bluuebunny/crossref_metadata_2025 huggingface dataset:
On the correlation between heterozygosity and fitness in natural populations
Authors: Hansson Bengt , Westerberg Lars
Published: March 2003

Three primary hypotheses currently prevail for correlations between heterozygosity at a set of molecular markers and fitness in natural populations. First, multilocus heterozygosity–fitness correlations might result from selection acting directly on the scored loci, such as at particular allozyme loci. Second, significant levels of linkage disequilibrium, as in recently bottlenecked‐and‐expanded populations, might cause associations between the markers and fitness loci in the local chromosomal vicinity. Third, in partially inbred populations, heterozygosity at the markers might reflect variation in the inbreeding coefficient and might associate with fitness as a result of effects of homozygosity at genome‐wide distributed loci. Despite years of research, the relative importance of these hypotheses remains unclear. The screening of heterozygosity at polymorphic DNA markers offers an opportunity to resolve this issue, and relevant empirical studies have now emerged. We provide an account of the recent progress on the subject, and give suggestions on how to distinguish between the three hypotheses in future studies.
Extremely low levels of genetic polymorphism in endosymbionts (Buchnera) of aphids (Pemphigus)
Authors: Abbot P. , Moran N. A.
Published: March 2003

Molecular evolutionary studies have suggested that vertically transmitted endosymbionts are subject to accumulation of deleterious mutations through genetic drift. Predictions of this hypothesis for patterns of intraspecific polymorphism were borne out in the single relevant study available, on the symbiont Buchnera aphidicola of Uroleucon ambrosiae. In order to examine the generality of this result, we surveyed DNA sequence variation in Buchnera of the distantly related aphid, Pemphigus obesinymphae. In contrast to Uroleucon species, Pemphigus species have complex life cycles with few dispersal stages. Despite these differences, P. obesinymphae showed patterns of variation at two Buchnera loci and one mitochondrial locus that were remarkably similar to those reported previously for Buchnera of U. ambrosiae. In the western US, Buchnera was nearly monomorphic, and in the eastern US, synonymous divergence ranged from 0.08 to 0.16%. Most polymorphisms involved rare alleles, consistent with a recent range of ancestral polymorphism, probably due to demographic fluctuations in aphid populations. These results support the generality of small effective population size in Buchnera and their aphid hosts.
Several Pleistocene refugia detected in the high alpine plant Phyteuma globulariifolium Sternb. & Hoppe (Campanulaceae) in the European Alps
Authors: Schönswetter P. , Tribsch A. , Barfuss M. , Niklfeld H.
Published: March 2003

Phyteuma globulariifolium is a high alpine plant species growing in the European Alps and the Pyrenees. In order to elucidate its glacial history, 325 individuals from 69 populations were analysed using the amplified fragment length polymorphism (AFLP) technique. A strongly hierarchical phylogeographical pattern was detected: Two major east–west vicariant groups can be separated along a gap in the distributional area. A further subdivision into at least four populational groups is in congruence with presumed peripheral glacial refugia. There is no indication for survival on unglaciated mountain tops (nunataks) in the interior of the Pleistocene ice shield covering the Alps. Our results favour glacial survival in peripheral, unglaciated or not fully glaciated areas. Populations of P. globulariifolium in the Pyrenees are the result of relatively recent long‐distance dispersal. Within the Alps, there is strong differentiation among groups of populations, whereas within them the differentiation is weak. This suggests high levels of gene‐flow over short to middle distances.
Palaeoclimatic changes explain Anatolian mountain frog evolution: a test for alternating vicariance and dispersal events
Authors: Veith M. , Schmidtler J. F. , Kosuch J. , Baran I. , Seitz A.
Published: March 2003

Holarctic biodiversity has been influenced by climatic fluctuations since the Pliocene. Asia Minor was one of the major corridors for postglacial invasions in the Palearctic. Today this area is characterized by an extraordinarily rich fauna with close affiliation to European, Asian and Indo‐African biota. However, exact scenarios of range expansion and contraction are lacking. Using a phylogeographical approach we (i) identify monophyletic lineages among Anatolian mountain frogs and (ii) derive a spatio‐temporal hypothesis for the invasion process in Anatolia. We sequenced 540 bp of the mitochondrial 16S rRNA gene from 40 populations of mountain frogs from Anatolia, the Elburz Mountains and the Caucasus. Our samples comprise all known species and subspecies: Rana macrocnemis macrocnemis, R. m. tavasensis, R. m. pseudodalmatina, R. camerani and R. holtzi. They include the type localities of four of these taxa. We used a nested clade analysis (NCA) to infer historical and recurrent events that account for the observed geographical distribution of haplotypes. None of the extant species is monophyletic. Based on a molecular clock calibration using homologous sequences of Western Palearctic water frogs of the same genus, we estimated that a basic radiation into three lineages c. 2 Mya was followed by several dispersal and fragmentation events. The geographical distribution of resident and widespread haplotypes allows us to infer and date scenarios of range expansion and fragmentation that are aligned with dramatic climatic oscillations that have occurred during the last 600 000 years. Consequently, Pliocene and Pleistocene climatic oscillations triggered the evolution of Anatolian mountain frogs through an interplay of vicariance and dispersal events.
Polymorphism of a complex resistance gene candidate family in wild populations of common bean (Phaseolus vulgaris) in Argentina: comparison with phenotypic resistance polymorphism
Authors: De Meaux J. , Cattan‐Toupance I. , Lavigne C. , Langin T. , Neema C.
Published: March 2003

Fifteen populations of wild bean (Phaseolus vulgaris), located in three provinces in Argentina, were analysed for their polymorphism for a complex resistance gene candidate (RGC) family clustered on the genome and for resistance phenotypes to strains of Colletotrichum lindemuthianum. Results indicate that RGC polymorphism is high. Population structure obtained for markers related to resistance was compared to population structure obtained for RAPD markers in order to infer the evolutionary forces driving polymorphism for resistance in wild populations at both molecular and phenotypic levels. Hierarchical analysis of differentiation showed that, within provinces, populations were differentiated for RAPD as well as for molecular and phenotypic markers of resistance. In contrast, provinces were differentiated only for RAPD and RGC markers and not for resistance phenotypes. The discrepancies found between diversity structures for molecular markers (RAPD and RGCs) and for resistance phenotypes suggest an effect of selection and indicate that diversity for resistance may not be driven by the same selective forces at the molecular and phenotypic levels. We further discuss whether specific selective forces are exerted on RGC markers and underline the importance of spatial scale of analysis for demonstrating an impact of selection.
Postglacial recolonization and cpDNA variation of silver birch, Betula pendula
Authors: Palmé A. E. , Su Q. , Rautenberg A. , Manni F. , Lascoux M.
Published: March 2003

Chloroplast PCR‐RFLP markers were used to reconstruct the history of the silver birch, Betula pendula Roth, in Europe since the last glacial maximum (LGM). In birch, fossil pollen maps do not reveal a clear chronological sequence of postglacial spread. If anything, the pollen record suggests that most of Europe was recolonized by birches as early as 10 000 bp, probably from populations that remained close to the ice sheets during the LGM. The geographical distribution of haplotypes supports a scenario of early colonization. Two of the 13 haplotypes that were observed are common, representing 35% and 49% of the total sample, respectively. Although one of the common haplotypes is predominant in the NW and the other in the SE, both are present throughout most of the investigated geographical area. Rare haplotypes are geographically restricted. The distribution of the haplotypes reveals five genetic boundaries between groups of haplotypes and allows us to infer patterns of postglacial recolonization. Europe was re‐occupied by two main waves of recolonization: one eastern and one western, with origins at intermediate latitudes. Populations in the Iberian Peninsula and in Italy did not take part in the postglacial recolonization of Europe.
A rapid population expansion retains genetic diversity within European rabbits in Australia
Authors: Zenger K. R. , Richardson B. J. , Vachot‐Griffin A‐M.
Published: March 2003

The well documented historical translocations of the European rabbit (Oryctolagus cuniculus) offer an excellent framework to test the genetic effects of reductions in effective population size. It has been proposed that rabbits went through an initial bottleneck at the time of their establishment in Australia, as well as multiple founder events during the rabbit's colonization process. To test these hypotheses, genetic variation at seven microsatellite loci was measured in 252 wild rabbits from five populations across Australia. These populations were compared to each other and to data from Europe. No evidence of a genetic bottleneck was observed with the movement of 13 rabbits from Europe to Australia when compared to French data. Within Australia the distribution of genetic diversity did not reflect the suggested pattern of sequential founder effects. In fact, the current pattern of genetic variation in Australia is most likely a result of multiple factors including mutation, genetic drift and geographical differentiation. The absence of reduced genetic diversity is almost certainly a result of the rabbit's rapid population expansion at the time of establishment in Australia. These results highlight the importance of population growth following a demographic bottleneck, which largely determines the severity of genetic loss.
Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa
Authors: Bitter Wilbert , Koster Margot , Latijnhouwers Maita , De Cock Hans , Tommassen Jan
Published: March 2003

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P. aeruginosa, was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C‐terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N‐terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ. Electron microscopic evaluation of the XcpQ multimers revealed large, ring‐shaped structures with an apparent central cavity of 95 Å. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 Å. The size of this cavity could allow for the transport of intact type IV pili.
The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers
Authors: Cornelissen Cynthia Nau , Kelley Meera , Hobbs Marcia M. , Anderson James E. , Cannon Janne G. , Cohen Myron S. , Sparling P. Frederick
Published: March 2003

Iron, an essential nutrient for most microorganisms, is sequestered by the host to decrease the concentration of iron available to bacterial pathogens. Neisseria gonorrhoeae, the causative agent of gonorrhoea, can acquire iron by direct interaction with human iron‐binding proteins, including the serum glycoprotein, transferrin. Iron internalization from host transferrin requires the expression of a bacterial receptor, which specifically recognizes the human form of transferrin. Two gonococcal transferrin‐binding proteins have been implicated in transferrin receptor function, TbpA and TbpB. We constructed a gonococcal transferrin receptor mutant without the introduction of additional antibiotic resistance markers and tested its ability to cause experimental urethritis in human male volunteers. The transferrin receptor mutant was incapable of initiating urethritis, although the same inoculum size of the wild‐type parent strain, FA1090, causes urethritis in >90% of inoculated volunteers. To our knowledge, this is the first experimental demonstration that a bacterial iron acquisition system is an essential virulence factor for human infection.
Classification and genetic characterization of pattern‐forming Bacilli
Authors: Rudner Rivka , Martsinkevich Oksana , Leung Wendy , Jarvis Erich D.
Published: March 2003

One of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi‐solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis, we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis. In this report, we show that these strains are actually not B. subtilis, but belong to a different class of Bacilli, group I. We show further that commonly used laboratory strains of B. subtilis can co‐exist as mixed cultures with group I Bacilli, and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli.
The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme
Authors: Sauvonnet Nathalie , Pugsley Anthony P.
Published: March 2003

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild‐type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.
The Escherichia coli ATP‐binding cassette (ABC) proteins
Authors: Linton Kenneth J. , Higgins Christopher F.
Published: March 2003

The recent completion of the Escherichia coli genome sequence (Blattner et al., 1997) has permitted an analysis of the complement of genomically encoded ATP‐binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E. coli. These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy‐generating domains of multicomponent membrane‐bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic‐binding protein‐dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.
The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)
Authors: Goverde Roos L. J. , Huis in't Veld Jos H. J. , Kusters Johannes G. , Mooi Frits R.
Published: March 2003

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature‐dependent adaptations. To investigate Y. enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy‐defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp. Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp, including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y. enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6‐fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold‐inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.
Inducer exclusion by glucose 6‐phosphate in Escherichia coli
Authors: Hogema Boris M. , Arents Jos C. , Bader Rechien , Eijkemans Kevin , Inada Toshifumi , Aiba Hiroji , Postma Pieter W.
Published: March 2003

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIAGlc as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIAGlc leads to ‘inducer exclusion’: inhibition of transport of a number of non‐PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non‐PTS carbon source glucose 6‐phosphate can also cause inducer exclusion. Glucose 6‐phosphate was shown to cause inhibition of transport of lactose and the non‐metabolizable lactose analogue methyl‐β‐D‐thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIAGlc or were insensitive to inducer exclusion because enzyme IIAGlc could not bind to the lactose carrier. Furthermore, we showed that glucose 6‐phosphate caused dephosphorylation of enzyme IIAGlc. In a mutant insensitive to enzyme IIAGlc‐mediated inducer exclusion, catabolite repression by glucose 6‐phosphate in lactose‐induced cells was much weaker than that in the wild‐type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose‐induced cells. We discuss an expanded model of enzyme IIAGlc‐mediated catabolite repression which embodies repression by non‐ PTS carbon sources.
The yopJ locus is required for Yersinia‐mediated inhibition of NF‐κB activation and cytokine expression: YopJ contains a eukaryotic SH2‐like domain that is essential for its repressive activity
Authors: Schesser Kurt , Spiik Ann‐Kristin , Dukuzumuremyi Jean‐Marie , Neurath Markus F. , Pettersson Sven , Wolf‐Watz Hans
Published: March 2003

Upon exposure to bacteria, eukaryotic cells activate signalling pathways that result in the increased expression of several defence‐related genes. Here, we report that the yopJ locus of the enteropathogen Yersinia pseudotuberculosis encodes a protein that inhibits the activation of NF‐κB transcription factors by a mechanism(s), which prevents the phosphorylation and subsequent degradation of the inhibitor protein IκB. Consequently, eukaryotic cells infected with YopJ‐expressing Yersinia become impaired in NF‐κB‐dependent cytokine expression. In addition, the blockage of inducible cytokine production coincides with yopJ‐dependent induction of apoptosis. Interestingly, the YopJ protein contains a region that resembles a src homology domain 2 (SH2), and we show that a wild‐type version of this motif is required for YopJ activity in suppressing cytokine expression and inducing apoptosis. As SH2 domains are found in several eukaryotic signalling proteins, we propose that YopJ, which we show is delivered into the cytoplasm of infected cells, interacts directly with signalling proteins involved in inductive cytokine expression. The repressive activity of YopJ on the expression of inflammatory mediators may account for the lack of an inflammatory host response observed in experimental yersiniosis. YopJ‐like activity may also be a common feature of commensal bacteria that, like Yersinia, do not provoke a host inflammatory response.
The function of auxiliary operators
Authors: Müller‐Hill Benno
Published: March 2003

Gene regulation by control of transcription has been analysed in great detail both in prokaryotes and in eukaryotes. The frequency of transcription may be decreased by repressors or increased by activators. A repressor may work by decreasing the concentration of RNA polymerase at a promoter capable of forming an open complex. An activator may work by increasing the concentration of RNA polymerase at a promoter capable of forming an open complex. For this purpose, a strategy is used over and over again. It is called increase in local concentration. How Escherichia coli uses this strategy efficiently is discussed.
Natural competence for DNA transformation in Helicobacter pylori : identification and genetic characterization of the comB locus
Authors: Hofreuter Dirk , Odenbreit Stefan , Henke Gabriele , Haas Rainer
Published: March 2003

The Gram‐negative bacterial pathogen Helicobacter pylori, an important aetiological agent of gastroduodenal disease in humans, belongs to a group of bacterial species displaying competence for genetic transformation. Here, we describe the comB gene locus of H. pylori involved in DNA transformation competence. It consists of a cluster of four tandemly arranged genes with partially overlapping open reading frames, orf2, comB1, comB2 and comB3, constituting a single transcriptional unit. Orf2 encodes a 37‐amino‐acid peptide carrying a signal sequence, whereas comB1, comB2 and comB3 produce 29 kDa, 38 kDa and 42 kDa proteins, respectively, as demonstrated by immunoblotting with specific antisera. For Orf2 and ComB1, no homologous proteins were identified in the database. For ComB3, the best homologies were found with TraS/TraB from the Pseudomonas aeruginosa conjugative plasmid RP1 and TrbI of plasmid RP4, VirB10 from the Ti plasmid of Agrobacterium tumefaciens and PtlG, a protein involved in secretion of pertussis toxin of Bordetella pertussis. Defined transposon knock‐out mutants in individual comB genes resulted in transformation‐defective phenotypes ranging from a 90% reduction to a complete loss of the natural transformation efficiency. The comB2 and comB3 genes show homology to HP0528 and HP0527, respectively, located on the cagII pathogenicity island of H. pylori strain 26695.
Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes
Authors: Lagier Béatrice , Pelicic Vladimir , Lecossier Denise , Prod'hom Guy , Rauzier Jean , Guilhot Christophe , Gicquel Brigitte , Hance Allan J.
Published: March 2003

A luminescence‐based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte‐derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand‐mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacteium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.
A glycyl radical solution: oxygen‐dependent interconversion of pyruvate formate‐lyase
Authors: Sawers Gary , Watson Gregory
Published: March 2003

Pyruvate formate‐lyase (PFL) catalyses the non‐oxidative dissimilation of pyruvate to formate and acetyl‐CoA using a radical–chemical mechanism. The enzyme is enzymically interconverted between inactive and active forms, the active form contains an organic free radical located on a glycyl residue in the C‐terminal portion of the polypeptide chain. Introduction of the radical into PFL only occurs anaerobically, and the activating enzyme responsible is an iron–sulphur protein that uses S‐adenosyl methionine as cofactor and reduced flavodoxin as reductant. As the radical form of PFL is inactivated by molecular oxygen it is safeguarded during the transition to aerobiosis by conversion back to the radical‐free, oxygen‐stable form. This reaction is catalysed by the anaerobically induced multimeric enzyme alcohol dehydrogenase. The genes encoding PFL and its activating enzyme are adjacent on the chromosome but form discrete transcriptional units. This genetic organization is highly conserved in many, but not all, organisms that have PFL. Recent studies have shown that proteins exhibiting significant similarity to PFL and its activating enzyme are relatively widespread in facultative and obligate anaerobic eubacteria, as well as archaea. The physiological function of many of these PFL‐like enzymes remains to be established. It is becoming increasingly apparent that glycyl radical enzymes are more prevalent than previously surmised. They represent a class of enzymes with unusual biochemistry and probably predate the appearance of molecular oxygen.
The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein
Authors: Barker Lucia P. , Brooks Diane M. , Small P. L. C.
Published: March 2003

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow‐growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter‐capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence‐activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.
DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence?
Authors: Mizrahi Valerie , Andersen Susan J.
Published: March 2003

The genome sequence of Mycobacterium tuberculosis was analysed by searching for homologues of genes known to be involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes necessary to perform nucleotide excision repair (NER), base excision repair (BER), recombination, and SOS repair and mutagenesis were identified. In particular, all of the genes known to be directly involved in the repair of oxidative and alkylative damage are present in M. tuberculosis. In contrast, we failed to identify homologues of genes involved in mismatch repair. This finding has potentially significant implications with respect to genome stability, strain variability at repeat loci and the emergence of chromosomally encoded drug resistance mutations.
Molecular analysis of Methanobacterium phage ΨM2
Authors: Pfister Peter , Wasserfallen Alain , Stettler Rolf , Leisinger Thomas
Published: March 2003

The methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the double‐stranded DNA phage ΨM2. The complete phage genome sequence of 26 111 bp was established. Thirty‐one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site‐specific recombinases. Analysis of the N‐terminal amino acid sequences of three phage‐encoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ΨM2 DNA sequence with parts of the sequence of the wild‐type phage ΨM1 suggests that ΨM2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.
Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments
Authors: Coulter Silvija N. , Schwan William R. , Ng Eva Y. W. , Langhorne Michael H. , Ritchie Heather D. , Westbrock‐Wadman Shannon , Hufnagle Wendy O. , Folger Kim R. , Bayer Arnold S. , Stover C. Kendall
Published: March 2003

The Gram‐positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature‐tag transposon mutagenesis screening of a 1520‐member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.
A novel PrfA‐regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice
Authors: Engelbrecht Fredi , Domínguez‐Bernal Gustavo , Hess Jürgen , Dickneite Carmen , Greiffenberg Lars , Lampidis Robert , Raffelsbauer Diana , Daniels Justin J. D. , Kreft Jürgen , Kaufmann Stefan H. E. , Vázquez‐Boland José‐Antonio , Goebel Werner
Published: March 2003

Several large, cell wall‐associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i‐inlFE. The two proteins encoded by this locus, i‐InlE and i‐InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i‐inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i‐InlE protein (198 amino acids) is secreted in large amounts into the brain–heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA‐dependent gene transcription, i‐inlE mRNA and i‐InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i‐inlF and i‐inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences (‘PrfA‐boxes’) were identified in the promoter regions of i‐inlF and i‐inlE. Non‐polar i‐inlE and i‐inlF deletion mutants and an i‐inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 × 107 bacteria per mouse. These data suggest an important role for i‐InlE and i‐InlF in L. ivanovii virulence.
Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus
Authors: Skrzypek Elzbieta , Cowan Clarissa , Straley Susan C.
Published: March 2003

The YopM virulence protein of Yersinia pestis has been described as binding human α‐thrombin and inhibiting thrombin‐induced platelet aggregation in vitro. However, recent studies have shown that a YopM–CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle‐associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.
The type II O‐antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence
Authors: DeShazer David , Brett Paul J. , Woods Donald E.
Published: March 2003

Melioidosis, an infection caused by the Gram‐negative bacterial pathogen Burkholderia pseudomallei, is endemic in south‐east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north‐east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10–30% NHS. We developed a simple screen for the identification of serum‐sensitive mutants based on this novel phenotype. Approximately 1200 Tn5‐OT182 mutants were screened, and three serum‐sensitive mutants were identified. The type II O‐antigenic polysaccharide (O‐PS) moiety of lipopolysaccharide was not present in the serum‐sensitive mutants. A representative serum‐sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5‐OT182 integrations in the serum‐sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O‐PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O‐PS is essential for B. pseudomallei serum resistance and virulence.
On the mechanism of FtsH‐dependent degradation of the σ32 transcriptional regulator of Escherichia coli and the role of the DnaK chaperone machine
Authors: Blaszczak Adam , Georgopoulos Costa , Liberek Krzysztof
Published: March 2003

The Escherichia coliσ32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ32 mutants lacking 15–20 C‐terminal amino acids had substantially increased half‐lives in vivo or in vitro, compared with wild‐type σ32. A truncated version of σ32, σ32CΔ, was purified to homogeneity and shown to be resistant to FtsH‐dependent degradation in vitro, suggesting that FtsH initiates σ32 degradation from its extreme C‐terminal region. Purified σ32CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild‐type σ32. However, in contrast to wild‐type σ32, σ32CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ32 protein was completely non‐functional in vivo, even when overproduced. Furthermore, it is shown that wild‐type σ32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.
Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii
Authors: Carruthers Vern B. , Sibley L. David
Published: March 2003

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore‐stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin–myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus‐coupled secretion system responsible for releasing adhesins involved in cell attachment.
Non‐iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria
Authors: Stojiljkovic Igor , Kumar Veena , Srinivasan Nithya
Published: March 2003

A new group of potent antibacterial compounds, non‐iron metalloporphyrins (MPs), is described. MPs possess a strong antibacterial activity against Gram‐positive bacteria, Gram‐negative bacteria and mycobacteria. Anaerobically grown bacteria and microorganisms that do not respire and/or express haem uptake systems were resistant to MPs. Antibacterial activity of MPs was not affected by known antibiotic resistance mechanisms operating in bacteria. The most potent MP against Y. enterocolitica, methicillin‐resistant S. aureus and M. smegmatis was gallium protoporphyrin IX (Ga‐PPIX). When tested alone, Ga ions and metal‐free porphyrins had approximately 100‐fold higher minimum inhibitory concentration (MIC) values for these organisms. Ga‐PPIX was not degraded by MP‐sensitive bacteria, indicating that the whole molecule is responsible for antibacterial activity. MPs are antibacterial ‘Trojan horses’, as they exploit haem transport systems of Gram‐negative bacteria as portals of entry into the cell. Bacterial mutants in superoxide dismutases, catalases and stationary‐phase sigma factors were hypersensitive to Ga‐PPIX. The extreme sensitivity of sod mutants to MPs and the requirement for active respiration for MP activity suggests that these compounds stimulate the production of reactive oxygen radicals in bacteria. Ga‐PPIX was not toxic to primary human fibroblasts, several established cell lines and experimental animals at concentrations > 100‐fold higher than the MIC for sensitive bacteria.
MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission
Authors: Poddar Atasi , Roy Nilanjan , Sinha Pratima
Published: March 2003

The minichromosome maintenance genes, MCM21 and MCM22, have been cloned and are shown to code for the ORFs YDR318W and YJR135C respectively. Mutations in these genes caused a decrease in the stability of the minichromosome. This decrease in stability was associated with an increase in the copy number of the minichromosome in cells carrying it. Small circular dicentric plasmids were maintained relatively stably and structurally intact in the mutants compared with the wild‐type strain. In the latter, such plasmids were mitotically unstable and, upon recovery, showed frequent rearrangements of their DNA. A centromere offered less obstruction to transcription in mutant cells than in the wild type, showing that both these mutants had a more relaxed kinetochore assembly. The mutant strains showed elevated rates of chromosome loss but not those of recombination. Both the mutations caused the cells to display a higher sensitivity towards the anti‐mitotic drug benomyl. All these observations suggest that MCM21 and MCM22 are important for chromosome segregation with a potential role in kinetochore function. These genes are non‐essential, as their deletions from chromosomes did not cause loss of cell viability. However, exponentially growing mutant cells carrying the deletion of the MCM21 gene had a significant population of large‐budded cells with a single nucleus at the neck. Furthermore, the DNA content of these cells showed a shift towards 2N, suggesting a temporary pause of cells in G2 or in an early phase of mitosis. The mcm21 and mcm22 mutations do not show synthetic lethality or any further enhancement of growth defects, implying that they could be carrying out non‐overlapping functions in chromosome segregation.
Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology
Authors: Forest Katrina T. , Dunham Steven A. , Koomey Michael , Tainer John A.
Published: March 2003

Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three‐dimensional structure and chemistry of the constituent pilin subunit. X‐ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 Å resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site‐specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O‐linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.
Domain III of the Bacillus thuringiensis delta‐endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N
Authors: De Maagd Ruud A. , Bakker Petra L. , Masson Luke , Adang Michael J. , Sangadala Sreedhara , Stiekema Willem , Bosch Dirk
Published: March 2003

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta‐endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS–PAGE‐separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N‐acetylgalactosamine (GalNAc) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta‐endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.