introductionPlanctomycetes bacteria have unique cell structure and seriously invaginated membrane, among whichT. ImmobilisThe subcellular proteome was examined by differential dissolution and subsequent LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis,A total of 1569 proteins were identified

introduction

Planctomycetes bacteria have unique cell structure and seriously invaginated membrane, among whichT. ImmobilisThe subcellular proteome was examined by differential dissolution and subsequent LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis,A total of 1569 proteins were identified.

Tris (tri (hydroxymethyl) aminomethane) soluble fraction mainly contains cytoplasmic protein, while inner membrane protein and outer membrane protein are respectively present in TritonX-100 (detergent) and SDS (sodium dodecyl sulfate) soluble fraction, but compared with Escherichia coli subcellular proteome, the predicted amount of cytoplasmic protein in SDS soluble fractionFive times higher.

This article will analyze and predict the expression patterns of proteins located in the cytoplasm, as well as the differential relationship between the inner and outer membranes of T. immobilis,Use a subcellular fractionation scheme based on differential protein solubility to identify unique changes in biological characteristics or different subcellular locations.

Based on FIB-SEM (Focused Ion Beam Scanning Electron Microscope) analysis, a three-dimensional (3D) model of T.immobis was reconstructed. The cells were partially reconstructed, and the reconstructed cells showed the presence of a tunnel system formed by invagination of the cell plasma membrane,It separates the non compartmentalized cytoplasmic space from the expanded periplasmic space.

This kind of cell structure was also observed in several other cells of similar volume for visual inspection. Cells with obvious closed structure in the cytoplasmic space of Acinetobacter showed obvious signs of membrane damage, intracellular debris, broken membrane vesicles and low contrast periplasmic space,These seemingly damaged cells are not considered to represent healthy and intact cell plans.

G. The nucleoids in obscuriglobus (dark algae bacteria) are more concentrated and distinct through FIB-SEM and other electron microscopy techniques, so they can be reconstructed. The nucleoids appear to be composed of a DNA complex, while the invaginated membrane and concentrated nucleoids exist in both the mother and daughter cells of a pair of budding cells,This cell pair is captured during the late stage of budding.

But this was only observed in the later stages, as in a previous study using low-temperature electron tomography,We observed a single spherical particle with a diameter of up to 400nm in almost every cell.

g = 8 K12 MG1655.

After ultrasound treatment of cell lysis, a series of protein extracts were performed,The first stage of subcellular division contains proteins soluble in TrisThe second stage contains proteins soluble in Tris/TritonX-100 (TX-100) and proteins soluble in Tris/SDS. For T.immobilis, up to 64% of cell lysates are recovered in the first stage,In the second stage, it is 8%, in the third stage, it is 1%, and the total production is 75%.

In contrast, in the insoluble portion of Tris,In addition to ribosome and multi ribosome, there are two different structural componentsSmall vesicles and non spherical irregular shaped structures TEM+.

After the second stage,The vesicle structure is no longer visible, indicating that the endometrial protein has been dissolved in Tris/TX-100.

SDS-PAGE analysis confirmed that there were different Protein mass spectrometry in the three subcellular components of Acinetobacter and Escherichia coli. For T. immibilis, S0 (lysate) and the Protein mass spectrometry of the first stage fraction were almost the same,This result is consistent with the calculated protein yield 65% Tris.

Analysis of the lysate confirmed high absorption at 410nm and 450 to 550nmS1 410 nm S2 410 nm 500 nm .

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The predicted subcellular positions of proteins identified in LC-MS/MS analysis were very similar in both species, with proteins assigned subcellular positions,Predicted 632-757 proteins are cytoplasmic proteins315-33514-46.

In these two species, we observed many predicted cytoplasmic proteins associated with component S1, while endometrial proteins (such as SEC translocation proteins) and outer membrane proteins generally exist in components S2 and S3,Lipoprotein is characterized by type II signal peptide/.

The grading and separation patterns of these proteins are similar,Cell envelope proteins were identified in all subcellular fractionsIn T. immibilis, 65 expressed proteins are classified into this category, while there are 133 proteins in E. coli, most of which are species specific,Only 17 homologous proteins exist in two species.

T. immobilis 28 BamB43 193 kDapI 5.1 9.3 3 S1bamB_4bamB_7bamB_28 S2 (bamB_17) S3 (bamB_1) .

The cytoplasmic enzyme KdsA involved in the synthesis of core oligosaccharides Kdo was identified in component S1 of two species,And the Kdo transferase WaaA, which connects the sugar subunits of polysaccharides to Kdo lipidA, is enriched in component S3.LpxK lipidA S3.

For MutS, we identified three collateral homologous genes in T.immobilis,Two of them were only found in the S3 component S2 S3 T. immobilis MutS MutS_V .

S3 S1 MutS .

The LigA homologues of DNA ligase contain different types and combinations of DNA_ Ligase domainS3.

T. ImmobilisS3 specific protein also includes ribonuclease, such as two collateral homologues of ribonuclease endonuclease and ribonuclease exonuclease Rbn,And 3 '-5' ribonucleic acid exonuclease YhaM Rbn RNase Z 3'- CCA tRNA .

In the tRNA modifying enzymes in the S3 class,We identified the proteins in the MnmE/MnmG complexThese complexes modify the oscillating uridine at position 34 in certain tRNAs, as well as the MiaAB complex, which methylthiolates the residue at position 37 in tRNA,Reading codons starts with uridine.

Several enzymes that modify rRNA molecules also show significant differences in the fractionation patterns of these two species,16SrRNA methyltransferase RsmH is recognized only in the S1 fraction of Escherichia coli RsmH S3 .

FIB-SEMT. immobilis

The study examined the membrane network of T.immobilis using FIB-SEM tomography, and analyzed its proteome through LC-MS/MS after a series of protein extraction,The reconstructed T.immobilis cells showed the presence of a tunnel like system formed by invagination of the cell plasma membraneIt separates the non compartmentalized cytoplasmic space from the expanded periplasmic space.

We studied the subcellular proteome of Acinetobacter (and Escherichia coli as the control) through differential dissolution and LC-MS/MS analysis,Identified over 1000 proteins in each species,.

In E. coli,Cytoplasmic proteins are mainly recovered in the first Tris EDTA fraction, Triton X-100 , SDS .

Although there is some overlap between the levels, the most significant difference between these two species is that approximately 50% of cytoplasmic proteins are only recognized in the third SDS soluble fraction of T.immobilis,Less than 10% of Escherichia coli .

A key observation in this study is that many predicted cytoplasmic proteins identified in the SDS soluble fraction of T.immobilis,It represents the collateral homologues of the recently evolved and highly differentiated large protein family,PlanctomycetalesGemmataceae.

T. The Ser/Thr kinase expressed in immobilis was identified in TritonX-100 and SDS soluble components, and the Pkinase domain was associated with various other domains,This may affect the grading mode Ser/Thr .

So the large number of SDS soluble proteins in the signal transduction category of Acinetobacter is due to the recovery of proteins unique to this species,Many Ser/Thr kinases may be involved in the recently evolved signaling pathways in Planctomycotes, Ser/Thr .

Two collateral homologues of RNA polymerase recycling factor RapA were identified in the SDS soluble part of T. immibilis,It was also found that they had different domain structure from the single RapA protein identified in Tris EDTA part of E. coli.

RapA protein in bacteria competes with sigma-70 factor to bind core RNA polymerase,The function of RapA protein in eukaryotes is to make tightly packed DNA more accessible to RNA polymerase and transcription factorsT. immobilis RapA .

SDS tRNArRNA .

The inner membrane of eukaryotic cell acts as a transport system,Enable different cellular processes to occur in different parts of the cellPlanctomycetes.

summary

The results of this study emphasize parallel logicalization and domain restructuring,The importance of mechanisms for functional innovation and adaptation to more complex cellular structures in Planctomycotes,.

The observed fractionation patterns are very complex, and this study can provide multiple explanations for these findings. Some proteins may interact with new molecular complexes,Other proteins may be embedded in vesicles or attached to the inner membrane of cells.

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Reference:

1. RNA
2. Xie Xinmeng, "Prediction of Protein Subcellular Localization Based on Machine Learning"
3. Wu Haofang, "Research on Protein Subcellular Localization Based on ResC-LSTM"

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