PubMed · 2497962
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R C Judd. 1989. Protein I: structure, function, and genetics.. https://doi.org/10.1128/cmr.2.suppl.s41
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In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidota, TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the function of TamL in Flavobacterium johnsoniae, an environmental Bacteroidota. Unlike its homologs in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we show that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe an overall decrease in the levels of β-barrel outer membrane proteins, nor substantial alterations in outer membrane lipid composition. By pull-down assays, we found TamL co-purifying with TamB in F. johnsoniae, suggesting an interaction. Furthermore, we found that while TamL and TamB monocistronic genes are conserved among Bacteroidota, only some species encode multiple TamL, TamB and TamA proteins. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria.
Reiterative transcription is the repetitive addition of nucleotides to the 3' end of a nascent transcript due to slippage between the transcript and DNA template. Recently, we showed that pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli occurs, in part, through a mechanism in which induction of UTP-dependent reiterative transcription within the initially transcribed region prevents downstream extension of the nascent transcript to include structural gene sequences. In this study we demonstrate that pyrimidine-mediated regulation of codBA operon expression in E. coli also involves UTP-dependent reiterative transcription during initiation; however, the mechanism is different from that of the pyrBI operon. The initially transcribed region of the codBA promoter contains the sequence GATTTTTTG (non-template strand). Our results show that transcription is initiated primarily at the first two bases designated G7 and A8 (counting from the -10 region). When transcripts are initiated at position A8, UTP-dependent reiterative transcription always occurs within the run of T residues in the initially transcribed region. The AUUUUn (where n = 1 to > 15) transcripts produced by this reaction are not extended productively to include downstream codBA sequences. In contrast, most transcripts initiated at position G7 do not engage in reiterative transcription and can be elongated normally. Characterization of a codBA promoter mutation that prevents reiterative transcription showed that this reaction is required for virtually all pyrimidine-mediated regulation of operon expression and that UTP levels control the selection of the G7 and A8 transcriptional start sites. These results suggest a model for regulation in which high intracellular levels of UTP favor transcriptional initiation at position A8 and thus the accompanying reiterative transcription, which together preclude initiation at position G7. Low levels of UTP inhibit initiation at position A8 and the associated reiterative transcription, thereby allowing high levels of initiation at position G7 and operon expression. Our results also indicate critical sequence requirements for reiterative transcription, which are important for understanding the mechanism of this reaction as well as for identifying other promoters at which this reaction may occur. Of particular interest is the indication that an RNA:DNA hybrid forms during transcriptional initiation and the strength of this hybrid controls the extent of reiterative transcription.
We have studied the kinetics of folding and membrane insertion of the outer membrane protein OmpA of Escherichia coli. In the native structure, its membrane-inserted domain forms a beta-barrel. The protein was unfolded in solubilized form in water/urea, and refolding was induced by dilution of urea and simultaneous addition of lipid vesicles. Three transitions along the folding pathway could be distinguished. Their characteristic times lie below a second, in the range of minutes, and in the range of an hour. The fast process corresponds to the transition from the unfolded state in water/urea to a misfolded state in water, the moderately slow process to a transition from the misfolded state to a partially folded state in the membrane, and the slow process to the transition from the partially folded to the native state. The partially folded state in the membrane is interpreted as the analogue of the molten globule state of soluble proteins.