虽然细菌肽聚糖(PG)是高度保守的,PG生物合成和结构的一些自然变化已经进化。了解这种变异的机制和限制将有助于我们对抗生素耐药性的理解。先天免疫,和细菌的进化。我们通过阻断费氏弧菌中PG生物合成的必要步骤,然后选择具有恢复的原养型的突变体,探索了对PG进化的限制。这里,我们尝试选择D-谷氨酸营养缺陷型murIracD突变体的原养型抑制因子。在未补充的溶源肉汤盐(LBS)上没有分离出抑制剂,尽管电镀>1011个细胞,通过甲磺酸乙酯的诱变也没有产生任何抑制剂。在补充有iso-D-gln的LBS上分离出单个抑制器,尽管iso-D-gln随后显得无关紧要。这种抑制剂具有通过创建新的连接而形成的基因组扩增,该连接将proB融合到编码推定的广谱外消旋酶的基因。fischeri,bsrF.缺乏推定分泌信号的工程化bsrF等位基因(ΔSS-bsrF)也抑制了D-glu营养缺陷型,导致PG与野生型无法区分。ΔSS-bsrF等位基因类似地抑制了alr突变体的D-丙氨酸营养缺陷型,并将原养型恢复为D-ala和D-glu的murIalr双突变体营养缺陷型。ΔSS-bsrF等位基因增加了对D-环丝氨酸的抗性,但对PG靶向抗生素青霉素的敏感性没有影响,氨苄青霉素,或者万古霉素.我们的工作有助于定义对PG进化的约束,并揭示了费氏弧菌中的周质广谱消旋酶,可以用于PG生物合成,伴随D-环丝氨酸抗性。重要氨基酸是由生物在生命的所有领域中使用和产生的,但通常,他们的起源和角色没有得到很好的理解。在细菌中,D-ala和D-glu是典型肽聚糖细胞壁的结构成分,由专用的消旋酶Alr和MurI产生,分别。其他细菌消旋酶的最新发现正在拓宽我们的视野并加深我们对D-氨基酸代谢的理解。这里,在探索费氏弧菌的其他PG生物合成途径时,我们意外地发现了一种不寻常的消旋酶,BsrF.我们的结果说明了抗生素耐药性演变的新机制,并为探索非规范消旋酶和D-氨基酸在细菌中的作用提供了新的途径。
Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance.
D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.