Tanel Tenson

The mechanism of action of macrolides, lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome.

The macrolide-lincosamide-streptogramin B class (MLS) of antibiotics contains structurally different but functionally similar drugs, that all bind to the 50S ribosomal subunit. It has been suggested that these compounds block the path by which nascent peptides exit the ribosome. We have studied the mechanisms of action of four macrolides (erythromycin, josamycin, spiramycin and telithromycin), one lincosamide (clindamycin) and one streptogramin B (pristinamycin IA). All these MLS drugs cause dissociation of peptidyl-tRNA from the ribosome. Josamycin, spiramycin and clindamycin, that extend to the peptidyl transferase center, cause dissociation of peptidyl-tRNAs containing two, three or four amino acid residues. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl-tRNAs containing six, seven or eight amino acid residues. Pristinamycin IA causes dissociation of peptidyl-tRNAs with six amino acid residues and telithromycin allows polymerisation of nine or ten amino acid residues before peptidyl-tRNA dissociates. Our data, in combination with previous structural information, suggest a common mode of action for all MLS antibiotics, which is modulated by the space available between the peptidyl transferase center and the drug.

Regulatory Nascent Peptides in the Ribosomal Tunnel

Accumulating evidence for nascent-peptide-mediated regulation of translation suggests that all nascent peptides do not necessarily interact with the ribosome in a similar manner. Recent studies have helped to elucidate the exit route of the nascent chain and its interactions with the ribosome.

Recent functional insights into the role of (p)ppGpp in bacterial physiology

The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p)ppGpp metabolism and (p)ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp. We also discuss the structural basis of transcriptional regulation by (p)ppGpp and the role of (p)ppGpp in GTP metabolism and in the emergence of bacterial persisters.

The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life

RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the “stringent” response and regulator of cellular metabolism. The classical “long” RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the “stringent response” to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.

Antibiotics and the ribosome

The ribosome is one of the main antibiotic targets in the cell. Recent years brought important insights into the mode of interaction of antibiotics with the ribosome and mechanisms of antibiotic action. Ribosome crystallography provided a detailed view of the interactions between antibiotics and rRNA. Advances in biochemical techniques let us better understand how the binding of small organic molecules can interfere with functions of an enzyme four orders of magnitude larger than the inhibitor. These and other achievements paved the way for the development of new ribosome‐targeting antibiotics, some of which have already entered medical practice. The recent progress, problems and new directions of research of ribosome‐targeting antibiotics are discussed in this review.

Cell division in Escherichia coli cultures monitored at single cell resolution

BackgroundA fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate.ResultsWe monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency.ConclusionIn principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli.

Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is highlighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

Age of Inoculum Strongly Influences Persister Frequency and Can Mask Effects of Mutations Implicated in Altered Persistence

The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and phoU deletions showed decreased persister levels only when the inocula were from aged cultures, while sucB and ygfA deletions had decreased persister levels irrespective of the age of the inocula. A dependency on culture conditions underlines the notion that during screening for mutants with altered persister frequencies, the exact experimental details are of great importance. Unlike ampicillin and norfloxacin, which always leave a fraction of bacteria alive, amikacin killed all cells in the growth resumption experiment. It was concluded that the frequency of persisters depends on the conditions of inoculum cultivation, particularly its age, and the choice of antibiotic.

The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non‐cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post‐initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase‐deficient mutants of eIF5B and IF2.

Translation initiation region sequence preferences in Escherichia coli

BackgroundThe mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.ResultsWe found that during bacterial growth at 37°C, a six-nucleotide SD (AGGAGG) is more efficient than shorter or longer sequences. The A/U-rich enhancer contributes strongly to the efficiency of initiation, having the greatest stimulatory effect in the exponential growth phase of the bacteria. The SD sequences and the A/U-rich enhancer stimulate translation co-operatively: strong SDs are stimulated by the enhancer much more than weak SDs. The bacterial growth rate does not have a major influence on the TIR selection pattern. On the other hand, temperature affects the TIR preference pattern: shorter SD sequences are preferred at lower growth temperatures. We also performed an in silico analysis of the TIRs in all E. coli mRNAs. The base pairing potential of the SD sequences does not correlate with the codon adaptation index, which is used as an estimate of gene expression level.ConclusionIn E. coli the SD selection preferences are influenced by the growth temperature and not influenced by the growth rate. The A/U rich enhancers stimulate translation considerably by acting co-operatively with the SD sequences.