Stephen G. Addinall

Molecular Evolution of FtsZ Protein Sequences Encoded Within the Genomes of Archaea, Bacteria, and Eukaryota

The FtsZ protein is a polymer-forming GTPase which drives bacterial cell division and is structurally and functionally related to eukaryotic tubulins. We have searched for FtsZ-related sequences in all freely accessible databases, then used strict criteria based on the tertiary structure of FtsZ and its well-characterized invitro and invivo properties to determine which sequences represent genuine homologues of FtsZ. We have identified 225 full-length FtsZ homologues, which we have used to document, phylum by phylum, the primary sequence characteristics of FtsZ homologues from the Bacteria, Archaea, and Eukaryota. We provide evidence for at least five independent ftsZ gene-duplication events in the bacterial kingdom and suggest the existence of three ancestoral euryarchaeal FtsZ paralogues. In addition, we identify “FtsZ-like” sequences from Bacteria and Archaea that, while showing significant sequence similarity to FtsZs, are unlikely to bind and hydrolyze GTP.

The Tubulin Ancester, FtsZ, Draughtsman, Designer and Driving Force for Bacterial Cytokinesis

We discuss in this review the regulation of synthesis and action of FtsZ, its structure in relation to tubulin and microtubules, and the mechanism of polymerization and disassembly (contraction) of FtsZ rings from a specific nucleation site (NS) at mid cell. These topics are considered in the light of recent immunocytological studies, high resolution structures of some division proteins and results indicating how bacteria may measure their mid cell point.

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

A Genomewide Suppressor and Enhancer Analysis of cdc13-1 Reveals Varied Cellular Processes Influencing Telomere Capping in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to “cap” chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology.

Colonyzer: automated quantification of micro-organism growth characteristics on solid agar

BackgroundHigh-throughput screens comparing growth rates of arrays of distinct micro-organism cultures on solid agar are useful, rapid methods of quantifying genetic interactions. Growth rate is an informative phenotype which can be estimated by measuring cell densities at one or more times after inoculation. Precise estimates can be made by inoculating cultures onto agar and capturing cell density frequently by plate-scanning or photography, especially throughout the exponential growth phase, and summarising growth with a simple dynamic model (e.g. the logistic growth model). In order to parametrize such a model, a robust image analysis tool capable of capturing a wide range of cell densities from plate photographs is required.ResultsColonyzer is a collection of image analysis algorithms for automatic quantification of the size, granularity, colour and location of micro-organism cultures grown on solid agar. Colonyzer is uniquely sensitive to extremely low cell densities photographed after dilute liquid culture inoculation (spotting) due to image segmentation using a mixed Gaussian model for plate-wide thresholding based on pixel intensity. Colonyzer is robust to slight experimental imperfections and corrects for lighting gradients which would otherwise introduce spatial bias to cell density estimates without the need for imaging dummy plates. Colonyzer is general enough to quantify cultures growing in any rectangular array format, either growing after pinning with a dense inoculum or growing with the irregular morphology characteristic of spotted cultures. Colonyzer was developed using the open source packages: Python, RPy and the Python Imaging Library and its source code and documentation are available on SourceForge under GNU General Public License. Colonyzer is adaptable to suit specific requirements: e.g. automatic detection of cultures at irregular locations on streaked plates for robotic picking, or decreasing analysis time by disabling components such as lighting correction or colour measures.ConclusionColonyzer can automatically quantify culture growth from large batches of captured images of microbial cultures grown during genome-wide scans over the wide range of cell densities observable after highly dilute liquid spot inoculation, as well as after more concentrated pinning inoculation. Colonyzer is open-source, allowing users to assess it, adapt it to particular research requirements and to contribute to its development.

New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109-->Ser109 for ftsZ6460, Ala129-->Thr129 for ftsZ972, Val157-->Met157 for ftsZ2066, Pro203-->Leu203 for ftsZ9124, and Ala239-->Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42 degrees C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42 degrees C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30 degrees C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42 degrees C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

Genome-Wide Analysis to Identify Pathways Affecting Telomere-Initiated Senescence in Budding Yeast

In telomerase-deficient yeast cells, like equivalent mammalian cells, telomeres shorten over many generations until a period of senescence/crisis is reached. After this, a small fraction of cells can escape senescence, principally using recombination-dependent mechanisms. To investigate the pathways that affect entry into and recovery from telomere-driven senescence, we combined a gene deletion disrupting telomerase (est1Δ) with the systematic yeast deletion collection and measured senescence characteristics in high-throughput assays. As expected, the vast majority of gene deletions showed no strong effects on entry into/exit from senescence. However, around 200 gene deletions behaving similarly to a rad52Δ est1Δ archetype (rad52Δ affects homologous recombination) accelerated entry into senescence, and such cells often could not recover growth. A smaller number of strains similar to a rif1Δ est1Δ archetype (rif1Δ affects proteins that bind telomeres) accelerated entry into senescence but also accelerated recovery from senescence. Our genome-wide analysis identifies genes that affect entry into and/or exit from telomere-initiated senescence and will be of interest to those studying telomere biology, replicative senescence, cancer, and ageing. Our dataset is complementary to other high-throughput studies relevant to telomere biology, genetic stability, and DNA damage responses.

The yeast copper response is regulated by DNA damage.

ABSTRACT Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast.

Clustering incorporating shortest paths identifies relevant modules in functional interaction networks

Many biological systems can be modeled as networks. Hence, network analysis is of increasing importance to systems biology. We describe an evolutionary algorithm for selecting clusters of nodes within a large network based upon network topology together with a measure of the relevance of nodes to a set of independently identified genes of interest. We apply the algorithm to a previously published integrated functional network of yeast genes, using a set of query genes derived from a whole genome screen of yeast strains with a mutation in a telomere uncapping gene. We find that the algorithm identifies biologically plausible clusters of genes which are related to the cell cycle, and which contain interactions not previously identified as potentially important. We conclude that the algorithm is valuable for the querying of complex networks, and the generation of biological hypotheses.

Expression, purification and crystallization of the cell-division protein YgfE from Escherichia coli

An open reading frame designated b2910 (ygfE) in the Escherichia coli K12-MG1655 genome sequence, identified as a possible homologue to the cell-division protein ZapA, was cloned into the high-expression plasmid pETDuet-1 and overexpressed in E. coli BL21 (DE3)-AI. The protein was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from a wide range of screened conditions, but principally from solutions containing 0.1 M HEPES pH 7.0, 18% PEG 6000, 5 mM CaCl2. Diffraction data to 1.8 A were collected at the European Synchrotron Radiation Facility (ESRF). The crystals belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 53.8, b = 53.8, c = 329.7 A, alpha = beta = 90, gamma = 120 degrees.