Departamento de Microbiologia, Universidade de São Paulo, São Paulo, Brazil.
BMC Microbiol. 2011 Nov 8;11:248. doi: 10.1186/1471-2180-11-248.
Microbiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor σ70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity.
We found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB.
Given that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.
微生物学研究经常涉及实验室和/或菌种中心之间的菌株交换。出于存档原因和确保可重复的实验结果，交换菌株的完整性至关重要。至少 50 年来，运输细菌最常用的方法之一是将细菌样本接种在琼脂斜面上。斜面上的长期培养会表现出遗传不稳定性，其中一种常见的不稳定性是 rpoS。当受到多种压力和处于静止期时，sigma 因子 RpoS 会积累。RpoS 积累的一个后果是与营养型 sigma 因子 σ70 竞争。在营养限制条件下，rpoS 或调节其表达的基因发生突变的倾向会积累。在这里，我们研究了在斜面上进行短期储存和邮寄培养是否会导致遗传异质性。
我们发现，我们实验室之间通过邮件三次交换的大肠杆菌 K-12 菌株 MC4100TF 的样本变得异质。重建研究表明，LB 斜面上的样品表现出在静止期 LB 肉汤中的 GASP 研究中发现的突变。至少 40%的重建库存和同等比例的实际邮寄库存都含有这些突变。在斜面上孵育 7 天内，低 RpoS 水平的突变体出现。对这 10 个分离株的序列分析表明，它们都含有三种不同的 rpoS 突变。这些突变体表现出缺乏 rpoS 的细菌的典型表型。MC4100TF 的遗传稳定性也在嵌入甘油的滤纸片上进行了测试。在这些条件下，GASP 突变体仅在 3 周后才出现。我们还证实，MC4100TF 中固有较高的 RpoS 水平主要是由于 rssB 中存在 IS1 插入。
鉴于许多大肠杆菌菌株含有类似于 MC4100TF 的高 RpoS 水平，因此在转移和储存过程中此类菌株的完整性值得怀疑。重要收藏的变化可能是由于存储转移相关问题引起的。这些结果对细菌档案和转移菌株的完整性提出了重要问题，解释了实验室之间 ECOR 收藏的变化，并表明需要开发更好的菌株转移方法。
