Molecular Microbiology

Pennaneach, Vincent; Putnam, Christopher D.; Kolodner, Richard D.

doi: 10.1111/j.1365-2958.2006.05026.xpmid: 16468981

Summary The repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerase‐dependent addition of telomeres at non‐telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA‐template (guide RNA) at short regions of homology is followed by extension of the nascent 3′‐end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single‐stranded DNA‐binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing–extension–dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae.

Andaluz, Encarnación; Ciudad, Toni; Gómez‐Raja, Jonathan; Calderone, Richard; Larriba, Germán

doi: 10.1111/j.1365-2958.2005.05038.xpmid: 16468988

Summary We have analysed the effect of RAD52 deletion in several aspects of the cell biology of Candida albicans. Cultures of rad52Δ strains exhibited slow growth and contained abundant cells with a filamentous morphology. Filamentation with polarization of actin patches was accompanied by the induction of the hypha‐specific genes (HSG) ECE1, HWP1 and HGC1. However, filament formation occurred in the absence of the transcription factors Efg1 and Cph1, even though disruption of EFG1 prevented expression of HSG. Therefore, expression of HSG genes accompanies but is dispensable for rad52Δ filamentation. However, deletion of adenylate cyclase severely impaired filamentation, this effect being largely reverted by the addition of exogenous cAMP. Filaments resembled elongated pseudohyphae, but some of them looked like true hyphae. Following depletion of Rad52, many cells arrested at the G2/M phase of the cell cycle with a single nucleus suggesting the early induction of the DNA‐damage checkpoint. Filaments formed later, preferentially from G2/M cells. The filamentation process was accompanied by the uncoupling of several landmark events of the cell cycle and was partially dependent on the action of the cell cycle modulator Swe1. Hyphae were still induced by serum, but a large number of rad52 cells myceliated in G2/M.

Hu, Guiqing; Lin, Gang; Wang, Ming; Dick, Lawrence; Xu, Rui‐Ming; Nathan, Carl; Li, Huilin

doi: 10.1111/j.1365-2958.2005.05036.xpmid: 16468986

Summary Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Å resolution reveals a substrate‐binding pocket with composite features of the distinct β1, β2 and β5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article (Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365‐2958.2005.05035.x). The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the α‐subunit N‐terminal residues. However, cryo‐electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative‐staining electron microscopy that depended on the presence of the N‐terminal octapetides of the α‐subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N‐(4‐morpholine)carbonyl‐β‐(1‐naphthyl)‐l‐alanine‐l‐leucine boronic acid (MLN‐273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb‐specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

Hohlfeld, Sabine; Pattis, Isabelle; Püls, Jürgen; Plano, Gregory V.; Haas, Rainer; Fischer, Wolfgang

doi: 10.1111/j.1365-2958.2006.05050.xpmid: 16469000

Summary Type IV secretion systems are increasingly recognized as important virulence determinants of Gram‐negative bacterial pathogens. While the examination of several type IV‐secreted proteins suggested that their secretion depends on C‐terminal signals, the nature of these signals and their conservation among different systems remain unclear. Here, we have characterized the secretion signal of the Helicobacter pylori CagA protein, which is translocated by the Cag type IV secretion apparatus into eucaryotic cells. The production of fusion proteins of CagA and green fluorescent protein (GFP) did not result in translocation of GFP to epithelial cells, but a fusion of GFP with the CagA C‐terminus exerted a dominant‐negative effect upon wild‐type CagA translocation. We show that CagA translocation depends on the presence of its 20 C‐terminal amino acids, containing an array of positively charged residues. Interestingly, these positive charges are neither necessary nor sufficient for CagA translocation, but replacing the C‐terminal region of CagA with that of other type IV‐secreted proteins reconstitutes CagA translocation competence. Using a novel type IV translocation assay with a phosphorylatable peptide tag, we show that removal of the N‐terminal part of the CagA protein renders the protein translocation‐incompetent as well. Thus, the Cag type IV secretion system seems to diverge from other systems not only with respect to its composition and architecture, but also in terms of substrate recognition and transport.

Van Nuland, An; Vandormael, Patrick; Donaton, Monica; Alenquer, Marta; Lourenço, Artur; Quintino, Edgar; Versele, Matthias; Thevelein, Johan M.

doi: 10.1111/j.1365-2958.2005.05043.xpmid: 16468990

Summary In the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose‐containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re‐addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re‐supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogen‐starved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non‐metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive site‐directed mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease‐based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant AtAmt1;1, AtAmt1;2, AtAmt1;3 and AtAmt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.

Traber, Katrina; Novick, Richard

doi: 10.1111/j.1365-2958.2006.04986.xpmid: 16468992

Summary agr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)‐induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the non‐haemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3′ end of agrA (agrA‐8A). This causes a frameshift that results in the addition of three amino acyl residues to the C‐terminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2–3 h, which results in failure to translate α‐ and δ‐haemolysins, and hence, in a non‐haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non‐haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non‐haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto‐inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate α‐ and δ‐haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non‐haemolytic phenotype of RN4220, and has solved the long‐standing mystery of the variable non‐haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.

Lin, Gang; Hu, Guiqing; Tsu, Christopher; Kunes, Yune Z.; Li, Huilin; Dick, Lawrence; Parsons, Thomas; Li, Ping; Chen, Zhiqiang; Zwickl, Peter; Weich, Nadine; Nathan, Carl

doi: 10.1111/j.1365-2958.2005.05035.x

Couturier, Etienne; Rocha, Eduardo P. C.

doi: 10.1111/j.1365-2958.2006.05046.xpmid: 16468991

Summary The bidirectional replication of bacterial genomes leads to transient gene dosage effects. Here, we show that such effects shape the chromosome organisation of fast‐growing bacteria and that they correlate strongly with maximal growth rate. Surprisingly the predicted maximal number of replication rounds shows little if any phylogenetic inertia, suggesting that it is a very labile trait. Yet, a combination of theoretical and statistical analyses predicts that dozens of replication forks may be simultaneously present in the cells of certain species. This suggests a strikingly efficient management of the replication apparatus, of replication fork arrests and of chromosome segregation in such cells. Gene dosage effects strongly constrain the position of genes involved in translation and transcription, but not other highly expressed genes. The relative proximity of the former genes to the origin of replication follows the regulatory dependencies observed under exponential growth, as the bias is stronger for RNA polymerase, then rDNA, then ribosomal proteins and tDNA. Within tDNAs we find that only the positions of the previously proposed ‘ubiquitous’ tRNA, which translate the most frequent codons in highly expressed genes, show strong signs of selection for gene dosage effects. Finally, we provide evidence for selection acting upon genome organisation to take advantage of gene dosage effects by identifying a positive correlation between genome stability and the number of simultaneous replication rounds. We also show that gene dosage effects can explain the over‐representation of highly expressed genes in the largest replichore of genomes containing more than one chromosome. Together, these results demonstrate that replication‐associated gene dosage is an important determinant of chromosome organisation and dynamics, especially among fast‐growing bacteria.

Sonnleitner, Elisabeth; Schuster, Martin; Sorger‐Domenigg, Theresa; Greenberg, Everett Peter; Bläsi, Udo

doi: 10.1111/j.1365-2958.2006.05032.xpmid: 16468994

Summary The Pseudomonas aeruginosa quorum‐sensing (QS) systems, Las and Rhl, control the production of several virulence factors and other proteins, which are important to sustain adverse conditions. A comparative transcriptome analysis of a rpoS – and a rpoS–hfq – strain indicated that the Sm‐like RNA‐binding protein Hfq affects approximately 5% of the P. aeruginosa O1 transcripts. Among these transcripts 72 were identified to be QS regulated. Expression studies revealed that Hfq does not control the master regulators of the Las system, LasR and LasI. Upon entry into stationary phase, Hfq exerted a moderate stimulatory effect on translation of the rhlR gene and on the qscR gene, encoding a LasR/RhlR homologue. However, Hfq considerably stimulated translation of the rhlI gene, encoding the synthetase of the autoinducer N‐Butyryl‐homoserine lactone (C4‐HSL). Correspondingly, the C4‐HSL levels were reduced in a hfq– strain. To elucidate the stimulatory effect of Hfq on rhlI expression we asked whether Hfq affects the stability of the regulatory RNAs RsmY and RsmZ, which have been implicated in sequestration of the translational repressor RsmA, which in turn is known to negatively regulate RhlI synthesis. We demonstrate that Hfq binds to and stabilizes the regulatory RNA RsmY, which is further shown to bind to the regulatory protein RsmA. A model for the Hfq regulatory network is presented, wherein an alleviation of the negative effect of RsmA accounts for the observed stimulation of rhlI expression by Hfq. The model is corroborated by the observation that a rsmY– mutant mimics the hfq – phenotype with regard to rhlI expression.

Schulze, Ryan J.; Zückert, Wolfram R.

doi: 10.1111/j.1365-2958.2006.05039.xpmid: 16468989

Summary Borrelia spirochaetes are unique among diderm bacteria in their abundance of surface‐displayed lipoproteins, some of which play important roles in the pathogenesis of Lyme disease and relapsing fever. To identify the lipoprotein‐sorting signals in Borrelia burgdorferi, we generated chimeras between the outer surface lipoprotein OspA, the periplasmic oligopeptide‐binding lipoprotein OppAIV and mRFP1, a monomeric red fluorescent reporter protein. Localization of OspA and OppAIV point mutants showed that Borrelia lipoproteins do not follow the ‘+2’ sorting rule which targets lipoproteins to the cytoplasmic or outer membrane of Gram‐negative bacteria via the Lol pathway. Fusions of mRFP1 to short N‐terminal lipopeptides of OspA, and surprisingly OppAIV, were targeted to the spirochaetal surface. Mutagenesis of the OspA N‐terminus defined less than five N‐terminal amino acids as the minimal secretion‐facilitating signal. With the exception of negative charges, which can act as partial subsurface retention signals in certain peptide contexts, lipoprotein secretion occurs independent of N‐terminal sequence. Together, these data indicate that Borrelia lipoproteins are targeted to the bacterial surface by default, but can be retained in the periplasm by sequence‐specific signals.