Methylobacillus flagellatus strain:A12
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PROVIDER: PRJNA666759 | ENA |
REPOSITORIES: ENA
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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.
2020-08-24 | GSE156714 | GEO
Project description:Tumebacillus flagellatus Genome sequencing
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| PRJNA473199 | ENA
Project description:Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, i.e. all proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa-protein without functional annotation. The protein comprises two characteristic PepSY domains and we thus termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays as well as protein ultrafiltration followed by inductively coupled plasma mass spectrometric analysis, we demonstrated the presence of four lanthanide binding sites that showed selectivity over the chemically similar Ca2+ ion. A similar arrangement of two PepSY domains is also present in the recently described uranium-binding protein UipA from Microbacterium spp. LanP and UipA thus represent the first members of PepSY domain proteins involved in metal binding. Although the physiological role of LanP is still unclear, its discovery has important implications for applications towards the sustainable purification and separation of lanthanides.
2023-01-31 | PXD032828 | Pride
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