Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress

Ribosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.

The activity of ribosome modulation factor during growth of Escherichia coli under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.

Calicivirus translation initiation requires an interaction between VPg and eIF4E

Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 50 end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF4E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF4E by 4E-BP1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF4E and the cap structure or 4E-BP1, suggesting that VPg binds to eIF4E at a different site from both cap and 4E-BP1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.

Permanent changes in growth and physiology of Bacillus subtilis ToC46 after loss of a recombinant plasmid

The physiology and growth of plasmid-bearing Bacillus subtilis carrying plasmid pPFF1, the non-transformed host, and cells after loss of the plasmid (so-called plasmid-cured cells) were investigated. It was found that, following plasmid loss, cells exhibited phenotypic characteristics different from those of the non-transformed host strains. Compared to plasmid-bearing cells and non-transformed host cells, an approximate 25% increase in the maximum specific growth rate and a more rapid increase in total RNA per unit cell mass were observed in plasmid-cured cells. The total enthalpy associated with irreversible denaturation events was determined in whole cells by differential scanning calorimetry. This showed higher enthalpies for plasmid-cured cells compared with the non-transformed host, which suggests increased ribosome numbers. The result from cellular DNA hybridisation suggests that there was no direct evidence of plasmid integration into the host chromosome. (C) 2004 Elsevier Inc. All rights reserved.

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Stops making sense: translational trade-offs and stop codon reassignment

Background Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature. Results In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences. Conclusions We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants.

Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC)

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.