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 2001, 4:103–113 Contents (chosen by) 103 Host–microbe interactions: bacteria (Blocker and Peppler) 104 Cell regulation (McFall-Ngai) 106 Ecology and industrial microbiology (Normand) 106 Microbial techniques (Grimont) 107 107 108 109 110 • ••
Host–microbe interactions: fungi (Spitzer) Host–microbe interactions: viruses (Rosenthal) Host–microbe interactions: parasites (Sibley) Genomics (Labedan and Forterre) Growth and development (Gold et al., Errington and Feucht) of special interest of outstanding interest
Host–microbe interactions: bacteria Selected by Ariel Blocker Sir William Dunn School of Pathology, Oxford, UK
Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in Gram-positive bacteria. Madden JC, Ruiz N, Caparon M: Cell 2001, 104:143-152. •• Significance: For decades, the many different families of bacterial pore-forming toxins have been assumed to damage or kill eukaryotic cells by generating holes in their plasma membrane after being secreted during infection. This paper shows that one of the most famous of these toxins, streptolysin O, in fact serves to translocate another bacterial protein in a contactdependent manner into the cytoplasm of host cells. The translocator and its effector are both required for cytotoxicity. A major paradigm shift follows from these findings. Findings: Streptococcus pyogenes (group A streptococcus) is the cause of numerous serious suppurative infections of the pharynx and soft tissues, as well as several systemic infections in humans. It secretes more than 20 different polypeptides: superantigens, proteases and hemolysins, for example. Despite their identified activities, the mechanisms by which each of these proteins contribute to disease are not understood. One of these factors, streptolysin O (SLO), is a member of the family of cholesterol-dependent cytolysins. The authors noticed that a commercial SLO antibody recognised not only SLO but also a second band in bacterial culture supernatants and infected host cell extracts. The 52kDa band was identified as S. pyogenes NAD-glycohydrolase (Spn), an enzyme capable of producing the potent second messenger cyclic ADP-ribose. Spn activity was detected in infected host cell extracts. spn is located adjacent to the gene encoding SLO in the S. pyogenes genome and the genes form an operon. The 52kDa band and Spn activity were not present in extracts of host cells infected with either an slo or an spn mutant. Remarkably, Spn was also not found in host cell extracts co-infected with the slo and spn mutants, implicating a contact-dependent transfer process. Spn translocation also required an S. pyogenes surface adhesin. The
authors next showed that during infection with wild-type S. pyogenes, 71% of Spn activity was found in host cell extracts, whereas with the slo mutants, 93% was found in the infection culture supernatant. The slo and the spn mutants had identical decreases in their cytotoxic effect on host cells, suggesting again that both proteins work synergistically in this process. A Toll-like receptor recognizes bacterial DNA. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K et al.: Nature 2001, 408:740-745. •• Significance: After the very recent identification of the Tolllike receptors (TLR) TLR2 and TLR4 as key cell surface players in the innate immune responses to peptidoglycan and lipopolysaccharide (LPS), respectively, this paper presents data showing that TLR9 plays an essential role in sensing bacterial DNA. This field continues to unfold at an exhilarating pace. Findings: Bacterial, but not mammalian, DNA specifically activates immune cells because of its high content in unmethylated CpG dinucleotides. The authors have identified a new Toll-like receptor, TLR9, and generated TLR9-deficient mice. They showed that TLR9-/- mice do not display the normal responses to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. The mice were resistant to the lethal effect of CpG DNA without any elevation in serum pro-inflammatory cytokine levels, and their CpG-DNA-mediated T-helper type I response was also abolished. TLR9 appeared to work through the recently identified IRAK and MyD88 to mediate NF-kB activation but, as with all other TLRs so far studied, the other probable key cell surface factor(s) acting upstream of it in “danger signal recognition” remain mysterious. Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB. Tezcan-Merdol D, Nyman T, Lindberg U, Haag F, Koch-Nolte F, Rhen M: Mol Microbiol 2001, 39:606-619. • Significance: Both the chromosomal SPI-2 type III secretion machinery and the virulence-plasmid-located spvRABCD gene cluster are required for systemic infection and intracellular growth of Salmonella species, although they do not seem to act synergistically. The function of the five spv genes (other than spvR, which encodes a LysR-type transcriptional activator) was completely mysterious. Recently, the authors showed that SpvB is a mono(ADP-ribosyl)transferase (Otto et al., Mol Microbiol 2000, 37:1106-1115). They now show that actin is a major host cell target for the SpvB enzyme during infection. This implies that SpvB becomes translocated into the host cell cytosol. Could this hint to a (potentially quite novel) function for the other three spv genes in SpvB translocation? Findings: The authors showed that a 43 kDa band was the major ADP-ribosylated protein in host cell extracts incubated with the recombinant catalytic domain of SpvB or with bacterial extracts containing full-length SpvB. This protein was also the major ADP-ribosylated protein in extracts of cells infected with wild-type Salmonella enterica but not with a S. enterica strain carrying a Tn5 insertion in spvB. Immunoprecipitation and immunoblotting identified the modified protein as actin. The SpvB catalytic domain specifically mediated actin modification
and prevented actin polymerisation in vitro. Infection with wildtype S. enterica, but not the insertional spvB mutant, led to a massive decrease in filamentous actin in MDCK cells and to complete rounding-up of the cells. Selected by Mark S Peppler University of Alberta, Edmonton, Alberta , Canada
Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II. Li Y, Li H, Dimasi N, McCormick JK, Martin R, Schuck P, Schlievert PM, Mariuzza RA: Immunity 2001, 14:93-104. • Significance: This is the first crystallographic data defining the critical high-affinity interaction between a superantigen and the zinc-dependent beta chain of the MHC class II molecule. Preventing such interactions could form the basis of new therapies for pyrogenic shock caused by bacterial toxin superantigens. Findings: Superantigens are molecules that trigger a polyclonal activation of T cells by binding to the MHC class II and T cell receptor (TCR) at the same time. This dual binding can result in dramatic pathological effects such as toxic-shock-like syndrome, in which streptococcal pyrogenic exotoxin C (SPE-C) is the activating superantigen. The authors co-crystallized SPE-C with MHC class II molecule HLA-DRA2a that contained a homogenous myelin basic protein (MBP) peptide bound in its peptide groove. Their results show that SPE-C binds to the beta chain of DRA2a through a zinc bridge, a result consistent with previous biochemical and mutagenesis experiments. It was a surprise, however, to find that SPE-C made numerous contacts with the bound MBP peptide. This closely resembles the normal interaction between the peptide/MHC class II and the TCR during antigen presentation, and may help explain how SPE-C triggers T cells with such efficiency. Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family. Nakamura K, Funakoshi H, Miyamoto K, Tokunaga F, Nakamura T: Biochem Biophys Res Commun 2001, 280:1028-1035. AND
Complement receptor 1/CD35 is a receptor for mannanbinding lectin. Ghiran I, Barbashov SF, Klickstein LB, Tas SW, Jensenius JC, Nicholson-Weller A: J Exp Med 2000, 192:1797-1808. • Significance: These papers describe the potential contribution of two distinct C-type lectins in innate immunity. The paper by Nakamura et al. gives the first description of human SRCL, a cell surface C-type lectin that is likely involved in clearing bacterial infections. The second paper identifies complement receptor 1 as the likely receptor on phagocytes for mannanbinding lectin, a soluble C-type lectin involved in bacterial opsonization. Findings: Scavenger receptors (SRs) are a family of cell surface glycoproteins that are thought to be important in a number of normal and pathological processes in the body. They are grouped on the basis of their structure, and those that have the C-type lectin structure are thought to be important in host defense. Nakamura et al. screened a human cDNA library and found clones expressing a new receptor that has structural properties of both C-type and class A type I SRs. This suggested that the new SR, which they named human scavenger receptor with C-type lectin (SRCL), may be involved in bacterial binding. They found that Escherichia coli and Staphylococcus aureus both bound to cells bearing cloned SRCL, most likely
through fucose, mannose or N-acetylglucosamine residues on the bacteria. Although no phagocytosis was observed, the authors still feel that this interaction may help remove invading organisms. Mannan-binding lectin (MBL) is a representative of the soluble C-type lectin family known as collectins. Ghiran et al. show that cloned complement receptor CR1/CD35 has a high (5 nM) affinity for MBL and is the likely receptor for MBL on polymorphonuclear leukocytes (PMNs). MBL alone was not sufficient to opsonize Salmonella montevideo, but it would opsonize if combined with subopsonic amounts of antiSalmonella IgG or if the PMNs were pre-incubated with soluble fibronectin. In both these instances, in which MBL could opsonize, polyclonal anti-CR1 Fabs would block opsonization, supporting the role of CR1 as the cell surface receptor for MBL. Role of serum amyloid P component in bacterial infection: protection of the host or protection of the pathogen? Noursadeghi M, Bickerstaff MC, Gallimore JR, Herbert J, Cohen J, Pepys MB: Proc Natl Acad Sci USA 2000, 97:14584-14589. • Significance: Serum amyloid P (SAP) increases susceptibility to infection with Neisseria meningitidis or Escherichia coli by inhibiting phagocytosis. However, a potential treatment that prevents SAP binding and restores the upper hand to the host exists. Findings: SAP is an acute-phase serum protein whose main function is to bind chromatin released from damaged cells. It is known that SAP binds to Streptococcus pyogenes and the authors explored the contribution of SAP to clearance of other bacterial infections. They were surprised to find SAP–/– mice to be more resistant to infection with E. coli and N. meningitidis, and that the resistance could be reversed by injecting SAP into the mice. SAP acted like an antiopsonin, but treating mice with the drug (R)-1-[6-(R)-2-carboxy-pyrrolidin-1-yl]-[6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (compound R) could reverse SAP binding, allowing normal phagocytosis. The authors suggest that this compound R could have therapeutic potential for those diseases caused by organisms known to bind SAP.
Cell regulation Selected by Margaret McFall-Ngai Pacific Biomedical Research Center, Kewalo Marine Laboratory, Honolulu, USA
QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. Chugani SA, Whiteley M, Lee KM, D’Argenio D, Manoil C, Greenberg EP: Proc Natl Acad Sci USA 2001, 98:2752-2759. •• Significance: The bacterium Pseudomonas aeruginosa is a generalist, occupying a wide variety of niches from the soil microbial community (in which it is a free-living constituent), to the lungs of patients with the genetic disease cystic fibrosis (in which it is a pathogen). The basis of the versatility of this microorganism remains largely unknown. The authors present evidence that modulation of P. aeruginosa quorum sensing, a behavior now recognized as common in bacteria, may be one mechanism by which the bacterium adjusts to different environmental conditions. Analogous, or even homologous, mechanisms may be present in other versatile microorganisms that exhibit quorum-sensing behavior. Findings: The authors describe a gene, qscR (quorum-sensingcontrol repressor), in P. aeruginosa and the effects of mutations in this gene. They provide evidence to show that the protein product, QscR, acts to repress the expression of one of the genes, lasI, associated with autoinduction, an activity that
results in a delay in the induction of genes governed by the lasI-associated quorum-sensing system. Mutations in the qscR gene lead to premature expression of lasI and the genes that the lasI product regulates. In addition, in experiments designed to investigate effects of this phenotype on virulence of the bacterium, wild-type strains and qscR mutants were fed to Drosophila melanogaster. Flies fed mutant strains died significantly more rapidly than those fed wild-type strains. The authors conclude that QrsC provides regulation of the timing of quorum-sensing behavior so that its associated genetic induction does not occur under inappropriate circumstances. Microbial modulation of innate defense: goblet cells and the intestinal mucus layer. Deplancke B, Gaskins HR: Amer J Clin Nutrit, in press. • Significance: This paper provides a valuable, comprehensive review of the current understanding of the interplay among the mucosal epithelium of the intestine, the immune system and the indigenous microbiota. Principal focuses of this paper include coverage of what is known about the development of mucosal layers of the mammalian gut through ontogeny and the use of germ-free animals to understand the role of microbes in control of the processes governing these systems. Findings: The authors first describe the structure and function of the mucus layer, the biology of the underlying goblet cells, and the dynamics of mucin synthesis and secretion. An emphasis is placed on what is known about the interface of bacteria with these tissues and their processes, as well as the various technical problems that have precluded a more complete understanding of these complex environments. The remainder of the article is devoted to reviewing studies of goblet cell ontogeny and what is understood about the roles of host and bacterial symbionts in defining developmental patterns and eventual function of mucosal layers of the intestine. Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development. Kannenberg EL, Carlson RW: Mol Microbiol 2001, 39:379-391. • Significance: Recent studies of the lipopolysaccharide (LPS) of bacterial pathogens have shown that LPS structure is often modified under the conditions of the microbe’s association with the host. This paper describes alterations in the LPS of a beneficial microbial partner under the conditions of symbiosis. Findings: The structure of Rhizobium leguminosarum LPS was characterized from cells grown under three different conditions: standard culture conditions traditionally used for the study of this species; culture conditions designed to mimic the root nodule environment; and the natural conditions of the root nodule, where the cells occur as highly modified bacteroids. The authors report that under reduced-oxygen culture conditions, similar to those that cells experience in the nodule, an LPS structure is produced that is significantly more hydrophobic. They present evidence that these changes are due to alternations in the lipid A and O-antigen constituents of the molecule. These modifications were reflected in several biochemical characteristics of these differently-derived molecules, including their extraction behavior in aqueous and organic solvents, their electrophoretic mobility, and their antigenic properties. Molecular analysis of commensal host–microbial relationships in the intestine. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI: Science 2001, 291:881-884.
•• Significance: The mammalian intestine maintains a complex consortium of bacteria throughout the host’s life history, although very little is known about the dynamics that occur between this community and host tissues. Using recently developed molecular methods, this contribution provides the first characterization of host gene expression during the initial stages of colonization of host intestinal tissues by bacterial symbionts. Findings: Gene expression of intestinal tissues of germ-free mice was compared with expression in these tissues in mice that had been colonized by the intestinal symbiont Bacteroides thetaiotaomicron. Using DNA microarrays and laser-capture microdissection coupled with quantitative RT-PCR, the authors showed that the bacterial population induces expression of a wide variety of genes, including ones associated with nutrient processing and mucosal immunity as well as genes involved in maturation of the intestine. They also compared gene expression induced by B. thetaiotaomicron with expression induced by colonization with either Bifidobacterium infantis or Escherichia coli, or with a complex mixture of bacteria derived from the intestine of conventionally raised mice. Their results indicate that bacteriainduced host gene expression is sensitive to the differences in the microbial species with which the intestine is interacting. A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration. Lee CA, Silva M, Siber AM, Kelly AJ, Galyov E, McCormick BA: Proc Natl Acad Sci USA 2000, 97:12283-12288. •• Significance: Salmonella typhimurium, which infects the mammalian intestine, is one of the pathogenic bacterial species with a type III protein secretion system, the components of which are thought to act in concert as a complex virulence system. The authors provide evidence to show that one protein of the system, SipA, is necessary and sufficient to induce proinflammatory responses in host tissues, i.e. the components associated with direct delivery of the protein into host cells are not required. The data suggest that the activity of elements of type III secretion systems is more versatile than previously thought. Findings: In neutrophil transmigration experiments with monolayers of cultured epithelial cells, strains of S. typhimurium mutant in sipA showed a diminished capacity to induce transepithelial migration of neutrophils. Purified SipA protein alone was capable of inducing this behavior and migration could be inhibited when experiments were performed in the presence of antibodies to SipA. In addition, an inhibitor of protein kinase C (PKC) blocked the Sip-A-induced transepithelial migration of neutrophils. Taken together, the data suggest that SipA directly activates PKC-dependent epithelial signaling pathways to affect transmigration of neutrophils. Studying interaction transcriptomes: coordinated analyses of gene expression during plant–microorganism interactions. Birch PRJ, Kamoun S: New Technologies for the Life Sciences: A Trends Guide 2000, 77-82. • Significance: One of the principal challenges in the study of a host–bacterium interaction is the accurate characterization of the complex reciprocal dialogue that occurs between the partners during the establishment and maintenance of the association. This contribution reviews the impact of the development of new molecular methods on the approach to these problems. Although the paper is directed specifically to methods that would enhance our understanding of plant–microbe interactions, the concepts that are discussed apply equally well to animal–microbe relationships.
Findings: The authors describe how the continued refinement of such methods and resources as RT-PCR, genomics and bioinformatics can be used to decipher the changes in gene expression that accompany the dynamics of host–microbe interactions. Emphasis is placed on mechanisms by which biologists may simultaneously analyze both partners during complex developmental events.
Ecology and industrial microbiology Selected by Philippe Normand Université Claude Bernard, Lyon, France
Gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication. Andersen JB, Heydorn A, Hentzer M, Eberl L, Geisenberger O, Christensen BB, Molin S, Givskov M: Appl Environ Microbiol 2001, 67:575-585. •• Significance: Bacteria have adapted to their various environments by developing ways to react to different stimuli, one of which is the quorum sensing response. This system, present in a variety of microbes, allows the cells to modulate their response as a function of their density, which brings the question of what functions are under the control of such a system and the control of interference between taxa. The present constructs, based on the ability to perform single-cell analysis and online studies of N-acylhomoserine lactone (AHL)-mediated communication among bacteria, should help elucidate this basic function. This green fluorescent AHL sensor provides an elegant and powerful tool for studies of communication between the individual cells present in mixed bacterial communities. Findings: The Vibrio fischeri quorum sensor encoded by luxR-PluxI was fused to mutant gfpmut3* genes that encode unstable green fluorescent proteins. Bacterial strains that harbor this construct reacted to small amounts of a broad spectrum of AHL molecules such as N-3-xohexanoyl-Lhomoserine lactone in their environment. In combination with epifluorescent microscopy, such reporter strains enable AHL detection at the single-cell level and allow for real-time measurements of fluctuations in AHL concentrations. Nitrogen fixation genes in an endosymbiotic Burkholderia strain. Minerdi D, Fani R, Gallo R, Boarino A, Bonfante P: Appl Environ Microbiol 2001, 67:725-732. • Significance: This report on the presence and expression of some nif (nitrogen fixation) genes in the Burkholderia strain present in the endomycorhizal fungus Gigaspora margarita opens up perspectives for the extension of the ability to fix nitrogen to a large number of plants. The endomycorhizal symbiosis is prevalent among land plants, and even though the real function of the Burkholderia endosymbiont is still unknown, it could be manipulated more easily than the fungus itself to fix nitrogen, among other things. Findings: This paper concerns the identification and characterization of a DNA region containing putative nif genes belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus, Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores containing also the bacterial genome was screened with Azospirillum brasilense nifHDK genes, which permitted the identification of a 6,413-bp region containing three open reading frames encoding putative proteins with a high degree of sequence similarity to the two subunits (NifD and NifK) of dinitrogenase and to dinitrogenase reductase (NifH) from different diazotrophs. Reverse transcriptase PCR experiments with primers targeting the Burkholderia nif genes performed on total RNA extracted from
spores demonstrated that gene expression was present during the germination phase. Burkholderia cepacia Genomovar III is a common plantassociated bacterium. Balandreau J, Viallard V, Cournoyer B, Coenye T, Laevens S, Vandamme P: Appl Environ Microbiol 2001, 67:982-985. • Significance: Due to environmental concerns about nitrogen in the water table and to economic pressures, plant growth promoting rhizobacteria (PGPR) are being used on an increasing basis. This study draws attention to the short genetic distance between ‘cepacia syndrome’ strains and potential PGPR strains, raising the question of what is a ‘safe’ bacterial strain. An animal model for cystic fibrosis would be necessary to determine the potential of a strain to cause cepacia syndrome. Findings: A polyphasic taxonomic study based on several techniques (DNA–DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis) led to the conclusion that a group of Burkholderia cepacia isolates obtained from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia Genomovar III, a genomic species associated with ‘cepacia syndrome’ in cystic fibrosis patients. Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). Wiedenmann J, Elke C, Spindler KD, Funke W: Proc Natl Acad Sci USA 2000, 97:14091-14096. • Significance: The green fluorescent protein (GFP) is becoming the tool of choice in several studies in environmental microbiology to follow and quantify the activity of microorganisms in situ, as it is not necessary to add a substrate. However, some of these environments have some level of autofluorescence, and it is useful to have a palette of differently-coloured related proteins to follow more than one strain or activity at a time. Findings: Two GFPs isolated from the sea anemone, Anemonia sulcata, were characterized: a fluorescent orange protein, and a nonfluorescent red protein. The two proteins seemed to represent two different states of the same protein. Furthermore, the nonfluorescent red protein and a GFP were cloned and the genes studied. Both proteins were found to be homologous to the GFP from the jellyfish Aequorea. The red protein was significantly smaller than other GFP homologues. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence.
Microbial techniques Selected by Francine Grimont Institut Pasteur, Paris, France
Portable system for microbial sample preparation and oligonucleotide microarray analysis. Bavykin SG, Akowski JP, Zakhariev VM, Barsky VE, Perov AN, Mirzabekov AD: Appl Envir Microbiol 2001, 67:922-928. •• Significance: The authors have developed a rapid, simple and inexpensive system for bacterial identification using biological microchip. The procedure combines: first, an universal syringeoperated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling and removal of excess free label and short oligonucleotides; second, immobilized oligonucleotide microarrays for 16S rRNA identification; and third, a portable imager for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. Findings: The process requires no vacuum filtration step, phenolchloroform extraction, CsCl fractionation or centrifugation.
Using the silica minicolumn, whole nucleic acids or fractionated DNA and RNA from Gram-negative bacteria can be isolated within several minutes. The procedure involves sequential washing of the column with different solutions. After hybridization, the microchip is analysed using a wide-field fluorescence microscope coupled with a cooled charge-coupled device (CCD) camera or a microchip imager. This system is capable of discriminating between Escherichia coli, Bacillus subtilis, Bacillus thuringensis and human HL60 cells. The procedure is rapid: it takes approximately 25 minutes to obtain labeled DNA and RNA samples and an additional 25 minutes to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager. This portable system can be successfully used in the laboratory for rapid bacterial identification in medical, agricultural or environmental applications. Monitoring of Lactobacillus fermentation process by using ion chromatography with a series piezoelectric quartz crystal detector. Zhang J, Xie Y, Dai X, Wei W: J Microbiol Methods 2001, 44:105-111. • Significance: An ion-exchange chromatographic technique (IC) combined with a series piezoelectric quartz crystal (SPQC) detector is presented in this paper. Findings: This IC/SPQC technique was successfully applied to monitoring and optimization of the Lactobacillus fermentation process. An automatic membrane dialyser was used for the pretreatment of the sample in online monitoring. A mixture of p-hydroxybenzoic acid and N,N-diethylethanolamine was used as mobile phase and its flow rate was 0.8 ml/min. The rate of production of lactic acid was studied at the optimal culture temperature of 45°C and the optimum pH of 7. This method is simple and convenient and the results obtained are accurate and reliable. In comparison with the conductivity detector, the SPQC detector has advantages of high sensitivity, low detection limit and simple fabrication. The proposed method can be applied to monitoring of metabolic processes of microorganisms.
Host–microbe interactions: fungi Selected by Eric D Spitzer Department of Pathology, SUNY at Stony Brook, New York, USA
Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct. Lengeler KB, Wang P, Cox GM, Perfect JR, Heitman J: Proc Natl Acad Sci USA 2000, 97:14455-14460. AND
Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the matingtype locus. Lengeler KB, Cox GM, Heitman J: Infect Immun 2001, 69:115-122. •• Significance: Most clinical and environmental isolates of Cryptococcus neoformans are mating type alpha (MATα). Furthermore, previous surveys have failed to detect MATa in any serotype A strains (also known as C. neoformans var. grubii), the predominant variety of C. neoformans. The first of these papers describes the cloning of a gene from the MATa locus and the discovery of the first MATa serotype A strain. The second paper describes several aneuploid or diploid serotype AD strains in which the MATa allele was contributed by a serotype A parent. These findings shed new light on the ecology of C. neoformans and will facilitate future genetic studies. Findings: Lengeler KB et al. (2000) isolated the gene for Ste20a kinase from the MATa locus of a serotype D strain by
using low-stringency PCR. The STE20a gene shared 70% identity with the STE20α gene. STE20a was detected in a serotype A clinical isolate from Tanzania. The STE20a genes from the serotype A and D strains were 95% identical to each other at the nucleotide level. Unfortunately, the serotype A MATa strain was sterile under all laboratory conditions tested. Lengeler KB et al. (2001) examined 10 serotype AD strains. Flow cytometry analysis revealed that all of them were aneuploid or diploid. This was confirmed by PCR analysis using serotype-specific primers that revealed heterozygosity at three separate loci in addition to the mating type loci. Several of the strains were typed as only A or D, suggesting that the AD phenotype is unstable. In four strains, the MATa locus was derived from a serotype A strain. These studies confirm the existence of a serotype A MATa locus and illustrate the importance of detailed genotypic analysis of clinical isolates. Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Luberto C, Toffaletti DL, Wills EA, Tucker SC, Casadevall A, Perfect JR, Hannun YA, Del Poeta M: Genes Dev 2001, 15:201-212. • Significance: Ipc1 is a fungus-specific enzyme that catalyzes the synthesis of essential sphingolipids. This is the first paper to present genetic evidence that Ipc1 contributes to the pathogenicity of C. neoformans. Decreased expression of IPC1 had pleiotropic effects on cell growth and metabolism. Findings: Mutants were constructed in which the IPC1 gene was placed under the control of an inducible GAL7 promoter. In the absence of galactose, the mutants exhibited decreased Ipc1 activity, decreased production of melanin, and decreased organism load compared to control yeasts in a rabbit chronic meningitis model. In contrast, intracellular growth of the mutant in a murine macrophage-like cell line increased significantly when IPC1 was induced by galactose. Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Cox GM, McDade HC, Chen SC, Tucker SC, Gottfredsson M, Wright LC, Sorrell TC, Leidich SD, Casadevall A, Ghannoum MA et al.: Mol Microbiol 2001, 39:166-175. • Significance: Previous work suggests that phospholipase contributes to the virulence of Candida albicans. This paper uses a genetic approach to extend these findings to C. neoformans. Findings: The C. neoformans phospholipase gene (PLB1) was cloned by using degenerate PCR primers, and a knockout mutant (plb1) was constructed. The plb1 mutant showed an almost complete deficiency of extracellular phospholipase B, lysophospholipase and lysophospholipase transacylase activities, but exhibited no growth defects in vitro. The plb1 mutant showed reduced virulence in a murine inhalation infection model. Mice infected with the mutant yeast survived an average of 62 days compared to 36 to 46 days for mice infected with yeast containing the wild-type PLB1 gene. The plb1 mutant also exhibited a substantially decreased organism load compared to control yeast in a rabbit chronic meningitis model.
Host–microbe interactions: viruses Selected by Kenneth L Rosenthal McMaster University Health Sciences Centre, Hamilton, Ontario, Canada
Isolation of primary HIV-1 that target CD8+ T lymphocytes using CD8 as a receptor. Saha K, Zhang J, Gupta A, Dave R, Yimen M, Zerhouni B: Nat Med 2001, 7:65-72.
• Significance: This paper provides further evidence to confirm the authors’ previous results showing that HLA supertypes are associated with protection against HIV-1 transmission. These results have implications for HIV vaccine design, as the frequency of this HLA supertype approaches 40% worldwide. Findings: HIV-1-producing CD8+ clones were isolated from two AIDS patients. Although HIV produced by CD8+ cells maintained ability to infect CD4+ T cells, these viruses infected CD8+ cells independent of CD4 or the chemokine co-receptors CCR5 and CXCR4. Evidence that CD8 serves as the receptor include CD8 downmodulation after infection, the ability of antiCD8 antibodies to block viral entry, and conversion of resistant cells to susceptibility after expression of CD8. Novel changes were observed in envelope sequences of CD8-tropic viruses. The HLA A2/6802 supertype is associated with reduced risk of perinatal human immunodeficiency virus type 1 transmission. MacDonald KS, Embree JE, Nagelkerke NJD, Castillo J, Ramhadin S, Njenga S, Oyug J, Ndinya-Achola J, Barber BH, Bwayo JJ et al.: J Infect Dis 2001, 183:503-506. • Significance: This paper contains an extension of the authors’ previous results showing that HLA supertypes are associated with differential susceptibility to HIV-1 transmission. Findings: Perinatal transmission occurred in 2.8% of 72 children with HLA A2/6802 supertype versus 17.2% of 99 children without the A2/6802 supertype (an estimated sevenfold protective effect). This effect was independent of the potent protection associated with maternal-child class I HLA discordance. Furthermore, possession of the A2/6802 supertype was not associated with protection from late breast milk transmission. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. Jackson RJ, Ramsay AJ, Christensen CD, Beaton S, Hall DF, Ramshaw IA: J Virol 2001, 75:1205-1210. • Significance: Virus-encoded interleukin 4 (IL-4) suppressed primary antiviral cell-mediated immune responses and also inhibited memory immune responses resulting in acute mousepox with high mortality. Findings: Expression of IL-4 by a thymidine-kinase-positive ectromelia virus suppressed cytolytic responses of NK and CTL and the expression of IFNγ. Genetically resistant mice infected with IL-4 expressing ectromelia virus developed acute mousepox and high mortality. Infection of immunized genetically resistant mice with virus expressing IL-4 also resulted in significant mortality. Protein design of an HIV-1 entry inhibitor. Root MJ, Kay MS, Kim PS: Science 2001, 291:884-888. • Significance: A small protein designed to bind the C-peptide region of HIV-1 gp41 displays potent inhibitory activity against diverse HIV-1 variants and may serve as the basis for a new class of antiviral agents. Findings: Agents that interfere with the formation of gp41 trimer-of-hairpins structure can inhibit HIV infection. In order to test the hypothesis that the C-peptide region of gp41 is a target for inhibition of HIV entry, a small protein, designated 5-Helix, was designed that binds strongly and specifically with the C-peptide region of HIV-1 gp41. 5-Helix protein potently inhibits HIV infectivity and cell–cell fusion. It inhibits infection by viruses pseudotyped with a variety of HIV-1 envelope proteins from clades A, B and D. Thus, 5-Helix is a potent broad-spectrum inhibitor of HIV entry.
Host–microbe interactions: parasites Selected by David Sibley Washington University School of Medicine, St Louis, Missouri, USA
Migration of Plasmodium sporozoites through cells before infection. Mota MM, Pradel G, Vanderberg JP, Hafalla JCR, Frevert U, Nussenzweig RS, Nussenzweig V, Rodriguez A: Science 2000, 291:141-144. • Significance: The observation that malaria sporozoites invade multiple host cells before establishing a replication-competent vacuole suggests that Plasmodium sporozoites actively select particular hepatocytes for parasitism. Findings: Plasmodium sporozoites added to hepatocytes in culture are able to migrate towards a potential host cell, invade it, and rapidly egress without a cycle of parasite replication. Entry into hepatocytes occurs by one of two processes: the breaching of the plasma membrane by parasites that subsequently reside freely in the cytosol, or the formation of a parasitophorous vacuole. Membrane breaching results in either plasma membrane repair or host cell death, whereas the welldescribed process of parasitophorous vacuole formation leaves the host cell momentarily intact. Finally, parasites may traverse through several hepatocytes, both in vitro and in vivo, prior to committing to a replicative pathway. A plastid segregation defect in the protozoan parasite Toxoplasma gondii. He CY, Shaw MK, Plethcer CH, Striepen B, Tilney LG, Roos DS: EMBO J 2001, 20:330-339. • Significance: The isolation of plastid-deficient and ‘super apicoplast’-containing parasites provides a new reagent to study the function and biochemistry of the apicoplast, which is needed for intracellular growth. Findings: Transient transfection of Toxoplama parasites with a chimeric recombinant protein that consisted of a nuclearencoded apicoplast protein (ACP) fused to the green fluorescent protein (GFP) and a portion of the rhoptry protein ROP1 (ACPGFP-mROP1) specifically disrupted apicoplast segregation during parasite division. Instead of dividing normally with one apicoplast per parasite, one parasite per vacuole contained an enlarged but functional apicoplast, whereas the remaining parasites lacked this organelle. Apicoplast-deficient parasites replicated normally in their first cycle of infection and were capable of reinvasion, but subsequent replication was prevented and host cell lysis did not occur. This data indicate that the apicoplast fulfils an essential function that can be complemented in trans (within the same vacuole). Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Salmon BL, Oksman A, Goldberg DE: Proc Natl Acad Sci USA 2001, 98:271-276. • Significance: The characterization of the requirement of proteolysis for normal exit from the host erythrocyte provides a convenient new method for the purification of merozoites and insight into a potential target for chemotherapeutic intervention. Findings: The authors treated synchronized cultures of Plasmodium falciparum middle-stage schizonts with E64 (an irreversible inhibitor of most cysteine proteases) and found that it leads to accumulation of merozoites clustered within thin membranes. Immunofluoresence and electron microscopy demonstrate that the merozoites are contained within membranes derived from the parasitophorous vacuole. The merozoite clusters can be purified by washing to remove residual E64, and they establish a highly synchronous wave of infection.
Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D: J Cell Biol 2001, 152:563-578. • Significance: This paper shows that the secretory process of adhesins implicated in host cell invasion and gliding motility of T. gondii is tightly regulated and sorting of these molecules involves complex formation of soluble and insoluble adhesins. Findings: The expression of the microneme proteins MIC1, MIC4 and MIC6 of Toxoplasma gondii was abrogated by gene disruption. Analysis of the mutants by immunofluorescence showed that the sorting of the two soluble proteins, MIC1 and MIC4, is dependent on the presence of the transmembrane protein MIC6. Immunoprecipitation experiments suggested the existence of a complex between the three proteins. This model potentially explains how soluble secretory adhesins can be sorted and accurately escorted to secretory organelles.
Genomics Selected by Bernard Labedan and Patrick Forterre Université Paris-Sud, Orsay, France
Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Perna NT, Plunkett G, Burland V, Mau B, Glasner JD, Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA et al.: Nature 2001, 409:529-533. • Significance: This paper emphasizes the power of comparing genomes from closely related strains to understand the biology of pathogenicity islands and the mechanism of lateral transfer. Findings: The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. The genome of E. coli O157:H7 has been sequenced by the whole genome shotgun approach. This and comparison with the genome of the nonpathogenic laboratory strain E. coli K-12 allowed the identification of candidate genes responsible for pathogenesis, the development of better methods of strain detection, and the advancement of our understanding of the evolution of E. coli. Lateral gene transfer has been found to be far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions, all of which could be targets for surveillance. The protein–protein interaction map of Helicobacter pylori. Rain JC, Selig L, Reuse HD, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, Schächter V et al.: Nature 2001, 409:211-215. • Significance: A proteome-wide approach has been designed to identify protein domain interactions, allowing a better understanding of protein function. Findings: A large-scale protein–protein interaction map of the human gastric pathogen Helicobacter pylori has been built, using a high-throughput strategy of the yeast two-hybrid assay. Two hundred and sixty-one H. pylori proteins have been screened against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, representing 46.6% of the proteome. The determination of a reliability score for every single protein– protein interaction and the identification of the actual interacting domains permitted the assignment of nonannotated proteins to biological pathways. As elementary constituents of
cellular protein complexes and pathways, protein–protein interactions are key determinants of protein function. The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Gomis-Rüth FX, Moncalián G, Pérez-Luque R, González A, Cabezón E, Cruz FDL, Coll M: Nature 2001, 409:637-641. •• Significance: This paper describes important findings about the molecular mechanism of bacterial conjugation, the main route for horizontal transfer and rapid acquisition of antibiotic resistance by pathogens. Findings: The transfer of DNA across membranes and between cells is a central biological process. However, its molecular mechanism remains unknown. Transkingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation, are special cases of conjugation. The 3-D structure of a soluble variant of TrwB, an integral membrane DNA-binding protein, has been determined. This large multimeric protein is responsible for recruiting the relaxosome-DNA–protein complex, and participates in the transfer of a single DNA strand during Escherichia coli R388 cell mating. The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel 20 Å in width traverses the hexamer. In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data. Edwards JS, Ibarra RU, Palsson BO: Nat Biotechnol 2001, 19:125-130. • Significance: The combination of in silico and experimental biology can be used to obtain a quantitative genotype–phenotype relationship for metabolism in bacterial cells. Findings: A significant goal in the post-genome era is to relate the annotated genome sequence to the physiological functions of a cell. Working from the annotated genome sequence, as well as biochemical and physiological information, it is possible to reconstruct complete metabolic networks. Furthermore, computational methods have been developed to interpret and predict the optimal performance of a metabolic network under a range of growth conditions. The hypothesis that Escherichia coli uses its metabolism to grow at a maximal rate has been tested in experiments designed to estimate the quantitative relationship between the rate of uptake of a primary carbon source (acetate or succinate), oxygen uptake rate, and maximal cellular growth rate. The experimental data obtained suggest that the E. coli MG1655 metabolic network is optimized to maximize growth under the experimental conditions considered. Comparative genomics of the restriction-modification systems in Helicobacter pylori. Lin LF, Posfai J, Roberts RJ, Kong H: Proc Natl Acad Sci USA 2001: in press and available at www.pnas.org (manuscript 10.1073/pnas.051612298). • Significance: Helicobacter pylori has extremely diversified restriction-modification (R-M) systems in different strains : the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones. Findings: More than 20 putative DNA R-M systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. Less than 30% of the Type II
R-M systems in 26695 are fully functional, and these findings are similar to those obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. A genomic approach to gene fusion technology. Van Dyk TK, Wei Y, Hanafey MK, Dolan M, Reeve MJG, Rafalski JA, Rothman-Denes LB, LaRossa RA: Proc Natl Acad Sci USA 2001, in press and available at www.pnas.org (manuscript 10.1073/pnas.041620498. • Significance: Partnering gene fusion and DNA microarray technology produces a powerful tool to discover promoters and define operons through analyses of transcriptional responses to cellular perturbation. Findings: A genome-wide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions is described. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of various stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. This genome-wide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin-C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these upregulated genes were used to confirm or contradict the DNA microarray results. Genetic architecture of thermal adaptation in Escherichia coli. Riehle MM, Bennett AF, Long AD: Proc Natl Acad Sci USA 2001, 98:525-530. •• Significance: Elucidation of the genetic basis of adaptation on a genome-wide scale has evaded biologists, but complete genome sequences and DNA high-density array technology make genome-wide surveys more tractable. Findings: Six strains of Escherichia coli adapted for 2,000 generations to a stressful high temperature of 41.5°C were examined on a genome-wide scale for duplication or deletion events by using DNA high-density arrays. A total of five duplication and deletion events were detected in three of the six strains. Three of the duplications were at 2.85 Mb of the E. coli chromosome, a region in which four candidate genes previously shown to play roles in stress and starvation survival were identified. Expression of the candidate genes examined was elevated compared to expression levels in the ancestral lines or the lines without duplication. In the two cases in which duplication at 2.85 Mb had been further characterized, the timing of the genome reorganization was coincident with significant increases in relative fitness. In both of these cases, the model for the origin of the duplication is a complex recombination event involving insertion sequences and repeat sequences. These results provide additional evidence for the idea that gene duplication plays an integral role in adaptation, specifically as a means for gene amplification.
Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: a laboratory model of molecular evolution. Yano T, Kagamiyama H: Proc Natl Acad Sci USA 2001, 98:903-907. •• Significance: An experimental model has been devised to simulate molecular evolution in the laboratory. It is based on the screening of natural expression libraries subjected to DNA shuffling. Findings: To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA. DNA shuffling was used to enhance the activity of this Pyrococcus furiosus clone by 50 rounds of directed evolution. Analysis of the evolved DNA fragments shows that two genetic regions have co-evolved: one region, which has no obvious open reading frame, is essential for the activity, whereas the other, which appears to encode a protein, is not essential but enhances the activity of the first region. Analysis of the evolutionary intermediates shows that negative mutations are effectively removed, whereas beneficial mutations accumulate, and illustrates how a protein has evolved over the course of the evolution experiments. Although the mechanism of the activity remains unclear, the evolved DNA fragments also confer resistance to other drugs that inhibit bacterial cell wall synthesis.
Growth and development Selected by Scott Gold, Mariola Garcia-Pedrajas, Alfredo D Martínez-Espinoza, John Duick Egan and Sricharan Bandhakavi University of Georgia, Athens, Georgia, USA
Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon. Hoffmann B, Eckert SE, Krappmann S, Braus GH: Genetics 2001, 157:141-147. • Significance: The homothalic sexual stage of Aspergillus nidulans (Emericella) has a mechanism with which it encourages outcrossing by preferentially forming premeiotic zygotes between genetically unlike nuclei. Findings: By crossing strains with increasingly divergent genetic background, the phenomenon known as relative heterothallism was confirmed in A. nidulans. In crosses between strains with very unlike nuclei, 100% of the ascospores were produced from heterozygous diploid nuclei. In crosses involving nuclei of intermediate dissimilarity, 85% of zygotes were heterozygous. In crosses between nearly isogenic nuclei, zygote formation was random. This indicates that the fungus has a mechanism with which it senses and encourage the fusion of genetically distinct nuclei over the fusion of like nuclei. Additionally, contrary to the assumption that all ascospores within a cleistothecium originate from a single prezygotic dikaryotic cell, both heterozygous and homozygous premeiotic zygotes could be produced in a single cleistothecium. The clp1 gene of the mushroom Coprinus cinereus is essential for A-regulated sexual development. Inada K, Morimoto Y, Arima T, Murata Y, Kamada T: Genetics 2001, 157:133-140. • Significance: The clampless 1 gene (clp1) located downstream of the homeodomain protein encoding A mating-type locus is critical for the sexual development program in mushrooms. Findings: The clp1 gene of Coprinus cinereus is required for clamp connection development, a critical feature in the normal sexual cycle of higher basidiomycetes. Constitutive transcription
of clp1 bypasses the requirement for heterozygosity at the A mating-type locus for clamp development. The putative Clp1 protein sequence provides no clues as to function. clp1 may be a direct target of the heterodimeric homeodomain transcriptional regulator encoded by the A genes. Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae. Mosch H-U, Köhler T, Braus GH: Mol Cell Biol 2001, 21:235-248. • Significance: The roles in morphogenesis of the essential yeast gene CDC42 were characterized more fully by production of single missense mutations, helping to implicate binding regions for specific effectors. Findings: Using plasmid shuffling, cdc42 single missense mutations were identified that reduced or increased diploid pseudohyphal growth, or reduced haploid invasive growth. Several of the mutants had little or no effect on growth rate or yeast morphogenesis, but had dramatic effects on filamentation. Mutations that affected invasive growth also affected pseudohyphal growth and vice versa. Based on the three dimensional model of Cdc42p, mutant analysis identified specific regions for binding of specific effector proteins and consequent morphogenetic function. Schizosaccharomyces pombe Rho2p GTPase regulates cell wall α-glucan biosynthesis through the protein kinase Pck2p. Calonge TM, Nakano K, Arellano M, Arai R, Katayama S, Toda T, Mabuchi I, Perez P: Mol Biol Cell 2000, 11:4393-4401. •• Significance: In the fission yeast, Schizosaccharomyces pombe, the GTPases Rho1p and Rho2p control morphogenesis, cell integrity and polarization of the actin cytoskeleton. Both GTPases interact with Pck1p and Pck2p, the two protein kinase C homologs of S. pombe. It is known that Rho1p regulates (1,3)β-D-glucan both directly and through Pck2p. In this paper, the authors report the presence of a second signaling pathway regulating the synthesis of α-D-glucan, the other major component of S. pombe cell wall. With intelligent use of their own results in this work and the information already available, they produce strong evidence that this pathway is controlled by Rho2p, which activates Pck2p, and that Pck2p in turn controls Mok1p, the main (1-3)α-D-glucan synthase. Findings: It is known that both GTPases Rho2p and Rho1p are involved in cell polarity and morphogenesis, but their functions appear to differ. Strains overproducing Rho2p have been shown to have an increase in α-glucan in their cell wall. This increase is not observed in Rho1p-overproducing strains, which suggest a major role for Rho2p as a positive regulator of α-glucan biosynthesis. Strains lacking Pck2p activity have similar cell wall defects as those lacking Rho2p activity. The same effect is not observed in strains lacking Pck1p. This suggests that rho2 and pck2 are in the same signaling pathway. Strong evidence that Rho2p signaling requires Pck2p is that the phenotype of strains overexpressing rho2, shown as lethality, round morphology, actin depolarization and an increase in α-glucan, is not observed in a pck2∆ background. The authors conclude that Rho1p regulates (1-3)β-D-glucan biosynthesis directly and through Pck2p, whereas Rho2p regulates α-D-glucan exclusively through Pck2p. And although it is known that Pck1p physically interacts with both GTPases, its role in maintaining cell integrity is still unknown.
The unfolded protein response represses nitrogen-starvation induced developmental differentiation in yeast. Schroder M, Chang JS, Kaufman RJ: Genes Dev 2000, 14:2962-2975. •• Significance: The importance of this paper is to connect aspects of the secretory pathways with the regulation of more cellular processes than have been expected. The authors demonstrate a novel function for the unfolded protein response (UPR) and the secretory pathway in controlling the global physiology of eukaryotic cells in response to nutrient availability. Findings: Nitrogen starvation in the presence of a fermentable carbon source induces morphological change from yeast to a filamentous or pseudohyphal growth form. In the presence of nonfermentable carbon sources nitrogen starvation induced meiosis and sporulation. It is not understood how the decision to undergo one or the other developmental stage is made at the molecular level. The UPR is a signal transduction pathway that transduces the stress signal for unfolded proteins in the endoplasmic reticulum (ER) to the nucleus through the action of the type I transmembrane kinase/endoribonuclease Ire1p and the mRNA transcription factor Hac1p. It was observed that in a nitrogen-rich media and independently of the carbon source, diploid ire1∆/ire1∆ and hac1∆/hac1∆ strains grew as pseudohyphae. The authors have proposed the following model: in a nitrogen-rich environment, protein unfolding is a byproduct of rapid translation, which results in activation of the UPR and subsequent repression of filamentous growth and meiosis. When nitrogen becomes limiting, the amount of unfolded protein in the ER drops to a level insufficient to activate the UPR, and repression of pseudohyphal growth and meiosis is relieved. Glucose depletion causes haploid invasive growth in yeast. Cullen PJ, Sprague GF: Proc Natl Acad Sci 2000, 97:13619-13624. • Significance: In Saccharomyces cerevisiae diploids, reduced environmental fixed nitrogen causes the transition (termed pseudohyphal growth) from round cells with a bipolar budding pattern to elongated cell forms with unipolar budding. In haploid cells, nutrient limitation causes a similar developmental switch that enables penetration of the agar called invasive growth. The authors developed a useful single-cell invasivegrowth assay and conclusively showed that the invasive growth was due to the loss of fermentable sugars such as glucose. Findings: YPD medium without glucose promoted constitutive invasive growth of haploid cells. Using a single-cell invasivegrowth assay, it was shown that individual cells adopted a nonaxial budding pattern and elongated form within the first few cell divisions, and invasive growth was observed in microcolonies with as few as 10 cells. In addition, the filamentous morphology of hyperinvasive mutants, STE11-4, pbs2, hsl7 and RAS2V19, was suppressed by glucose. Glucose-controlled proteins are important for proper invasiveness. Snf1, a protein required for derepression of glucose-repressed genes, is necessary for invasive growth. Sip4, a transcription factor that interacts with Snf1 and is induced during the diauxic shift from fermentation to respiration, had an inhibitory role on invasive growth, suggesting multiple mechanisms for glucose-depletion- dependent invasion. The inducible N-acetylglucosamine catabolic pathway gene cluster in Candida albicans: discrete N-acetylglucosamine-inducible factors interact at the promoter of NAG1. Kumar MJ, Jamaluddin MS, Natarajan K, Kaur D, Datta A: Proc Acad Natl Sci USA 2000, 97:14218-14223.
• Significance: The catabolic pathway of N-acetylglucosamine (GlcNAc) in Candida albicans is important for its pathogenicity and morphogenesis. This paper conclusively shows the presence of a gene cluster for the GlcNAc-catabolic pathway and demonstrates a coordinated regulation through a bidirectional promoter. Findings: Adjacent to NAG-1 (glucosamine-6-phosphate deaminase), two additional genes were found: one encoding the GlcNAc-6-phosphate deacetylase (DAC1), and one encoding a hexokinase (HXK1). NAG1 and DAC1 are expressed in opposite orientations and regulated by a single promoter. Promoter analysis showed important regions for GlcNAc induction located at –200 and –400bp upstream of the NAG1 ATG start codon. Gel-mobility-shift assays and DNA footprinting revealed two regions that are bound by at least two inducible activator proteins directing the regulation of gene expression. DNA replication-independent silencing in S. cerevisiae. Kirchmaier AL, Rine J: Science 2001, 291:646-650. AND
Establishment of transcriptional silencing in the absence of DNA replication. Li YC, Cheng TH, Gartenberg MR: Science 2001, 291:650-653. •• Significance: Previous evidence supported the hypothesis that the passing of the DNA replication fork was the cell cycle event required for the establishment of gene silencing in yeast. Through the creation of nonreplicating HMR loci with chimeric silencer binding sites, gene silencing still occurs in the absence of replication. Findings: The authors took similar approaches to show that gene silencing occurs in the absence of DNA replication. Modifications were made to the homeodomain regulatory protein (HMR) locus by removing the origin recognition complex site and substituting single binding sites for Rap1p and Abf1p and multiple binding sites for either Gal4p (Kirchmaier and Rine) or LexAp (Li et al.). By flanking the HMR locus with recombinase target sites, the locus could be excised from the chromosome and not replicated. A chimeric Gal4–Sir1p (Kirchmaier and Rine) or LexA–Sir1p (Li et al.) was used to tether the silencing machinery (Sir4p) to the HMR locus. The HMR locus was silenced efficiently, regardless of whether the locus was in the chromosome or excised as a covalently-closed, nonreplicated circular DNA molecule. Multigenerational cortical inheritance of the Rax2 protein in orienting polarity and division in yeast. Chen T, Hiroko T, Chaudhuri A, Inose F, Lord M, Tanaka S, Chant J, Fujita A: Science 2000, 290:1975-1978. • Significance: Rax2–GFP can be used as an inheritable cortical protein marker to couple a cell’s history to its future morphogenetic development. Findings: Rax2p is required for the maintenance of diploid bipolar budding patterns in yeast cells. Rax2–GFP is a late cellcycle protein that is produced just before cytokinesis. Through a series of experiments, a Rax2–GFP ring was shown to split and be inherited from the mother to the daughter cell over several generations. The Rax2 protein was shown to be very stable, with a half-life of six hours, and could remain detectable for up to 24 hours after repression of its expression. Rax2 rings contribute to the maintenance of the bipolar budding pattern, and these rings possibly remain unaltered on the surface of the cell throughout its lifetime.
A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. Jensen S, Segal M, Clarke DJ, Reed SI: J Cell Biol 2001, 152:27-40. •• Significance: The mechanism by which chromatid separation during anaphase A (which follows the degradation of the cohesin protein, Scc1, by Esp1 at the metaphase–anaphase transition) is coupled with movement of the anaphase spindle (anaphase B) is not well understood. This work fills a critical void in this area. Findings: Yeast cells lacking both Scc1 and Esp1 function do not undergo elongation of the spindle, indicating that the lengthening of the anaphase spindle (anaphase B) does not result directly from the release of cohesive forces that hold the sister chromatids together (Scc1), but also requires Esp1 function after the degradation of Scc1. Additionally, Esp1 localizes to the spindle apparatus during anaphase in a Pds1-dependent fashion. Role of Hsp90 in salt stress tolerance via stabilization and regulation of calcineurin. Imai J, Yahara I: Mol Cell Biol 2000, 20:9262-9270. • Significance: Hsp90 is a major eukaryotic chaperone that is involved in folding and stabilizing the activity of several protein kinases and steroid receptors. Now, a new role for Hsp90 in maintaining the activity of calcineurin, a calcium-calmodulin-activated Ser/Thr protein phosphatase, is reported. Findings: Overexpression of Hsc82, one of the yeast homologues of Hsp90, made yeast cells hypersensitive to salt stress (a phenotype also seen in calcineurin-defective mutants). On further investigation, the authors found that Hsc82 physically interacted with and stabilized the catalytic subunit of calcineurin, Cna2, and that its release from Hsc82 was required for its activation. Selected by Jeff Errington and Andrea Feucht University of Oxford, Oxford, UK
Global analysis of the genetic network controlling a bacterial cell cycle. Laub MT, McAdams HH, Feldblyum T, Fraser CM, Shapiro L: Science 2001, 290:2144-2148. •• Significance: This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Findings: DNA microarrays, containing about 90% of all Caulobacter crescentus genes, were constructed and variations in mRNA levels of synchronized swarmer cells were examined during the cell cycle. This analysis identified 553 genes (19% of the genome) whose mRNA levels varied as a function of the cell cycle. A single regulatory factor, CtrA, a member of the twocomponent signal transduction family, is directly or indirectly involved in the control of 26% of these cell-cycle-regulated genes. The authors conclude that in bacteria, as in yeast, the following occurs: genes involved in a given function are activated at the time of execution of that function; genes encoding proteins that form multiprotein complexes are co-expressed; and multiprotein structure biogenesis is controlled by temporal cascades of gene expression. Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Burkholder WF, Kurtser I, Grossman AD: Cell 2001, 104:269-279. •• Significance: The authors identified a signaling pathway that prevents initiation of sporulation in Bacillus subtilis when replication initiation is impaired.
Findings: Mutations allowing a replication initiation mutant (dnaA1) to sporulate were isolated and found to lie in a small open reading frame, sda. Its product, Sda was overexpressed in dnaA1 and other replication initiation mutants. Genetic and biochemical experiments showed that Sda inhibits sporulation by preventing activation of the transcription factor SpoOA. Thus, Sda couples activation of SpoOA and initiation of sporulation to the function of replication initiation proteins. Movement of replicating DNA through a stationary replisome. Lemon KP, Grossman AD: Mol Cell 2000, 6:1321-1330. • Significance: Results presented here indicate that during replication in Bacillus subtilis, DNA moves through a stationary replisome located at or near midcell. Findings: The location of the left stringent terminus region (LSTer) of the chromosome was visualised in living cells, in which replication was blocked by induction of the stringent response. The LSTer was found to be coincident with DNA polymerase at or near midcell. After release from replication arrest, the region was duplicated and the two copies moved to opposite halves of the cell. Localisation of a second region of the chromosome (the 270° region – located midway between origin and terminus) showed that this region moved to the DNA polymerase just prior to duplication. The data suggest that the polymerase at the replication forks is stationary and the DNA is threaded through the polymerase during duplication and then extruded from the polymerase after duplication. The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle. Fu X, Shih YL, Zhang Y, Rothfield RI: Proc Natl Acad Sci USA 2001, 98:980-985. • Significance: This paper has important implications for how the correct midcell site for cell division in E. coli is selected by the MinE protein. Findings: Previously it has been shown that MinE–GFP fusion protein assembles into a ring-like structure near midcell. Time-lapse fluorescence microscopy of MinE–GFP now shows that the MinE ring is not fixed in position but is a
dynamic structure that undergoes a repetitive cycle of movement to the cell pole, followed by dissolution, reformation at a new midcell site, and movement to the opposite pole. The results suggest that the ability of MinE to move the MinCD division inhibitor away from midcell and to the cell poles may impart topological specifity to MinCD. Identification and characterisation of the dif site from Bacillus subtilis. Sciochetti SA, Piggot JP, Blakely GW: J Bacteriol 2001, 183:1058-1068. • Significance: The dif site of Bacillus subtilis was identified and shown to be involved in the resolution of chromosome dimers generated by homologous recombination. Findings: The authors present genetic and biochemical evidence that a 28bp sequence of DNA (dif), located in the B. subtilis replication terminus region, is the site at which the recombinases RipX and CodV catalyse recombination reactions required for normal chromosome partitioning. Suprisingly, the resolution of chromosome dimers did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. Additionally, they show that the SPβ bacteriophage-encoded gene yopP affects chromosome dimer resolution in vivo. FtsK functions in the processing of a Holliday junction intermediate during bacterial chromosome segregation. Barre FX, Aroyo M, Colloms SD, Helfrich A, Cornet F, Sherratt DJ: Genes Dev 2000, 14:2976-2988. • Significance: Chromosome dimer resolution requires the activity of FtsK, a septum-located protein which coordinates cell division and chromosome segregation in Escherichia coli. Here it is shown to activate, through its carboxyl terminus, the XerD recombinase when a chromosome dimer is present during bacterial chromosome segregation. Findings: The soluble, carboxy-terminal domain of FtsK is sufficient to activate an Xer recombination reaction. FtsKc controls Xer recombination at the XerD-mediated strand exchange step, allowing both intra- and intermolecular recombination reactions to go to completion. Furthermore, proper positioning of dif in the chromosome and of FtsK at the septum appear to be required to restrict Xer reactions to dimeric chromosomes.