Microbiology

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Microbiology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in microbiology. Current Opinion in Microbiology 1999, 2:453–462 Contents (chosen by) 453 Host–microbe interactions: bacteria (Blocker and Peppler) 454 Cell regulation (Stock) 455 Ecology and industrial microbiology (Normand) 456 Microbial techniques (Grimont) 456 Host–microbe interactions: fungi (d’Enfert) 457 Host–microbe interactions: viruses (Rosenthal) 457 Host–microbe interactions: parasites (Sibley) 458 Genomics (Labedan and Forterre) 459 Antimicrobials (Chopra) 460 Growth and development (Casselton and Errington) • ••

of special interest of outstanding interest

Host–microbe interactions: bacteria Selected by Ariel Blocker Institute Pasteur, Paris, France

Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Brightbill HD, Libraty DH, Krutzik SR, Yang R-B, Belisle JT, Bleharski JR, Maitland M, Norgard MV, Plevy SE, Smale ST et al.: Science 1999, 285:732-735. AND

Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Aliprantis AO, Yang R-B, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, Zychlinsky A: Science 1999, 285:736-739. AND

HMG-1 as a late mediator of endotoxin lethality in mice. Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, Frazier A, Yang H, Ivanova S, Borovikova L et al.: Science 1999, 285:248-251. •• Significance: These papers present major developments in our understanding of early and late innate responses of immune cells to bacterial lipoproteins (BLPs) and lipolysaccharide (LPS), and they highlight once again the importance of the recent discovery of the LPS receptor, hTLR2. Findings: The first and second papers show that human Tolllike receptor-2 (hTLR2) is not only a receptor for LPS but also for BLPs. BLPs are characterised by a unique, amino-terminal lipo-amino acid, N-acyl-S-diacylglyceryl cysteine, and are ideal targets for innate immune surveillance because they are produced by all bacteria. These papers show that, via activation of hTLR2, BLPs potently stimulate NF-κB, IL-12 release, transcription of nitric oxide synthase and nitric oxide production (a powerful microbicidal pathway), the respiratory burst and apoptosis. The third paper presents evidence for a delayed response to LPS (also triggered through hTLR-2), occurring well after the initial burst of TNF and IL-1, and of the importance of this delayed response in the development of endotoxic shock. They then demonstrate that high mobility group-1 protein (HMG-1) is a major mediator of this response. HMG-1 was released by cultured mouse macrophages more than eight hours after exposure to LPS, TNF or IL-1. HMG-1 is

a highly conserved protein initially identified as nuclear protein that bound cruciform DNA. Extracellular HMG-1 interacts directly with plasminogen and tissue plasminogen activator. Like other macrophage products (e.g. TNF, IL-1β and MIF), HMG-1 lacks a classic secretion signal, so the mechanism of extracellular release (which is not cell lysis) remains to be determined. Septic patients who succumbed to infection had increased HMG-1 levels. Role of bacterial intimin in colonic hyperplasia and inflammation. Higgins LM, Frankel G, Connerton I, Gonçalves NS, Dougan G, Mac Donald TT: Science 1999, 285:588-591. •• Significance: Citrobacter rodentium is the mouse equivalent of Enteropathogenic Escherichia coli (EPEC), which is a major cause of infantile diarrhea in humans. EPEC causes disease via a type III secretion system that allows it to colonize the surface of epithelial cells in the intestine by causing an attaching and effacing lesion. C. rodentium causes colonic hyperplasia in young mice. This paper shows that this effect is likely to be mediated via a bacterial outer membrane protein intimin. The increased epithelial renewal in the intestine had until now been considered to be controlled by the host. The authors suggests that intimin-induced epithelial proliferation would benefit the bacterium by providing an increased colonization area, a continuing supply of uninfected cells, and increased enterocyte shedding and, hence, transmission of the organism. Findings: Bacterial intimin was found to co-stimulate submitogenic signals through the T cell receptor. Dead intimin-expressing C. rodentium or EPEC also induced mucosal hyperplasia, as well as a massive T helper cell-type 1 immune response in the colonic mucosa. This indicates that active bacterial processes, such as type III secretion, are not involved in causing hyperplasia. In support of this a mutation at cysteine 397 of intimin, which reduces its interaction with β1 integrins, reduced co-stimulatory activity in vitro and prevented immunopathology in vivo. The mucosal changes induced by C. rodentium were IFN-γ-dependent. A Salmonella virulence protein that inhibits cellular trafficking. Uchiya K, Barbieri MA, Funato K, Shah AH, Stahl PD, Groisman EA: EMBO J 1999, 18:3924-3933. •• Significance: The SPI-2 pathogenicity island of Salmonella encodes a type III secretion system which is active intracellularly, when the bacteria are internalized within macrophages. To survive and proliferate within macrophages, Salmonella inhibits phagosome–lysosome fusion and resides within phagosomes that have diverged from the normal degradative pathway of the macrophages. This paper identifies the SPI-2 encoded protein SpiC as an effector of this secretion system and provides evidence that SpiC is delivered into host cytosol where it interferes with intracellular trafficking. Findings: In J774 macrophages, wild-type Salmonella, but not a spiC mutant or dead bacteria, inhibited fusion of Salmonellacontaining phagosomes with endosomes and lysosomes, and interfered with trafficking of vesicles devoid of the microorganism. Expression of SpiC within eukaryotic cells interfered with normal trafficking of the transferrin receptor and addition of purified SpiC protein inhibited endosome–endosome fusion in vitro.

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Selected by Mark S Peppler University of Alberta, Edmonton, Alberta, Canada

Glucose metabolism in Chlamydia trachomatis: the ‘energy parasite’ hypothesis revisited. Iliffe-Lee ER, McClarty G: Mol Microbiol 1999, 33:177-187. • Significance: Chlamydia trachomatis may not be solely dependent on host cell ATP for its energy, as has been generally accepted. Findings: Four functional enzymes involved in glycolysis were found in Chlamydia trachomatis that could complement analogous genes in E. coli mutants and were shown by RT-PCR to be expressed optimally in the middle of the chlamydial developmental cycle. The chlamydial pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase showed high amino acid homology with other bacterial enzymes of similar function but, unlike most bacteria, the chlamydial enzymes were found scattered around the genome and not in an operon. Coinfection with influenza B virus does not affect association of Neisseria meningitidis with human nasopharyngeal mucosa in organ culture. Read RC, Goodwin L, Parsons MA, Silcocks P, Kaczmarski EB, Parker A, Baldwin TJ: Infect Immun 1999, 67:3082-3086. • Significance: The epidemiological association of influenza B virus with meningococcal infection does not appear to be a result of virally enhanced adherence of pathogenic bacteria. Findings: Cells from the nasopharyngeal mucosa of 19 human donors were infected with influenza B virus and then subsequently infected at different times with serogroup B Neisseria meningitidis. On the basis of electron microscopy and viable counts, even seven days of influenza infection had no effect on bacterial attachment or invasion. On the basis of western blots, no new receptors were up-regulated by viral infection, instead the major receptors were lost with time after influenza B infection. Dissemination of Listeria monocytogenes by infected phagocytes. Drevets DA: Infect Immun 1999, 67:3512-3517. • Significance: This paper provides the first experimental evidence that blood-borne Listeria monocytogenes can enter the central nervous system, to cause meningitis, by carriage within phagocytes. Findings: Peripheral blood leukocytes (PBL) were obtained from bacteremic mice, and 22.2% of monocytes and 1.6% of neutrophils were found to contain bacteria. These cells were then introduced to unifected mice and were shown to cause brain infections more effectively than in vitro-infected macrophage cell lines or broth grown organisms. Infected PBLs could also spread infection to endothelial cells in vitro suggesting that infected phagocytes could be a vehicle for delivering virulent Listeria monocytogenes across the blood–brain barrier. Elderly humans show prolonged in vivo inflammatory activity during pneumococcal infections. Bruunsgaard H, Skinhoj P, Qvist J, Pedersen BK: J Infect Dis 1999, 180:551-554. • Significance: Inability to control pneumococcal infection in the elderly may be attributed to prolonged inflammatory activity. Findings: Age-associated differences were noted in the levels of several cytokines in sera collected from 22 hospitalized patients with pneumococcal infection when compared to ageand sex-matched controls. Upon admission, the levels of most cytokines were similarly elevated in elderly (68–91 years) and young (37–55 years) patients. Levels of TNF-alpha and soluble

TNF receptor I were significantly elevated in the elderly patients one week after admission, and levels of other inflammatory mediators, such as IL-10, were positively correlated with age.

Cell regulation Selected by Jeff Stock Princeton University, New Jersey, USA

Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F. Feher VA, Cavanagh J: Nature 1999, 400:289-293. • Significance: A central problem in protein science is to measure structural dynamics and to determine its functional significance. This report shows that a simple enzyme that catalyzes the transfer of a phosphoryl group between two proteins exhibits flexibility on a millisecond time scale in residues that correlate with portions of the molecule that are important for phosphotransfer. Findings: Spo0A is a doubly wound α/β protein. Using 15N NMR relaxation measurements it was determined that maximum flexibility is largely confined to loops at the active site as well as one of the five α-helices. These regions correlate with functionally important residues identified by alanine scanning mutagenesis. Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor. Lloyd SA, Whitby FG, Blair DF, Hill CP: Nature 1999, 400:472-475. •• Significance: The bacterial flagellar basal structure functions as a proton motive force driven rotary motor. This paper reports the high-resolution structure of the central energy-transducing element within this motor, the carboxy-terminal domain of FliG. The results, together with previous molecular genetic studies, indicate a molecular mechanism for rotation. The model also provides a basis for understanding how changes in FliG orientation could reverse the sense of rotation to control motility and allow chemotaxis. Findings: The carboxy-terminal domain of FliG from Thermotoga maritima was crystallized and its structure determined by standard X-ray methods. The protein has a novel fold consisting of six tightly packed α-helices. The energy-transducing surface is a distinct wedge-like edge that is thought to interact with the proton conducting Mot proteins. Detecting protein function and protein–protein interactions from genome sequences. Marcotte EM, Pellegrini M, Ng H-L, Rice DW, Yeates TO, Eisenberg D: Science 1999, 285:751-753. •• Significance: One of the greatest challenges of modern molecular biology is to find ways to use genome sequences to determine the structure and function of proteins. This paper reports a simple and powerful method to predict likely protein–protein interactions. Findings: Pairs of independently encoded non-homologous proteins in one genome are mapped to homologues in other genomes where the proteins are linked in a single gene. Over 6000 such pairs were found among the 4290 proteins in Escherichia coli. These are strongly predicted to interact at least at a functional level. Overlapping pair wise interactions determined by this method also flag groups of proteins that work together in complexes or pathways. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Mazmanian SK, Liu G, Ton-That H, Schneewind O: Science 1999, 285:760-763.

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• Significance: Gram-positive bacteria covalently attach essential proteins to their surfaces through peptide crosslinks to the cell wall polyglycines that are otherwise employed to crosslink murien. The transpeptidation reaction is catalyzed by sortase. This paper reports the identification and characterization of the sortase gene. Given the close relationship of the sortase reaction to that catalyzed by the targets of penicillin, sortase provides an excellent target for the design of novel antibiotics. Findings: A sortase mutant, srtA, was selected by looking for temperature-sensitive lethal mutations that accumulate outer membrane protein precursors. Homologues of srtA occur in a wide range of different Gram-positive bacteria. DNA protection by stress-induced biocrystallization. Wolf SG, Frenkiel D, Arad T, Finkel SE, Kolter R, Minsky A: Nature 1999, 400:83-85. •• Significance: Dps is an Escherichia coli ferritin homologue that binds iron and forms ring-like dodecamers about 9 nm in diameter. It binds nucleic acids and is induced by oxidative stress and stationary phase. This paper shows that during stationary phase Dps co-crystallizes with DNA — packaging it into an ironclad structure. Dps/ferritin homologues are common in bacteria so this may be a general mechanism for DNA protection and sequestration. Findings: Purified Dps rapidly associates with single or double stranded, relaxed or supercoiled, DNA or RNA to form crystalline arrays whose unit cells contain nucleic acids sandwiched between protein dodecamers. The same crystals can be detected in stationary phase cells. Bacterial photoreceptor with similarity to photoactive yellow protein and plant phytochromes. Jiang ZY, Swem LR, Rushing BG, Devanathan S, Tollin G, Bauer CE: Science 1999, 285:406-409. • Significance: Phytochromes mediate plant and cyanobacterial responses to red light. They are composed of a photosensory domain coupled to a histidine protein kinase (HPK) ATP-binding domain. This paper reports that a photoreceptor for blue light in Rhodospirillum cetenum, Ppr, which functions as a two-component sensor kinase, is a common ancestor of all phytochromes. Findings: Ppr is composed of a photoactive yellow protein (PYP) domain, a central domain homologous to the phytochrome photosensory domain, and an HPK domain. Blue light acts through the PYP domain to inhibit kinase activity and control the transcription of light-responsive genes. Polypeptide flux through bacterial Hsp79:DnaK cooperates with trigger factor in chaperoning nascent chains. Teter SA, Houry WA, Ang D, Tradler T, Rockabrand D, Fischer G, Blum P, Georgopoulos C, Hartl FU: Cell 1999, 97:755-765. •• Significance: Although some proteins can spontaneously fold, in vitro studies indicate that efficient folding generally requires chaperones. Are chaperones invariably involved in folding in vivo, or are they only used under some conditions? The results presented here indicate that most Escherichia coli nascent chains interact with either DnaK and/or trigger factor (TF) as they are leaving the ribosome. Findings: DnaK tends to interact with larger polypeptides (>30 kD), whereas TF, which is ribosome bound, interacts with smaller chains. In a TF mutant the number of different polypeptides that interact with DnaK is doubled. Mutants lacking DnaK and TF are not viable.

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Oxidative protein folding is driven by the electron transport system. Bader M, Muse W, Ballou DP, Gassner C, Bardwell JCA: Cell 1999, 98:217-227. •• Significance: The periplasm of Escherichia coli contains several enzymes that act to insure rapid and correct disulfide bond formation in secreted proteins. Two of these enzymes, DsbA and DsbB, are responsible for the rapid oxidation of protein sulfhydryl groups to form disulfide bonds. A reactive disulfide in DsbA exchanges with sulfhydryl groups in secreted proteins. A disulfide in DsbB is then used to reoxidize DsbA. To complete the cycle DsbB must be reoxidized. This report shows the mechanism. Findings: Reduced DsbB donates its electrons to ubiquinone which funnels them into the electron transport system and ultimately to oxygen, or to an alternative electron acceptor, under anaerobic conditions.

Ecology and industrial microbiology Selected by Philippe Normand Université Claude Bernard, Lyon, France

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment. Niemann S, Dammann-Kalinowski T, Nagel A, Pühler A, Selbitschka W: Arch Microbiol 1999, 172:22-30. • Significance: ERIC-PCR is often used to discriminate between closely related strains or isolates, and it is widely assumed that the targets of the primers used are repetitive intergenic sequences. This work shows that the targets are neither repetitive nor intergenic but that, nevertheless, they provide a valuable discriminating fingerprint. Findings: The ERIC-PCR method was employed to generate genomic amplicons of S. meliloti strain 2011 that were sequenced. Eleven such amplicon sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Differences between Sinorhizobium species were noted. Diversity of microorganisms isolated from amber. Greenblatt CL, Davis A, Clement BG, Kitts CL, Cox T, Cano RJ: Microbial Ecology 1999, 38:58-68. • Significance: Archaeomicrobiology aims at reconstructing the evolution of bacteria using different tools. Amber analysis, made famous by the book and the movie “Jurassic Park”, has been attacked on the basis that contamination by ‘modern’ bacteria may obscure any ‘ancient’ isolate. The present paper by the same group who characterized a Bacillus from ancient amber aims at using two complementary techniques to show that the organisms grown from such ancient amber deposits have their DNA present and do not result from contamination events. Findings: Bacteria present in 120 million years old amber from Israel were cultured and found by FAME and 16S rRNA sequence analysis to belong to several distinct types. Direct characterization without culture confirmed the presence of five of these cultivated organisms. Composition of toluene-degrading microbial communities from soil at different concentrations of toluene. Hubert C, Shen Y, Voordouw G: Applied Environ Microbiol 1999, 65:3064-3070.

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• Significance: Bioremediation aims at reducing or eliminating pollutants from environmental samples by using bacteria with precise metabolic properties. It is often assumed that one bacterium will be able to eliminate all of the pollutant. The present paper highlights the fact that some of these pollutants such as toluene are toxic for bacteria and that consequently assemblages of bacteria may be more appropriate for an heterogeneous environment such as the soil. Findings: Toluene-degrading bacteria were isolated from an hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was maintained at 100, 10, or 1% (wt/wt). Different phylogenetically close Pseudomonas isolates were obtained at the different toluene vapor pressures and found to have different response curves to toluene. Restoration of culturability of starvation-stressed and lowtemperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds. Mizunoe Y, Wai SN, Takade A, Yoshida S: Arch Microbiol 1999, 172:63-67. • Significance: VBNC (viable but nonculturable state) is a hotly debated issue in environmental microbiology. Many bacteria respond to changes in environmental conditions by entering the VBNC state. The present paper suggests that temperature shifts that reduce the culturability of bacterial cells could do so by generating H2O2 and that the removal of that compound would permit the cells to grow normally. Findings: It was found that Escherichia coli became nonculturable in sterilized distilled water microcosms at 4°C but that plate counts reverted to 1/10 of the values from before the thermal shock when catalase containing media were used. Similar results were obtained with nonenzyme peroxide-degrading compounds such as sodium pyruvate or ketoglutaric acid.

genetic analysis and molecular biology applications. Haapa S, Taira S, Heikkinen E, Savilahti H: Nucleic Acids Res 1999, 27:2777-2784. • Significance: A genetic analysis methodology is described that is based on bacteriophage Mu DNA transposition and circumvents current limitations. The Mu transposition tool is composed of only a few components, and utilizes a higly efficient and accurate in vitro DNA transposition reaction with relatively even distribution of target sites. Findings: The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposonbased techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins. The Mu system proved to perform optimally by all three criteria critical for efficient utilization of in vitro transposition technology: efficiency, accuracy, and even distribution of target sites. The described Mu in vitro reaction differs from the authentic Mu transposition reaction in vivo in that the two-step process of cleavage and strand transfer is reduced to the latter step only.

Microbial techniques

BACTOX, a rapid bioassay that uses protozoa to assess the toxicity of bacteria. Schlimme W, Marchiani M, Hanselmann K, Jenni B: Appl Environ Microbiol 1999, 65:2754-2757. • Significance: A new type of semi-quantitative bioassay to assess overall bacterial toxicity based on the protozoan Tetrahymena pyriformis fed with naturally occurring or genetically modified bacteria is presented here. Findings: This type of bioassay is of ecological relevance, since it monitors a trophic interaction at the first level of the food web. This simple and rapid test is able to detect toxicant-producing bacteria, which may present a biohazard. It can also be used for the risk assessment of microbes designed for deliberate release.

Selected by Francine Grimont Institut Pasteur, Paris, France

Host–microbe interactions: fungi

Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry. Zubkov MV, Fuchs BM, Eilers H, Burkill PH, Amann R: Applied Environ Microbiol 1999, 65:3251-3257. • Significance: An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. This method was calibrated by using five species of bacteria isolated from seawater samples and grown in culture at different temperatures. Findings: The intensity of SYPRO-protein fluorescence of five genera (Arcobacter sp., Cytophaga sp., Oceanospirillum sp., Pseudomonas sp., and Vibrio sp.) was strongly correlated with their protein content, which was measured by the bicinchoninic acid method to be in the range of 60–330 fg of protein per cell. This method is an improvement over flow cytometric methods for determining microorganism biomass, because it is a direct method for determination of protein content of individual cells without biases due to species and/or shape. It does not require preliminary knowledge of individual populations of bacterioplankton and, therefore, can be directly applied to the analysis of natural samples. According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell cell. An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional

Selected by Christophe d’Enfert Institut Pasteur, Paris, France

An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Cormack B, Ghori N, Falkow S: Science 1999, 285:578-582. •• Significance: A signature-tagged mutagenesis strategy identifies a lectin of Candida glabrata that is required for adhesion of yeast cells to human epithelial cells. Findings: A collection of 96 C. glabrata ura3 mutants was generated that each carry a specific tag and then subjected to insertional mutagenesis by DNA-mediated transformation, thus yielding a collection of 9600 tagged insertional mutants. Analysis of the mutations in 16 strains that had a defect in adherence to a monolayer of human epithelial cells revealed that 14 had an insertion in the 5′-untranslated region of the EPA1 gene, which was shown to encode a lectin recognizing asialo-lactosyl-containing carbohydrates. Disruption of EPA1 confers a non-adherent phenotype to C. glabrata cells whereas expression of EPA1 confers an adherent phenotype to otherwise non-adherent Saccharomyces cerevisiae cells. Avirulence of Candida albicans CaHK1 mutants in a murine model of hematogenously disseminated candidiasis. Calera JA, Zhao X-J, de Bernardis F, Sheridan M, Calderone R: Infect Immun 1999, 67:4280-4284. AND

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Flocculation of hyphae is associated with a deletion in the putative CaHK1 two-component histidine kinase gene from Candida albicans. Calera JA, Calderone R: Microbiology 1999, 145:1431-1442. • Significance: Inactivation of a two-component histidine kinase impairs Candida albicans virulence under specific conditions suggesting that it might be involved in controlling the expression of virulence determinants in response to specific environmental cues. Findings: The CaHK1 genes encode one of three histidine kinases identified in C. albicans. Heterozygous and homozygous null mutants at the CaHK1 locus have been constructed by gene replacement. These mutants show a striking flocculation phenotype at neutral pH that is due to an interaction between the hyphae. Furthermore, the homozygous null mutant is avirulent in a murine model of disseminated candidiasis despite an ability to form hyphae in infected tissues. In contrast, the homozygous null mutant is virulent in a rat model of vaginal candidiasis, suggesting that expression of several virulence factors may be restricted to a host-specific niche.

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able to protect. Further, the rapid protective capacity of the memory CTL correlated with persistence of antigen in vivo. RANTES regulates Fas ligand expression and killing by HIV-specific CD8 cytotoxic T cells. Hadida F, Vieillard V, Mollet L, Clark-Lewis I, Baggiolini M, Debre P: J Immunol 1999, 163:1105-1109. • Significance: The results in this paper show that RANTES acts through CCR3 to regulate Fas ligand (FasL) expression on HIV-specific CD8+ T cells that kill through the Fas/FasL pathway. Thus, RANTES can enhance the cytotoxicity of HIV-specific CTLs, or alternatively, contribute to pathogenesis by accelerating the decline of CD4+ or CD8+ T cells that express Fas. Findings: Anti-HIV CTL activity was markedly enhanced by RANTES and suppressed by anti-RANTES or anti-CCR3 monoclonal antibodies. RANTES enhanced FasL expression in a time- and concentration-dependent manner, whereas antiRANTES antibody markedly decreased FasL expression. Cell surface expression of FasL was also upregulated by eotaxin, a selective ligand for CCR3.

Host–microbe interactions: viruses Selected by Kenneth L Rosenthal McMaster University Health Sciences Centre, Hamilton, Ontario, Canada

Modelling T-cell memory by genetic marking of memory T cells in vivo. Jacob J, Baltimore D: Nature 1999, 399:593-597. •• Significance: This paper describes a transgenic mouse model system that irreversibly marks memory T cells and their precursors using a reporter gene for unambiguous identification. This capability will be a valuable tool for the study of T cell responses to pathogens and vaccine evaluation. Findings: A transgenic mouse model using a Cre-recombinase-mediated genetic recombination event to irreversibly mark specifically activated T cells with human placental alkaline phosphastase (PLAP) was used with the classic model of lymphocytic choriomeningitis virus (LCMV) infection to analyze acute and memory CD8+ T cell responses. Although 70–90% of CD8+ T cells were activated eight days after LCMV infection, PLAP expression was only observed in a subset (8–10%) of CD8+ T cells. Both populations of CD8+ T cells were LCMVspecific and cytolytic. Adoptive transfer experiments showed that only the PLAP+ CD8+ T cells were precursors for longlived LCMV-specific memory T cells. A comparison of T cell memory against the same antigen induced by virus versus intracellular bacteria. Ochsenbein AF, Karrer U, Klenerman P, Althage A, Ciurea A, Shen H, Miller JF, Whitton JL, Hengartner H, Zinkernagel RM: Proc Natl Acad Sci USA 1999, 96:9293-9298. •• Significance: This paper documents a striking difference in protective T cell memory between viral and bacterial vaccines, and indicates that rapid T cell-dependent immune protection correlates with antigen persistence. Findings: Cytotoxic T lymphocyte (CTL) memory was analyzed following infection of mice with lymphocytic choriomeningitis virus (LCMV) and recombinant Listeria monocytogenes (rLM) expressing the complete nucleoprotein of LCMV or its immunodominant epitope. Although immunization with either LCMV or rLM induced a long-lived increase in anti-viral CTL precursor (CTLp) frequency, only mice immunized with LCMV maintained memory CTLs that rapidly cleared a reinfection, whereas rLM induced memory CTLs required reactivation before they were

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anit-retroviral therapy. Chun T-W, Engel D, Mizell SB, Hallahan CW, Fischette M, Park S, Davey RT, Dybul M, Kovacs JA, Metcalf JA et al.: Nature Med 1999, 5:651-655. • Significance: Intermittent administration of interleukin-2 (IL-2) with continuous highly active anti-retroviral therapy (HAART) may lead to substantial reduction in the pool of resting CD4+ T cells that contain replication competent HIV-1. Findings: A nonrandomized, cross-sectional study comparing HIV-infected individuals with undetectable viral loads (<50 copies/ml) treated with HAART plus intermittent IL-2 or HAART alone. Infectious virus was not detected in resting CD4+ T cells from six of 14 patients receiving IL-2 plus HAART, whereas HIV was isolated from all 12 patients receiving HAART alone. HIV was not detected in three of the six patients using a high-input co-culture assay. In two of the three patients from whom virus could not be isolated, replication-competent virus could not be isolated from cultures of their inguinal lymph nodes.

Host–microbe interactions: parasites Selected by David Sibley Washington University School of Medicine, St Louis, Missouri, USA

Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Urban BC, Ferguson JP, Pain A, Wilcox N, Plebanski M, Austyn JM, Roberts DJ: Nature 1999, 400:73-77. • Significance: This report demonstrates a novel process is used by Plasmodium falciparum to inhibit dendritic cell function and, consequently, to down-regulate the host immune response. Findings: Intact malaria-infected erythrocytes adhere tightly to dendritic cells altering or preventing their activation and reducing their capacity to stimulate T cells. Dendritic cell down-regulation required binding of intact-infected erythrocytes as it was not induced by lysates of infected-erythrocytes or by erythrocytes infected with a non-adherent parasite line. Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite. Pandey AV, Tekwani BL, Singh RL, Chauhan VS: J Biol Chem 1999, 274:19383-19388.

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• Significance: Endoperoxide drugs offer an important hope against malarial strains that have developed resistances to current antimalarial drugs. Findings: The endoperoxide drugs, artemisinin and arteether (analogue of artemisinin), have been found to interfere with two important post Plasmodium-invasion processes: hemoglobin digestion and heme polymerization. Artemisinin was found to inhibit malarial cysteine proteases to a similar extent as E64. Both artemisinin and arteether were shown to significantly inhibit heme polymerization. In addition, artemisinin was also shown to be faster acting than quinoline antimalarial drugs. Induction of an acrosomal process in Toxoplasma gondii: visualization of actin filaments in a protozoam parasite. Shaw MK, Tilney LG: Proc Natl Acad Sci USA 1999, 96:9095 9099. • Significance: This paper represents the first visualization of actin filaments in apicomplexan parasites which rely on an actin-myosin motor for motility and cell invasion. Findings: Jasplakinolide, a membrane-permeable drug that stabilizes actin filaments, caused filaments to polymerize at the anterior end of Toxoplasma gondii tachyzoites in association with the conoid; there they often formed a prominent membraneenclosed apical projection. These filaments decorated with myosin subfragment S1, proving that they were actin. Tachyzoites treated with jasplakinolide also were unable to invade host cells and this effect was reversible, after the drug was washed out. A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum. Ben Mamoun C, Gluzman I, Goyard S, Beverley S, Goldberg D: Proc Natl Acad Sci USA 1999, 96:8716-8720. • Significance: This paper describes two new selection markers for P. falciparum that will allow better utilization of genetic manipulations to study gene function. Findings: The genes BSD and NEO, which confer resistance to blasticidin S and G418, respectively, were studied for use as selection markers in P. falciparum. Enzyme assays were performed for both markers to ensure translation of the resistance gene. Plasmid number was dependent on drug concentration, as determined by plasmid recovery. The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii. Hager KM, Striepen B, Tilney LG, Roos DS: J Cell Sci 1999, 112:2631-2638. • Significance: This paper suggests that the nuclear envelope associated with the T. gondii apical surface is functionally distinct from that of the basal domain, and forms an integral component of a polarized protein secretion pathway. Findings: Microscopic analysis indicates that the single T. gondii Golgi apparatus associates with the anterior end of the nucleus, where numerous coatomer-coated vesicles are localized. Interference with traffic from the endoplasmic reticulum (ER) to Golgi with brefeldin A, microtubule inhibitors, or dithiothreitol resulted in both disruption of the Golgi and swelling of the nuclear envelope. The carboxy-terminal retention signal of BiP, an ER chaperone protein associated with folding and exit of proteins, caused a surface protein to be retained intracellularly and localize primarily to the apical end of the nucleus. Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic

reticulum in Leishmania mexicana. Llgoutz SC, Mullin KA, Southwell BR, McConville MJ: EMBO J, 1999, 18:3643-3654. • Significance: This paper provides evidence that glycosylphosphatidylinositol (GPI) biosynthetic enzymes are localized to a tubular subcompartment of the endoplasmic reticulum (ER) of Leishmania mexicana promastigotes. Integral molecules in this subcompartment are potential targets for development of antiparasitic compounds. Findings: A green fluorecent protein (GFP)–dolichol-phosphate-mannose synthase (DPMS) chimera and subcellular fractionation identified compartmentalization of GPI biosynthetic enzymes in the ER of Leishmania. DPMS, a key enzyme in GPI biosynthesis in Leishmania, was localized to a distinct and novel tubular membrane subdomain of the ER, termed the DPMS tubule. The DPMS tubule is important in glycolipid biosynthesis and may be connected to both the ER and the Golgi apparatus. Disruption of the DPMS tubule structure prevents further processing of lypophosphoglycan (LPG) and protein anchor intermediates in the Golgi apparatus or transfer to protein in the ER. It is suggested that the DPMS tubule corresponds to a stable transitional ER where proteins and lipids are packaged into vesicles for transport to the Golgi apparatus.

Genomics Selected by Bernard Labedan and Patrick Forterre Université Paris-Sud, Orsay, France

Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont and Wolbachia. Heddi A, Grenier A-M, Khatchadourian C, Charles H, Nardon P: Proc Natl Acad Sci USA 1999, 96:6814-6819. •• Significance: Acquisition of bacterial genomes by eukaryotic cells continues today, and the successive steps allowing the formation of a complex endosymbiosis can be traced back to the original genetic integration. Findings: The weevil was already known to contain, beside mitochondria, another endosymbiont, the so-called SOPE (Sitophilus orysae principal endosymbiont), belonging to the Enterobacteriacae family (gamma subclass of proteobacteria). SOPE is supplying the animal cell with vitamins enhancing flight ability of adults through mediation on mitochondrial oxidative metabolism. SOPE is present in all weevil species and is transmitted only vertically. In more than half of strains belonging to three different species, another Gram-negative prokaryote has been found and identified as Wolbachia, an alpha proteobacterium close to the Rickettsia (the ancestors to mitochondria). Wolbachia is present in high density in germ cells causing nucleocytoplasmic incompatibility. Its role on insect metabolism does not appear as crucial as SOPE. It is proposed that Wolbachia, which can be transmitted horizontally as well as vertically, is still in the process of genetic integration. Mutation, recombination and incipient speciation of bacteria in the laboratory. Vulic M, Lenski RE, Radman M: Proc Natl Acad Sci USA 1999, 96:7348-7351. • Significance: The mismatch repair system can influence bacterial speciation dynamics though its simultaneous effects on mutation and recombination. Findings: The role of the methyl-directed mismatch repair (MMR) has been investigated in various lines of Escherichia coli deriving from a common ancestor and evolved for 20,000 generations. Two E. coli lines that have retained functional MMR have been compared to two lines that became defective in their MMR around generation 3,000 and since remained

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mutators. These defective lines have undergone more sequence divergence than the lines with functional MMR, and the obtained results support an important role of MMR in speciation — even if the magnitude of the barrier to recombination, which has been created in less than 10 years in the laboratory, is much smaller than that separating Salmonella and E. coli (i.e. 130 million years). Evolution of DNA base composition under no-strand-bias conditions when the substitutions rates are not constant. Lobry JR, Lobry C: Mol Biol Evol 1999, 16:719-723. • Significance: The no-strand-bias conditions were violated during the course of evolution of the DNA sequence to a state in which A = T and G = C within a strand suggesting that either mutation, or selection, or both were not symmetric with respect to the two DNA strands. Findings: A mathematical model has been built to understand the history of DNA base composition evolution. Two phenomena may be distinguished: first, the G+C content tends to equilibrium controlled by four parameters; and second, the evolution to the equifrequencies A = T and G = C can be observed in the 16 completely sequenced prokaryotic genomes presently available. However, local deviations have been reported meaning that the substitution rates are dependent on mutation and selection. This is true, for example, for the second codon positions (where T>A and C>G) in the coding sequences for integral membrane proteins in Escherichia coli because of the selective pressure to avoid polar or charged amino acids. This is equally true of the third codon positions in Borrelia burgdorferi as a consequence of an asymetric mutation pressure between the leading and lagging strands of replication. Archaea sister group of bacteria? Indications from tree reconstruction artifacts in ancient phylogenies. Brinkmann H, Philippe H: Mol Biol Evol 1999, 16:817-825. •• Significance: The root of the universal tree of life could be placed in the eukaryotic branch, instead of the bacterial branch as in the present textbook universal tree. This would have important implications for people trying to infer the nature of the last universal common ancestor from comparative genomics. Findings: The authors have revisited the phylogeny of the signal recognition particles and their paralogous receptor (SR α) in the three domains of life, focusing on slowly evolving positions. The trees based on only slowly evolving positions slightly favor the eukaryotic rooting, whereas those including rapidly evolving positions (noise) favor the bacterial rooting. Analysis of the data suggests that the bacterial rooting obtained with all positions is an artifact of long branch attraction due to rapidly evolving positions. Comparative genomics of the Archaea (Euryarchaeota): evolution of conserved protein families, the stable core, and the variable shell. Makarova KS, Aravind L, Galperin MY, Grishin NV, Tatusov RL, Wolf YI, Koonin EV: Genome Res 1999, 9:608-628. • Significance: Comparative genomics allows identification of specific features in the genomes of euryarchaeota that suggest the existence of two classes of genes with different origins: first, those that diverged by vertical inheritage, and second, those that are likely to have originated from Bacteria by lateral gene transfer. Findings: The first complete analysis of the four available euryarchaeal genomes identifies a core of 31%–35% genes

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present in all these Archaea. New putative homologues are identified in Eukaryotes or in Bacteria for some of these euryarchaeal orthologues. In general, most eukaryotic-like genes in euryarchaea belong to the core genes, which includes the great majority of genes coding for proteins involved in genome replication and expression. Many of the bacterial-like genes are only present in a subset of euryarchaeal species, suggesting loss or lateral gene transfer from Bacteria. A number of features typical for euryarchaea are identified such as unique protein families or expension of some protein families present in other domains. Is there a phylogenetic signal in prokaryote proteins? Teichmann SA, Mitchinson G: J Mol Evol 1999, 49:98-107. •• Significance: Traditional methods of tree reconstruction cannot recover the correct phylogeny for orthologous proteins between major bacterial lineages, indicating that it may not be possible to reconstruct the prokaryote phylogeny using standard sequence-based methods Findings: The 32 proteins found in single copy in nine completely sequenced microbial genomes (seven bacteria and two Archaea) were used to constuct a concatenated tree. Three cases of putative horizontal transfer giving robust nodes were detected and removed from the analysis. The tree obtained with the 29 remaining proteins consists almost entirely of noise at the level of bacterial phylum division. The only robust nodes are those between Archaea and Bacteria, and those grouping Escherichia coli and Haemophilus influenzae. The authors suggest that their conclusion also apply to rRNA phylogeny. Detecting protein function and protein–protein interactions from genome sequences. Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D: Science 1999, 285:751753. • Significance: The modular composition of proteins in some genomes (mainly eukaryotes) can be used to predict protein–protein interactions and help in functional assignement of genes in other genomes Findings: It has been previously noticed that some pairs of nonhomologous proteins in some genomes (e.g. GyrA and GyrB in bacteria) are fused into a single protein in other genomes (in this case, Topo II in eukaryotes). Using such fused proteins as the ‘Rosetta Stone’, the authors identified 6809 such putative protein interactions in Escherichia coli and 45,502 in yeast. In some cases, interacting networks can be suggested. Several tests confirm the validity of this new computational method to identify new pathways and protein complexes in organisms whose genome has been completely sequenced.

Antimicrobials Selected by Ian Chopra Antimicrobial Research Centre, University of Leeds, Leeds, UK

Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Mazmanian SK, Liu G, Ton-That H, Schneewind O: Science 1999, 285:760-763. •• Significance: It has been known for several years that S. aureus and other pathogenic Gram-positive bacteria contain a number of virulence proteins covalently attached to the cell wall. A specific mechanism, involving recognition of an LPXTG motif near the carboxyl terminus of the proteins, is responsible for their linkage to the cell wall. Until the publication of this paper the postulated enzyme, sortase, responsible for protein attachment had not been identified. Adopting a genetic approach the present authors have identified in S. aureus the

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novel gene, srtA, which appears to encode sortase. Homologues of the gene were also found in other Gram-positive organisms such as the enterococci and streptococci. In view of growing concerns that therapeutic options for the treatment of certain Gram-positive pathogens, such as S. aureus and enterococci, are becoming limited due to the emergence of resistance to current agents, sortase may represent a new drug target in these organisms. Findings: A S. aureus mutant defective in the anchoring of surface proteins was isolated and shown to carry a mutation in a novel gene defined as srtA (surface protein sorting A). The strA gene encodes a protein of 206 amino acids with a potential amino-terminal signal sequence and a presumed active-site cysteine at position 184. The assumption of an active-site cysteine is consistent with the observation that protein anchorage in vivo is susceptible to reagents that modify sulphydryl groups. Overexpression of srtA increased the rate of surface protein anchoring in S. aureus. Database searches revealed that homologues of srtA occur in a number of other Gram-positive bacteria including streptococci and enterococci. A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49. Tanabe K, Kondo T, Onodera Y, Furusawa M: FEMS Microbiol Lett 1999, 176:191-196. •• Significance: Recently, naturally occurring hyper-mutable strains of Escherichia coli and Salmonella typhimurium have been implicated as sources for the rapid evolution and emergence of antibiotic resistance genes. However, studies have not been performed to establish whether the patterns of resistance arising in hyper-mutable strains in the laboratory correspond to those observed in resistant clinical isolates. A correlation would provide evidence that hyper-mutable strains are indeed significant in the natural evolution of resistance to antibacterial agents. This paper has addressed this issue by examining the molecular basis of ofloxacin resistance in mutants selected in a mutator strain of E. coli. It is suggested that the mutator strain demonstrates under laboratory conditions the history of resistance acquisition observed in clinical isolates. Findings: The E. coli mutator strain dnaQ49, which has a modified 3′-5′ exonuclease subunit in DNA polymerase III, gave rise to various ofloxacin-resistant mutants following successive rounds of selection with increasing concentrations of the antibiotic. In several mutants a single amino acid change corresponding to a serine-83 to leucine-83 modification was observed in the quinolone resistance-determining region (QRDR) of the gyrase A subunit. In addition, a further two resistant mutants, expressing higher levels of ofloxacin resistance, contained double mutations, one at serine-83 (to leucine) in gyrA and a second in the related region of parC (a topisomerase IV subunit). The order and sites of quinolone resistance are coincident with those reported from clinical isolates of quinolone-resistant E. coli. Reversal of tetracycline resistance mediated by different bacterial tetracycline resistance determinants by an inhibitor of the Tet(B) antiport protein. Nelson ML, Levy SB: Antimicrob Agents Chemotherapy 1999, 43:1719-1724. • Significance: Much interest has recently been expressed in discovering and developing inhibitors of bacterial efflux pumps that remove antibiotics from the cell and, hence, confer phenotypic resistance. It is envisaged that such agents would be used in a manner similar to that of the β-lactamase inhibitors which, when combined with certain β-lactam antibiotics,

restore the activity of the antibiotic against β-lactamase producing strains. The tetracyclines are an important group of antibiotics whose clinical utility has declined due to the emergence of resistant isolates expressing tetracycline efflux (antiport) mechanisms. This paper describes the properties of a C-13 substituted semi-synthetic tetracycline analogue that blocks the activity of different tetracycline efflux systems. Findings: A number of semi-synthetic tetracycline analogues substituted at the carbon-6 position on the C ring of the tetracycline molecule were previously shown by the present authors to block the efflux of tetracycline mediated by the class B tetracycline resistance determinant found commonly in the enterobacteriaceae. More detailed studies, reported here, have now been conducted on a representative analogue, 13cyclopentylthio-5-OH-TC (13-CPTC). 13-CPTC competetively inhibited tetracycline efflux by the TetB system, blocking the uptake of tetracycline into everted E. coli membrane vesicles containing the TetB transporter. When used in combination with doxycycline, 13-CPTC exhibited a synergistic response against tetracycline resistant strains of E. coli carrying TetA or TetB efflux pumps. The inhibition of efflux protein activity demonstrates the ability to disrupt transport processes and modulate the tetracycline resistance phenotype.

Growth and development Selected by Lorna Casselton University of Oxford, Oxford, UK

Ploidy regulation of gene expression. Galitski T, Saldanha AJ, Styles CA, Lander ES, Fink GR: Science 1999, 285:251-254. •• Significance: Increased ploidy can markedly affect the size, physiology and behaviour of cells even though there is no change in relative gene dosage. The authors used budding yeast and whole genome expression analysis to demonstrate that there are genes whose expression is ploidy-regulated. Their findings have important implications with respect to cell ploidy changes that occur normally during eukaryotic development and tumour cell growth. Findings: The authors generated isogenic yeast strains having 1–4 sets of chromosomes and used oligonucleotide-probe microarrays to look for changes in gene expression. Amongst 10 ploidy-induced genes was CTS1, which encodes an endochitinase involved in cell separation. Seven ploidy-repressed genes included FLO11, which is required for invasive growth, and the G1 cyclin encoding genes CLN1 and PC11, whose decrease in expression may explain why polyploid cells are larger. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Uhlmann F, Lottspeich F, Nasmyth K: Nature 1999, 400:37-42. •• Significance: Cohesin is a protein complex in budding yeast that establishes cohesion between sister chromatids during DNA replication. Cohesion is necessary for chromosome alignment at mitotic metaphase but must be released when chromatids separate at anaphase. This paper shows that cohesion is destroyed when one of the cohesin subunits is cleaved by a protein known as Esp1. Significantly homologues of this yeast protein are found in other eukaryotes indicating that it has a highly conserved function in mitosis. Findings: The cohesin subunit Scc1 disappears at the metaphase–anaphase transition and this has previously been correlated with the activity of Esp1p. The authors developed an in vitro assay of Esp1p activity and showed that it can cleave Scc1p at two sites. Expression of a cleavage-resistant Scc1p

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was lethal to cells because it bound to chromosomes and failed to dissociate at the onset of anaphase. As a result sister chromatids failed to separate and cells with abnormal DNA content were formed. A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Klein F, Mahr P, Galova M, Buonomo SBC, Michaelis C, Nairz K, Nasmyth K: Cell 1999, 98:91-103. • Significance: This paper looks at the role of cohesins in meiosis and shows that they play a central role in the entire process. When chromosomes exit mitotic division sister chromatids separate to opposite poles of the spindle. At meiosis, however, there are two divisions, and sister chromosomes must stay associated at their centromeres during the first division and separate only at the second division. Proper segregation of chromatids is shown to relate to the presence of a meiosis-specific cohesin, Rec8p, which replaces the mitotic cohesin Scc1p. Findings: Using mutants and tagged proteins, the authors demonstrate that Rec8p is meiosis specific. Together with cohesin Smc3p, Rec8p is shown to localise to pachytene chromosomes and to be essential for proper chromatid behaviour at the first meiotic division. Both proteins persisted at the centromeric regions until the onset of anaphase II when sister chromatids separate. Mutants for either protein mis-segregated sister chromatids, lacked a synaptonemal complex and were defective in double-strand break repair and recombination. Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Watanabe Y, Nurse P: Nature 1999, 400:461-464. • Significance: The authors of this paper studied the role of the fission yeast cohesin Rec8, the homologue of the budding yeast Rec8p, and showed that it is essential for maintaining the correct segregation of sister chromatids at meiosis. Findings: The authors demonstrate the cohesin properties of Rec8 and show that deletion of the Rec8-encoding gene causes equational instead of reductional segregation of sister chromatids. GFP-labelled Rec8 was localised to the centromere and adjacent chromosomal regions during meiosis I, but disappeared at anaphase of meiosis II when equational segregation normally occurs. They propose that persistence of Rec8 during meiosis I maintains sister chromatid cohesion and in some way co-ordinates orientation of their kinetochores towards the same pole. Interaction of the yeast γ-tubulin complex-binding protein Spc72p with Kar1p is essential for microtubule function during karyogamy. Pereira G, Grueneberg U, Knop M, Schiebel E: EMBO J 1999, 18:4180-4195. • Significance: When haploid yeast cells respond to mating pheromone, they orient their growth towards each other and cell fusion is followed closely by nuclear fusion (karyogamy). Nuclei move along cytoplasmic microtubules (CMs) organised by the spindle pole body (SPB) embedded in the nuclear envelope and the orientation of these is critical. The Kar1 protein, a component of the so-called half bridge substructure of the SPB, is essential for karyogamy. This report shows that, in response to pheromone, Kar1p recruits the protein complex necessary for microtubule assembly and causes a rearrangement of CMs at the SPB. Findings: Using a two-hybrid screen and GST-association assay, an amino-terminal region of Kar1p was identified that

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interacts with the carboxyl terminus of γ-tubulin-complex-binding protein Spc72. Evidence from studies with mutants indicates that Spc72p location fluctuates within the SPB during the cell cycle and that the Kar1p–Spc72p interaction has minor significance in vegetative growth but is essential during mating. Selected by Jeff Errington University of Oxford, Oxford, UK

Gene silencing via protein-mediated subcellular localisation of DNA. Kim S-K, Wang JC: Proc Natl Acad Sci USA 1999, 96:8557-8561. •• Significance: The SopB plasmid segregation protein of Escherichia coli F plasmid exhibits a gene silencing effect, which was previously thought to occur by the formation of a nucleoprotein filament. The new data challenge the nucleoprotein model and provide evidence for a sub-cellular sequestration mechanism of gene silencing of a more universal nature. Findings: Segments of SopB were fused to the yeast GAL4 specific DNA-binding domain, and the ability of the fusion protein to silence a reporter gene linked to the GAL4-binding site was tested. Remarkably, silencing could be achieved with a fusion protein containing only the amino-terminal 82 amino acids of SopB, which does not have DNA-binding activity, but is the segment required for the localisation of the protein to the 1/4 positions in the cell. The results suggest a model whereby patches of sequence-specific DNA-binding domains are formed by targeting of the amino terminus of SopB to a specific cellular location. These patches are then able to non-specifically bind DNA in their vicinity, which prevents transcription. Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly. Harry EJ, Rodwell J, Wake RG: Mol Microbiol 1999, 33:33-40. • Significance: A link between the early stages of DNA replication and formation of a central cell division site was identified. Findings: Spore germination was used to characterise the dependence of formation of an FtsZ ring (an early step in formation of the cell division machinery) on progression of a round of DNA replication. Replication was controlled using a mutant temperature-sensitive for the initiation of DNA replication, by withholding thymine, or by use of an inhibitor of DNA polymerase III. Centrally located FtsZ rings were found to assemble under conditions that permitted initiation of the first round of DNA replication, followed by limited chain elongation. Central Z rings did not form if DNA replication was completely blocked by an inhibitor, suggesting that the early stages of a round of DNA replication are required for positioning of the Z ring at mid-cell. Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics and implications for peptidoglycan synthesis. Denome S, Elf PK, Henderson TA, Nelson DE: J Bacteriol 1999, 181:3981-3993. • Significance: A demonstration that most of the multiple genes encoding penicillin-binding proteins (PBPs) of E. coli can be disrupted without a serious effect on cell viability. The mutant strain collection will provide an excellent base on which to test current models for peptidoglycan biosynthesis and to answer fundamental questions about cell wall metabolism. Findings: A large set of E. coli mutants was generated in which the genes encoding all possible combinations of a set of PBPs (1a, 1b, 4, 5, 6, and 7, AmpC and AmpH) were disrupted. The

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mutants were made by an ingenious method in which part of the gene was replaced with a kanamycin resistance cassette, which was then excised by site-specific recombination. Of the 12 known PBPs, eight could be deleted to give viable strains, confirming the redundancy that exists for their particular biological function under laboratory conditions. When two further PBPs were inhibited by antibiotics (PBPs 2 and 3), leaving only two PBPs active (1b and 1c), the cells remained viable, although cell morphology was perturbed (the cells were enlarged and sperical). A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli. Templin MF, Ursinus A, Höltje J-V: EMBO J 1999, 18:4108-4117. • Significance: Cloning and characterization of the lcd gene encoding an important enzyme involved in recycling of the precursors of cell wall synthesis. Disruption of the gene leads to

cell lysis under some growth conditions, making it a possible target for new antibiotics. Findings: The gene lcd encoding an enzyme purified on the basis of L,D-carboxypeptidase activity (bonds found only in bacterial cell wall material, peptidoglycan or murein) was identified in an E. coli expression library. The predicted sequence was similar to that of a family of proteins of previously unknown function present in a broad range of bacteria. The enzyme was shown to localise in the cytoplasm, and its substrate specificity and susceptibility to β-lactam antibiotics were determined. The effects of lcd mutation on murein composition and intracellular pools of murein precursors were also studied. Cells containing a disruption of lcd grew normally but lysed in stationary phase. This effect could be suppressed by mutation of the ampD gene, consistent with a role for Lcd in murein precursor recycling and with the accumulation of a dead-end intermediate, UDPMurNAc-tetrapeptide, as the basis for the defect in wall structure in stationary phase cells.