Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.

Project description: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.

Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high. Detection of gene expression variance among Frankia HfpCci3 (Cci3) cells grown in ammonium chloride for three days, five days and HfpCci3 cells grown in nitrogen fixing conditions for three days using mRNA-seq

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Streptococcus sp. NM strain:NM

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