΔagaIΔnagB，以研究这些酶在 Aga/Gam 和 GlcNAc 途径之间是否存在相互作用。结果表明，Aga 利用不受ΔagaA 突变体的影响，但ΔagaAΔnagA 突变体在 Aga 和 GlcNAc 利用中被阻断。大肠杆菌 CΔnagA 不能在 GlcNAc 上生长，但当 aga/gam 调控子被组成型表达时，它可以生长。用 agaA 或 nagA 互补ΔagaAΔnagA 突变体，可在 Aga 和 GlcNAc 上生长。还发现，ΔagaI、ΔnagB 和ΔagaIΔnagB 突变体在利用 Aga 和 Gam 时不受影响。重要的是，ΔagaS 突变体在利用 Aga 和 Gam 时被阻断。通过实时 RT-PCR 对这些具有不同遗传背景的菌株中相关基因的表达分析支持了这些观察结果。
ΔagaA 突变体中 Aga 的利用不受影响，因为 nagA 被表达并替代了 agaA。用 agaA 或 nagA 互补ΔagaAΔnagA 突变体也表明，agaA 和 nagA 可以相互替代。ΔagaI、ΔnagB 和ΔagaIΔnagB 突变体在利用 Aga 和 Gam 时不受影响，表明 agaI 和 nagB 都不参与 Gam-6-P 的脱氨和异构化。我们提出 agaS 在 Aga/Gam 途径中编码 Gam-6-P 脱氨酶/异构酶。
