Ribosome biogenesis is temperature‐dependent and delayed in <i>Escherichia coli</i> lacking the chaperones DnaK or DnaJ<sup>†</sup>

Refaii, Abdalla Al; Alix, Jean‐Hervé

doi:10.1111/j.1365-2958.2008.06561.x

Cited by 57 publications

(55 citation statements)

References 82 publications

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“…The formation of m 3 C was shown to occur on the level of the 70S ribosomes (Ero et al 2008). Therefore, the 11.4-min peaks of the 35S and 45S particles probably contain mostly m 5 C. Moreover, z80% of the free 50S subunits are incompletely assembled precursors of the large ribosome subunit Al Refaii and Alix 2009). Thus, the 11.4-min peak of the free 50S can contain only 20% of mature 23S rRNA and the corresponding amount of m 3 C. Therefore, m 5 C is present in the free 50S particles by z100%.…”

Section: Discussionmentioning

confidence: 99%

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Siibak¹,

Rèmme²

2010

RNA

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Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

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Section: Discussionmentioning

confidence: 99%

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Siibak¹,

Rèmme²

2010

RNA

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“…Ribosome dissociation is considered a major event in the thermal death of E. coli, and the 30S subunit is more temperature sensitive than the 50S subunit (28,33). DnaK, a heat shock chaperone (2,6), is also essential for ribosome assembly in heat-stressed E. coli (3). Simultaneous overexpression of 30S and 50S ribosomal subunits, DnaK, and the 30S ribosomal subunit-associated cold shock protein Yfia indicates that the ribosome stability of E. coli GGGF10 under heat shock conditions is lower than that of E. coli AW1.7.…”

Section: Discussionmentioning

confidence: 99%

Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7

Ruan

Pleitner

Gänzle

et al. 2011

Appl Environ Microbiol

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This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50-to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins.Escherichia coli is a common contaminant of the food supply. The majority of the strains of this species are not pathogenic; however, their relatively high resistance to environmental insults and the occurrence of virotypes with a low infectious dose, particularly enterohemorrhagic E. coli, make E. coli an organism of major concern in the production of minimally processed foods, particularly produce and fresh beef (11).Most strains of E. coli have a D 60 value (the duration of heat treatment at 60°C required to reduce the number of microorganisms to 1/10 of the initial value) of less than 1 min. However, the heat resistance of E. coli is highly variable among different strains and individual strains exhibit D 60 values of up to 6.5 min (8,21,25,37). The heat resistance of individual strains of E. coli relates to their ability adapt to heat stress by the homoviscous adaptation of the plasma membrane, as well as the synthesis of heat shock proteins (20, 43). The 32 -induced expression of heat shock proteins after sublethal thermal stress increases resistance to lethal heat treatment (43; for a review, see reference 6). Increased basal expression of the heat shock proteins DnaK, Lon, and ClpX was linked to the increased heat resistance of E. coli mutants LMM1010, LMM1020, and LMM 1030 (1, 21). The S -mediated general stress response additionally contributes to acid, heat, pressure, and salt resistance in E. coli (2,14,22,3...

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“…Freshly formed ribosome subunits are ''immature'' and enter the translating ribosome pool after an additional 1-2 min have passed (Lindahl 1975). Therefore, the rate-limiting step of ribosome assembly is the final maturation of subunits after the majority, if not all, of the ribosomal proteins have already bound to rRNA Al Refaii and Alix 2009). Conformational rearrangements and a subset of rRNA processing and modification events are likely to take place during that time period (Holmes and Culver 2004;Kaczanowska and Rydén-Aulin 2007).…”

Section: Introductionmentioning

confidence: 99%

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Ero¹,

Leppik²,

Liiv³

et al. 2010

RNA

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Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K M for substrate 70S ribosomes is 0.51 6 0.06 mM, and for cofactor S-adenosyl-L-methionine 27 6 3 mM; the k cat values are 4.95 6 1.10 min À1 and 6.4 6 1.3 min À1, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K M value for substrate 50S subunits is 0.98 6 0.18 mM and the k cat value is 1.97 6 0.46 min À1. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD-and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.

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Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ^†

Cited by 57 publications

References 82 publications

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

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Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ†

Cited by 57 publications

References 82 publications

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

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Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ^†