Unraveling new roles for minor components of the E. coli RNA degradosome

Kaberdin, Vladimir R.; Lin‐Chao, Sue

doi:10.4161/rna.6.4.9320

Cited by 29 publications

(22 citation statements)

References 27 publications

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“…We examined whether other organelle-like assemblies in bacteria had enrichment patterns similar to those mentioned previously. Although individual proteins were enriched in fraction F4-p, this was not generally observed for the tRNA synthetase complex [83], the mRNA degadosome [35] and the purinosome. Purinosome (purine biosynthesis) subunits enriched in F4-p were Add, PurC, PurM and PurT.…”

Section: Resultsmentioning

confidence: 75%

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Characterizing the Escherichia coli O157:H7 Proteome Including Protein Associations with Higher Order Assemblies

et al. 2011

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BackgroundThe recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.Methodology/Principal FindingsWe examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate Mr ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high Mr fraction. Hundreds of proteins were enriched in a Mr range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.SignificanceNearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.

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Section: Resultsmentioning

confidence: 75%

“…Even for proteins attributed to have a specific function, novel functions continue to emerge, e.g. the roles of elongation factor Tu in cell shape maintenance [34] and enolase as a component of the mRNA degradosome [35]. The first genome of an E. coli serotype O157:H7 strain was published by Perna et al [2].…”

Section: Introductionmentioning

confidence: 99%

Characterizing the Escherichia coli O157:H7 Proteome Including Protein Associations with Higher Order Assemblies

et al. 2011

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“…4) and microarray profiling consistently show no activation of the σ H -controlled heat-shock regulon in the protein-overexpressing cells, even in those expressing partially (NBD-EcMsbA) or mostly (EcYojI) misfolded proteins. Remarkably, overexpression of Ec Enolase* strongly suppresses expression of σ H -dependent genes, presumably reflecting a uncharacterized feature of the biology of this protein that has been reported previously to have functions beyond its enzymatic activity in glycolysis (48, 49). However, expression of σ H -dependent genes including dnaK (Hsp70) and groELS (Hsp60) continues unchanged in cells overexpressing the other target proteins, demonstrating that the heat-shock regulon remains active but is not enhanced in its activity by misfolding of some of our target proteins at 37 °C in vivo .…”

Section: Discussionmentioning

confidence: 75%

Physiological Response to Membrane Protein Overexpression in E. coli

Gubellini

Verdon

Karpowich

et al. 2011

Molecular & Cellular Proteomics

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Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization.

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“…Interestingly, Bd3461 (rhlB) is one part of the degradosome, a large multiprotein complex involved in RNA degradation and decay in Esche-richia coli (2). The interaction of different proteins of the degradosome is required for efficient degradation of RNA, and the disruption of the complex has stabilizing effects on bacterial transcripts (19). No further experimental evidence was found for a role of Bd3464 coding for the poly(A) polymerase PcnB; however, it is intriguing that this gene also encodes an enzyme that catalyzes RNA polyadenylation at the 3Ј end, which leads to its faster degradation (24).…”

Section: Discussionmentioning

confidence: 99%

Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

et al. 2011

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Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Wholegenome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.

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Unraveling new roles for minor components of the E. coli RNA degradosome

Cited by 29 publications

References 27 publications

Characterizing the Escherichia coli O157:H7 Proteome Including Protein Associations with Higher Order Assemblies

Characterizing the Escherichia coli O157:H7 Proteome Including Protein Associations with Higher Order Assemblies

Physiological Response to Membrane Protein Overexpression in E. coli

Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

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