A single genetic locus senses and protects the beneficial oral bacterium Streptococcus sp. A12 from a spectrum of antimicrobial peptides

BACKGROUND: While substantial advances have been made to understand the nature and significance of interspecies interaction in oral microbial communities, much remains to be learned about how commensal species contribute to the maintenance of health-associated biofilm communities. We report that successful competition of Streptococcus sp. A12 with the dental pathogen Streptococcus mutans requires many different factors. While A12 can directly inhibit the growth of S. mutans, A12 harbors several genes (i.e. pcfFEGRK) that are required to be able to tolerate antagonistic factors of S. mutans, and these gene products are critical to the competitive fitness of A12. Here, we delve deeper in the role of the pcfFEG, a predicted lantibiotic immunity transporter, and the pcfRK, its genetically-linked two-component system (TCS) in A12. METHODS: RNA-Seq was utilized to compare the transcriptomes of A12 wild-type strain and A12 lacking the response regulator (pcfR) or histidine kinase (pcfK) of pcfRK, a TCS directly regulating pcfFEG. Each strains were prepared in 3 biological replicates. Strains were grown to OD600 nm = 0.4 in BHI medium before harvest. Deep sequencing was performed at the University of Florida ICBR facilities (Gainesville, FL). Approximately 15 million short-reads were obtained for each sample. After removing adapter sequences from each short-read and trimming of the 3’-ends by quality scores, the resulting sequences were mapped onto the reference genome of strain Streptococcus sp. A12 (NCBI Reference Sequence: NZ_CP013651.1) using the short-read aligner. Mapped short-read alignments were then converted into readable formats using SAMTOOLS. RESULTS: Using an optimized data analysis workflow, we mapped 13-16 million reads per sample to the genome of A12. For viewing of the mapped reads aligned to the genome, .bam files were uploaded into the Integrative Genomics Viewer (IGV – version 2.3.55). A .csv file containing raw read counts for each replicate (3) was then uploaded to Degust (http://degust.erc.monash.edu/) and edgeR analysis performed to determine Log2 fold change and a false discovery rate (FDR). When compared to the wild-type A12 strain, 61 genes were differentially expressed in A12 lacking pcfR and 35 genes were differentially expressed in A12 lacking pcfK (Log2 fold change > (-)1.5, -log10 P-value > 4). Interestingly, a subset of the same genes was found to be upregulated in both pcfR and pcfK deletion strains, including ATM98_04215, ATM98_04220 and ATM98_03625, annotated as a protease, a dipeptidase (both co-transcribed), and an aminopeptidase, respectively. Another interesting finding was a cluster of genes predicted to be the mannose PTS system was downregulated in the pcfK mutant strain, and a two-component ABC transporter annotated as ABC-type CcmA multidrug resistance system was found to be upregulated in the pcfR mutant strain. The pcfK mutant strain showed an upregulation of the pcfFEG genes and the cognate response regulator gene, pcfR. In the pcfR mutant, pcfK was also upregulated, compared to the parental A12 strain. CONCLUSIONS: Transcriptional profiling of pcfR and pcfK mutant strains revealed the scope of the pcfRK regulon. When supplemented with functional genomics, we uncovered additional genes shown to function independently or cooperatively with PcfFEGRK in tolerating the lantibiotic nisin. Together these results highlight additional mechanisms beneficial species may be utilizing to remain competitive when existing in complex microbial communities. Transcriptional profiling of wild-type strain A12 and A12 lacking the response regulator (pcfR) or histidine kinase (pcfK) of pcfRK, a TCS directly regulating pcfFEG, a LanFEG-type ABC transporter shown to enhance tolerance to bacteriocins

背景：尽管我们在理解口腔微生物群落种间互作的本质与意义方面已取得长足进展，但关于共生菌如何助力维持健康相关生物被膜群落，仍有诸多问题亟待阐明。本研究报道，链球菌属（Streptococcus）菌株A12要与致龋病原菌变形链球菌（Streptococcus mutans）成功竞争，需要多种不同的调控因子。尽管A12可直接抑制变形链球菌的生长，但其自身携带多个基因（即pcfFEGRK），这些基因是A12耐受变形链球菌拮抗因子所必需的，且这些基因产物对A12的竞争适应度至关重要。本研究进一步深入探究了预测为羊毛硫抗生素（lantibiotic）免疫转运体的pcfFEG，以及与其遗传连锁的双组分系统（two-component system, TCS）pcfRK在A12中的作用。
实验方法：本研究利用RNA测序（RNA-Seq）比较野生型A12菌株，以及缺失直接调控pcfFEG的双组分系统pcfRK的应答调节因子pcfR或组氨酸激酶pcfK的A12菌株的转录组。所有菌株均设置3次生物学重复。将菌株在脑心浸液培养基（BHI）中培养至OD600 nm=0.4后收集样本。深度测序工作在美国佛罗里达大学ICBR设施（佛罗里达州盖恩斯维尔）完成，每个样本约获得1500万条短读长序列。对每条短读长序列去除接头序列，并基于质量值对3'端进行修剪后，利用短读长比对工具将处理后的序列比对至链球菌属菌株A12的参考基因组（NCBI参考序列号：NZ_CP013651.1）。随后使用SAMTOOLS工具将比对得到的短读长比对结果转换为可读格式。
实验结果：通过优化后的数据分析流程，我们将每个样本的1300万至1600万条读长序列比对至A12基因组。为查看比对至基因组的读长序列，将.bam文件上传至整合基因组浏览器（Integrative Genomics Viewer, IGV）版本2.3.55。将包含3次生物学重复原始读长计数的.csv文件上传至Degust平台（http://degust.erc.monash.edu/），并进行edgeR分析以计算对数2倍变化值（Log2 fold change）与错误发现率（false discovery rate, FDR）。与野生型A12菌株相比，缺失pcfR的A12菌株中有61个基因存在差异表达，缺失pcfK的A12菌株中有35个基因存在差异表达（筛选标准：Log2倍变化绝对值>1.5，-log10 P值>4）。有趣的是，在pcfR缺失株与pcfK缺失株中均有部分相同基因被上调，包括分别注释为蛋白酶、二肽酶（二者共转录）以及氨肽酶的ATM98_04215、ATM98_04220和ATM98_03625。另一项重要发现为：在pcfK突变株中，一组预测为磷酸转移酶系统（phosphotransferase system, PTS）甘露糖亚型的基因被下调；而在pcfR突变株中，注释为ABC型CcmA多药耐药系统的双组分ABC转运体被上调。pcfK突变株中，pcfFEG基因及其同源应答调节因子基因pcfR均呈现上调表达；与亲本A12菌株相比，pcfR突变株中pcfK的表达同样被上调。
研究结论：对pcfR与pcfK突变株的转录组分析明确了pcfRK调控子的调控范围。结合功能基因组学分析，我们还发现了更多可独立或与PcfFEGRK协同发挥作用的基因，这些基因参与增强菌株对羊毛硫抗生素乳链菌肽（lantibiotic nisin）的耐受性。综上，本研究结果揭示了有益微生物在复杂微生物群落中维持竞争优势的额外潜在机制。本研究对野生型A12菌株，以及缺失直接调控pcfFEG的双组分系统pcfRK的应答调节因子pcfR或组氨酸激酶pcfK的A12菌株进行了转录组分析——其中pcfFEG为LanFEG型ABC转运体，已被证实可增强菌株对细菌素的耐受性。