bluuebunny/crossref_metadata_2025 huggingface dataset:
A genetic analysis of the functions of LuxN: a two‐component hybrid sensor kinase that regulates quorum sensing in Vibrio harveyi
Authors: Freeman Jeremy A. , Lilley Brendan N. , Bassler Bonnie L.
Published: March 2003

The bioluminescent marine bacterium Vibrio harveyi controls light production using two parallel quorum‐sensing systems. V. harveyi produces two autoinducers (AI‐1 and AI‐2), which are recognized by cognate membrane‐bound two‐component hybrid sensor kinases called LuxN and LuxQ respectively. Under conditions of low cell density, in the absence of autoinducer, the hybrid sensors are kinases, and under conditions of high cell density, in the presence of autoinducer, the sensors are phosphatases. These activities allow LuxN and LuxQ to modulate the level of phosphorylation of the response regulator protein LuxO. LuxO, in turn, controls the transcription of the genes encoding luciferase. The phosphorelay protein LuxU is required for signalling to LuxO. In this report, we present a genetic analysis of the activities of the AI‐1 sensor LuxN. Point mutations and in frame deletions were constructed in luxN and recombined onto the chromosome of V. harveyi for in vivo phenotypic analysis. We show that the conserved histidine (H471) in the sensor kinase domain of LuxN is required for kinase activity but not for phosphatase activity. In contrast, the conserved aspartate (D771) in the response regulator domain of LuxN is required for both activities. Furthermore, the LuxN phosphatase activity is localized to the response regulator domain. Our results indicate that the LuxN kinase activity is regulated by the presence of AI‐1, whereas the LuxN phosphatase activity is constitutive. We also show that signalling from the two V. harveyi quorum‐sensing systems is not equivalent. AI‐1 and LuxN have a much greater effect on the level of LuxO phosphate and therefore Lux expression than do AI‐2 and LuxQ.
Evolutionary conservation of prion‐forming abilities of the yeast Sup35 protein
Authors: Chernoff Yury O. , Galkin Alexey P. , Lewitin Eugene , Chernova Tatiana A. , Newnam Gary P. , Belenkiy Slava M.
Published: March 2003

Saccharomyces cerevisiae prion [PSI ] is a self‐propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions – Sup35N, which serves as a prion‐forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI + ]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a ‘species barrier’ in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion‐forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the ‘inducer’ and ‘inducee’ sequences, and can overcome the ‘species barrier’.
Regulation of quorum sensing in Vibrio harveyi by LuxO and Sigma‐54
Authors: Lilley Brendan N. , Bassler Bonnie L.
Published: March 2003

The bioluminescent marine bacterium Vibrio harveyi controls light production (lux) by an elaborate quorum‐sensing circuit. V. harveyi produces and responds to two different autoinducer signals (AI‐1 and AI‐2) to modulate the luciferase structural operon (luxCDABEGH) in response to changes in cell‐population density. Unlike all other Gram‐negative quorum‐sensing organisms, V. harveyi regulates quorum sensing using a two‐component phosphorylation–dephosphorylation cascade. Each autoinducer is recognized by a cognate hybrid sensor kinase (called LuxN and LuxQ). Both sensors transduce information to a shared phosphorelay protein called LuxU, which in turn conveys the signal to the response regulator protein LuxO. Phospho‐LuxO is responsible for repression of luxCDABEGH expression at low cell density. In the present study, we demonstrate that LuxO functions as an activator protein via interaction with the alternative sigma factor, σ54 (encoded by rpoN). Our results suggest that LuxO, together with σ54, activates the expression of a negative regulator of luminescence. We also show that phenotypes other than lux are regulated by LuxO and σ54, demonstrating that in Vibrio harveyi, quorum sensing controls multiple processes.
Frequent interspecific genetic exchange between commensal neisseriae and Neisseria meningitidis
Authors: Linz Bodo , Schenker Martin , Zhu Peixuan , Achtman Mark
Published: March 2003

Natural sequence variation was investigated among serogroup A subgroup IV‐1 Neisseria meningitidis isolated from diseased patients and healthy carriers in The Gambia, West Africa. The frequencies of DNA import were analysed by sequencing fragments of four linked genes encoding the immunogenic outer membrane proteins TbpB (transferrin binding protein B) and OpaA (an adhesin) plus two housekeeping enzymes. Seventeen foreign tbpB alleles were independently imported into the 98 strains tested, apparently due to immune selection. The median size of the imported DNA fragments was 5 kb, resulting in the occasional concurrent import of linked housekeeping genes by hitchhiking. Sequences of tbpB from other strains of N. meningitidis as well as commensal Neisseria lactamica and Neisseria spp. isolated from the same geographical area revealed that these species share a common tbpB gene pool and identified several examples of interspecific genetic exchange. These observations indicate that recombination can be more frequent between related species than within a species and indicate that effective vaccination against serogroup B meningococcal disease may be difficult to achieve.
Lewis antigens in Helicobacter pylori: biosynthesis and phase variation
Authors: Wang Ge , Ge Zhongming , Rasko David A. , Taylor Diane E.
Published: March 2003

The lipopolysaccharides (LPS) of most Helicobacter pylori strains contain complex carbohydrates known as Lewis antigens that are structurally related to the human blood group antigens. Investigations on the genetic determinants involved in the biosynthesis of Lewis antigens have led to the identification of the fucosyltransferases of H. pylori, which have substrate specificities distinct from the mammalian fucosyltransferases. Compared with its human host, H. pylori utilizes a different pathway to synthesize the difucosylated Lewis antigens, Lewis y and Lewis b. Unique features in the H. pylori fucosyltransferase genes, including homopolymeric tracts mediating slipped‐strand mispairing and the elements regulating translational frameshifting, enable H. pylori to produce variable LPS epitopes on its surface. These new findings have provided us with a basis to further examine the roles of molecular mimicry and phase variation of H. pylori Lewis antigen expression in both persistent infection and pathogenesis of this important human gastric pathogen.
Identification and localization of a methyl‐accepting chemotaxis protein in Rhodobacter sphaeroides
Authors: Wadhams George H. , Martin Angela C. , Armitage Judith P.
Published: March 2003

Genes coding for a classical membrane spanning chemoreceptor (mcpG) and a response regulator (cheY4) were identified in a region of Rhodobacter sphaeroides DNA unlinked to either of the two previously identified chemosensory operons. Immunogold electron microscopy had shown that the expression of chemoreceptors in R. sphaeroides varies with growth conditions. Using GFP fused to the newly identified McpG, we examined the targeting of this single methyl‐accepting chemotaxis protein (MCP) under different growth conditions. The gene encoding the C‐terminal McpG–GFP fusion was introduced by homologous recombination into the chromosome, replacing the wild‐type gene. The resultant protein localized to the poles of the cell under aerobic, photoheterotrophic and anaerobic dark conditions, demonstrating that this MCP is expressed under all three growth conditions. More protein was always found at one pole than the other. The polar fluorescence increased during the cell cycle, with protein becoming evident at the second pole around the time of septation. At division, each daughter cell had a label at one pole, but the intensity of fluorescence was higher in the daughter cell containing the original labelled pole. McpG localization was not altered in a che Operon 1 deletion strain, lacking CheW1 and CheA1, but a che Operon 2 deletion strain, lacking CheW2, CheW3 and CheA2, showed significantly reduced polar localization. This observation indicates that polar localization of McpG depends on Che proteins encoded by Operon 2, but not homologues encoded by Operon 1.
Plasmid and chromosome partitioning: surprises from phylogeny
Authors: Gerdes Kenn , Møller‐Jensen Jakob , Jensen Rasmus Bugge
Published: March 2003

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid‐encoded par loci specify three components: a cis‐acting centromere‐like site and two trans‐acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par‐encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host‐encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter‐cell positions). Two types of partitioning ATPases are known: the Walker‐type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin‐like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid‐encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome‐encoded ATPases, including members from both Gram‐negative and Gram‐positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.
Paradigms of plasmid organization
Authors: Thomas Christopher M.
Published: March 2003

Plasmids are extrachromosomal elements built from a selection of generally quite well understood survival and propagation functions, including replication, partitioning, multimer resolution, post‐segregational killing and conjugative transfer. Evolution has favoured clustering of these modules to form plasmid cores or backbones. Co‐regulation of these core genes can also provide advantages that favour retention of the backbone organization. Tumour‐inducing and symbiosis‐determining plasmids appear to co‐regulate replication and transfer in response to cell density, both being stimulated at high density. Broad‐host‐range plasmids of the IncP‐1 group, on the other hand, have autogenous control circuits, which allow a burst of expression during establishment in a new host, but a minimum of expression during maintenance. The lessons that plasmids have for clustering and co‐regulation may explain the logic and organization of many small bacterial genomes currently being investigated.
Genetic analysis of Escherichia coli K1 gastrointestinal colonization
Authors: Martindale J. , Stroud D. , Moxon E. R. , Tang C. M.
Published: March 2003

Strains of Escherichia coli expressing the K1 polysaccharide capsule colonize the large intestine of newborn infants, and are the leading cause of Gram‐negative septicaemia and meningitis in the neonatal period. We used signature‐tagged mutagenesis (STM) to identify genes that E. coli K1 requires to colonize the gastrointestinal (GI) tract. A total of 2140 mTn5 mutants was screened for their capacity to colonize the GI tract of infant rats, and 16 colonization defective mutants were identified. The mutants have transposon insertions in genes affecting the synthesis of cell surface structures, membrane transporters, transcriptional regulators, enzymes in metabolic pathways, and in genes of unknown function, designated dgc (defective in GI colonization). Three dgcs are absent from the whole genome sequence of E. coli K‐12, although related sequences are found in other pathogenic strains of E. coli and in Shigella flexneri. Additionally, immunohistochemistry was used to define the nature of the colonization defect in five mutants including all dgc mutants. STM was successfully applied to examine the factors involved in E. coli K1 colonization, and the findings are relevant to the pathogenesis of other enteric infections.
The R‐type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F‐type is related to lambda phage
Authors: Nakayama Keisuke , Takashima Kayoko , Ishihara Hiroshi , Shinomiya Tomoyuki , Kageyama Makoto , Kanaya Shigehiko , Ohnishi Makoto , Murata Takahiro , Mori Hirotada , Hayashi Tetsuya
Published: March 2003

Pseudomonas aeruginosa produces three types of bacteriocins: R‐, F‐ and S‐type pyocins. The S‐type pyocin is a colicin‐like protein, whereas the R‐type pyocin resembles a contractile but non‐flexible tail structure of bacteriophage, and the F‐type a flexible but non‐contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R‐type pyocin is derived from a common ancestral origin with P2 phage and the F‐type from λ phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.
Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target
Authors: Kubo Aya , Stephens Richard S.
Published: March 2003

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome‐swelling assay was used to determine whether this protein had pore‐forming capabilities. PorB had pore‐forming activity and was shown to be different from MOMP porin activity.
Genome organization and characterization of mycobacteriophage Bxb1
Authors: Mediavilla José , Jain Shruti , Kriakov Jordon , Ford Michael E. , Duda Robert L. , Jacobs William R. , Hendrix Roger W. , Hatfull Graham F.
Published: March 2003

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow‐growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.
N‐mediated transcription antitermination in lambdoid phage H‐19B is characterized by alternative NUT RNA structures and a reduced requirement for host factors
Authors: Neely Melody N. , Friedman David I.
Published: March 2003

Gene expression in lambdoid phages in part is controlled by transcription antitermination. For most lambdoid phages, maximal expression of delayed early genes requires an RNA polymerase modified by the phage N and host Nus proteins at RNA NUT sites. The NUT sites (NUTL and NUTR) are made up of three elements: BOXA, BOXB and an intervening spacer sequence. We report on N antitermination in H‐19B, a lambdoid phage carrying shiga toxin 1 genes. H‐19B N requires NusA, but not two other host factors required by λ N, NusB and ribosomal protein S10. The H‐19B NUT site BOXA is not required, whereas the BOXB is required for N action. H‐19B nut sites have dyad symmetries in the spacer regions that are not in other nut sites. Changes in one arm of the dyad symmetry inactivate the NUT RNA. Compensating changes increasing the number of mutant nucleotides but restoring dyad symmetry restore activity. Deletion of the sequences encoding the dyad symmetry has little effect. Thus, the specific nucleotides composing the dyad symmetry seem relatively unimportant. We propose that the RNA stem–loop structure, called the ‘reducer’, by sequestering nucleotides from the linear RNA brings into proximity sites on either side of the dyad symmetry that contribute to forming an active NUT site.
A collagen‐like surface glycoprotein is a structural component of the Bacillus anthracis exosporium
Authors: Sylvestre Patricia , Couture‐Tosi Evelyne , Mock Michèle
Published: March 2003

Bacillus anthracis
, the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino‐terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen‐like surface protein was named BclA (for
Bacillus
c
ollagen‐
l
ike protein of
anthracis
). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor
σ
K
, which is required for a variety of events late in sporulation, was found upstream from the
bclA
gene. A
bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.
An extended hydrophobic interactive surface of Yersinia pestis Caf1M chaperone is essential for subunit binding and F1 capsule assembly
Authors: MacIntyre Sheila , Zyrianova Irina M. , Chernovskaya Tatiana V. , Leonard Mary , Rudenko Elena G. , Zav'yalov Vladimir P. , Chapman David A. G.
Published: March 2003

A single polypeptide subunit, Caf1, polymerizes to form a dense, poorly defined structure (F1 capsule) on the surface of Yersinia pestis. The caf‐encoded assembly components belong to the chaperone–usher protein family involved in the assembly of composite adhesive pili, but the Caf1M chaperone itself belongs to a distinct subfamily. One unique feature of this subfamily is the possession of a long, variable sequence between the F1 β‐strand and the G1 subunit binding β‐strand (FGL; F1 β‐strand to G1 β‐strand long). Deletion and insertion mutations confirmed that the FGL sequence was not essential for folding of the protein but was absolutely essential for function. Site‐specific mutagenesis of individual residues identified Val‐126, in particular, together with Val‐128 as critical residues for the formation of a stable subunit–chaperone complex and the promotion of surface assembly. Differential effects on periplasmic polymerization of the subunit were also observed with different mutants. Together with the G1 strand, the FGL sequence has the potential to form an interactive surface of five alternating hydrophobic residues on Caf1M chaperone as well as in seven of the 10 other members of the FGL subfamily. Mutation of the absolutely conserved Arg‐20 to Ser led to drastic reduction in Caf1 binding and surface assembled polymer. Thus, although Caf1M–Caf1 subunit binding almost certainly involves the basic principle of donor strand complementation elucidated for the PapD–PapK complex, a key feature unique to the chaperones of this subfamily would appear to be capping via high‐affinity binding of an extended hydrophobic surface on the respective single subunits.
Mycobacterium smegmatis laminin‐binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin‐binding haemagglutinin
Authors: Pethe Kevin , Puech Virginie , Daffé Mamadou , Josenhans Christine , Drobecq Hervé , Locht Camille , Menozzi Franco D.
Published: March 2003

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin‐binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast‐growing mycobacteria, Mycobacterium smegmatis was studied using anti‐HBHA monoclonal antibodies (mAbs). A cross‐reactive protein was detected by immunoblotting of M. smegmatis whole‐cell lysates. However, the M. tuberculosis HBHA‐encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti‐HBHA mAbs was purified by heparin–Sepharose chromatography, and its amino‐terminal sequence was found to be identical to that of the previously described histone‐like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone‐like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone‐like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin‐binding protein (MS‐LBP). MS‐LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin‐mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin‐binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.
Iron‐free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport
Authors: Schalk Isabelle J. , Hennard Christophe , Dugave Christophe , Poole Keith , Abdallah Mohamed A. , Pattus Franc
Published: March 2003

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co‐purification of FpvA with iron‐free PaA and reported similar binding affinities of iron‐free pyoverdin and ferric‐pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron‐free PaA and the FpvA receptor here reveals the existence of an FpvA–pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin‐loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric‐pyoverdin, it is shown that iron‐free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric‐pyoverdin displaces the bound iron‐free pyoverdin on FpvA–PaA to form FpvA–PaA‐Fe complexes. In vivo, the kinetics of formation of this FpvA–PaA‐Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric‐pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17‐fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA‐Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.
Co‐ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis
Authors: Mamoun Choukri Ben , Gluzman Ilya Y. , Hott Christian , MacMillan Sandra K. , Amarakone Aloka S. , Anderson Dustin L. , Carlton Jane M.‐R. , Dame John B. , Chakrabarti Debopam , Martin Rodger K. , Brownstein Bernard H. , Goldberg Daniel E.
Published: March 2003

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage‐specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co‐ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut‐off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.
The relationship between visible intracellular aggregates that appear after overexpression of Sup35 and the yeast prion‐like elements [PSI+] and [PIN+]
Authors: Zhou Ping , Derkatch Irina L. , Liebman Susan W.
Published: March 2003

Overproduced fusions of Sup35 or its prion domain with green fluorescent protein (GFP) have previously been shown to form frequent dots in [PSI+] cells. Rare foci seen in [psi−] cells were hypothesized to indicate the de novo induction of [PSI+] caused by the overproduced prion domain. Here, we describe novel ring‐type aggregates that also appear in [psi−] cultures upon Sup35 overproduction and show directly that dot and ring aggregates only appear in cells that have become [PSI+]. The formation of either type of aggregate requires [PIN+], an element needed for the induction of [PSI+]. Although aggregates are visible predominantly in stationary‐phase cultures, [PSI+] induction starts in exponential phase, suggesting that much smaller aggregates can also propagate [PSI+]. Such small aggregates are probably present in [PSI+] cells and, upon Sup35–GFP overproduction, facilitate the frequent formation of dot aggregates, but only the occasional appearance of ring aggregates. In contrast, rings are very frequent when [PSI+] cultures, including those lacking [PIN+], are grown in the presence of GuHCl or excess Hsp104 while overexpressing Sup35–GFP. Thus, intermediates formed during [PSI+] curing seem to facilitate ring formation. Surprisingly, GuHCl and excess Hsp104, which are known to promote loss of [PSI+], did not prevent the de novo induction of [PSI+] by excess Sup35 in [psi−][PIN+] strains.
Lipid A and O‐chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development
Authors: Kannenberg Elmar L. , Carlson Russell W.
Published: March 2003

Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea‐nodulating strain Rhizobium leguminosarum 3841. The LPSs extracted by hot phenol–water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases. Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced‐oxygen conditions, or when isolated from root nodules as bacteroids. Compared with the water‐extracted LPSs, the phenol‐extracted LPSs contained greater degrees of glycosyl methylation and O‐acetylation, increased levels of xylose, glucose and mannose and increased amounts of long‐chain fatty acids attached to the lipid A moiety. The water‐ and phenol‐phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns. Phenol‐extracted LPSs from rhizobia grown under reduced‐oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e. mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour. This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low‐oxygen conditions. The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats.
Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 Å resolution
Authors: Campos Andrés , Zhang RongGuang , Alkire Randal W. , Matsumura Philip , Westbrook Edwin M.
Published: March 2003

FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA‐binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno‐methionine for natural sulphur‐methionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple‐energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly α‐helical. The C‐termini of both FlhD monomers (residues 83–116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C‐terminus structure in chain A (residues 83–98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA‐binding, helix–turn–helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA‐binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA‐binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys‐65) of FlhD native homodimers. Alanine substitution at Cys‐65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.
Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery
Authors: Subtil Agathe , Parsot Claude , Dautry‐Varsat Alice
Published: March 2003

Chlamydia spp. are strictly intracellular pathogens that grow inside a vacuole, called an inclusion. They possess genes encoding proteins homologous to components of type III secretion machineries, which, in other bacterial pathogens, are involved in delivery of bacterial proteins within or through the membrane of eukaryotic host cells. Inc proteins are chlamydial proteins that are associated with the inclusion membrane and are characterized by the presence of a large hydrophobic domain in their amino acid sequence. To investigate whether Inc proteins and other proteins exhibiting a similar hydropathic profile might be secreted by a type III system, we used a heterologous secretion system. Chimeras were constructed by fusing the N‐terminal part of these proteins with a reporter, the Cya protein of Bordetella pertussis, and these were expressed in various strains of Shigella flexneri. We demonstrate that these hybrid proteins are secreted by the type III secretion system of S. flexneri, thereby providing evidence that IncA, IncB and IncC are secreted by a type III mechanism in chlamydiae. Moreover, we show that three other proteins from Chlamydia pneumoniae, all of which have in common the presence of a large hydrophobic domain, are also secreted by S. flexneri type III secretion machinery.
Type III export: new uses for an old pathway
Authors: Plano Gregory V. , Day James B. , Ferracci Franco
Published: March 2003

Gram‐negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.
TrlR, a defective TraR‐like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrlR:TraR dimers
Authors: Chai Yunrong , Zhu Jun , Winans Stephen C.
Published: March 2003

Octopine‐type Ti plasmids of Agrobacterium tumefaciens require the quorum‐sensing proteins TraR and TraI and the diffusible pheromone 3‐oxooctanoyl homoserine lactone (AAI) to regulate genes required for conjugal transfer. TraR activity is inhibited by a protein called TrlR, which closely resembles amino acids 1–181 of TraR but is truncated as a result of a shift in the reading frame at codon 182. This frameshift does not affect synthesis of the amino‐terminal domain, which is thought to bind autoinducer and mediate protein dimerization, but abolishes translation of the carboxyl‐terminal, DNA‐binding domain. In this study, we show that TrlR, like TraR, requires AAI for solubility when overexpressed in Escherichia coli. TrlR bound one molecule of AAI per protein monomer, supporting the prediction that the amino‐terminal domain of TraR contains the AAI binding site. Purified TrlR blocked TraR for both specific DNA binding and transcription of a tra promoter, supporting previous studies performed with whole cells. When TrlR and a TraR fusion protein were co‐expressed in E. coli, these proteins readily formed heterodimeric complexes that were inactive in DNA‐binding activity. These data support the hypotheses that (i) the amino‐terminal half of TraR binds AAI and mediates protein dimerization; (ii) both DNA‐binding domains in a TraR dimer are required for stable DNA binding; and (iii) TrlR blocks TraR by direct protein–protein interactions.
Experimental genome evolution: large‐scale genome rearrangements associated with resistance to replacement of a chromosomal restriction–modification gene complex
Authors: Handa N. , Nakayama Y. , Sadykov M. , Kobayashi I.
Published: March 2003

Type II restriction enzymes are paired with modification enzymes that protect type II restriction sites from cleavage by methylating them. A plasmid carrying a type II restriction–modification gene complex is not easily replaced by an incompatible plasmid because loss of the former leads to cell death through chromosome cleavage. In the present work, we looked to see whether a chromosomally located restriction–modification gene complex could be replaced by a homologous stretch of DNA. We tried to replace the PaeR7I gene complex on the Escherichia coli chromosome by transducing a homologous stretch of PaeR7I‐modified DNA. The replacement efficiency of the restriction–modification complex was lower than expected. Some of the resulting recombinant clones retained the recipient restriction–modification gene complex as well as the homologous DNA (donor allele), and slowly lost the donor allele in the absence of selection. Analysis of their genome‐wide rearrangements by Southern hybridization, inverse polymerase chain reaction (iPCR) and sequence determination demonstrated the occurrence of unequal homologous recombination between copies of the transposon IS3. It was strongly suggested that multiple rounds of unequal IS3–IS3 recombination caused large‐scale duplication and inversion of the chromosome, and that only one of the duplicated copies of the recipient PaeR7I was replaced.
Regulation of late flagellar gene transcription and cell division by flagellum assembly in Caulobacter crescentus
Authors: Muir Rachel E. , Gober James W.
Published: March 2003

Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a trans‐acting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a trans‐acting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain‐of‐function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD–bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.
Codon‐usage based regulation of colicin K synthesis by the stress alarmone ppGpp
Authors: Kuhar Irena , Van Putten Jos P. M. , Žgur‐Bertok Darja , Gaastra Wim , Jordi Bart J. A. M.
Published: March 2003

The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG‐regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3′,5′‐bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post‐transcriptional regulatory mechanism. Reporter (β‐galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5′ end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine‐encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post‐transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.
Ectopic transposition of a group II intron in natural bacterial populations
Authors: Muñoz Estefanía , Villadas Pablo J. , Toro Nicolás
Published: March 2003

Self‐splicing group II introns are thought to be the evolutionary progenitors of eukaryotic spliceosomal introns. The invasion of novel (ectopic) sites by group II introns is considered to be a key mechanism by which spliceosomal introns may have become widely dispersed. However, the dynamics of these events in populations are unknown. In bacteria, only two group II introns have been shown to splice and to be mobile in vivo. One of these introns, RmInt1 from Sinorhizobium meliloti, which encodes a protein with no endonuclease domain, has been shown to invade the ectopic oxi1 site independently of recombinase. In this study, we analysed ectopic transposition of the RmInt1 intron in a natural population of S. meliloti. We characterized S. meliloti isolates by polymerase chain reaction amplification of a gene, dapB, which is found only on the pRmeGR4b plasmid diagnostic of GR4‐type strains. The diversity within this specific field population of bacteria was analysed by restriction fragment length polymorphism using ISRm2011‐2 (homing site of RmInt1) and RmInt1 as probes. We found that ectopic transposition of RmInt1 to the oxi1 site occurred in this natural bacterial population. This ectopic transposition was also the most frequent genetic event observed. This work provides further evidence that the ectopic transposition of group II introns is an important mechanism for their spread in natural bacterial populations.
Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA
Authors: Herbig Andrew F. , Helmann John D.
Published: March 2003

The inducible response to H2O2 stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism. PerR was purified in both an inactive, metal‐dependent form and an active, metal‐containing form as determined using DNA‐binding assays. Active PerR contains both zinc and iron and is designated PerR:Zn,Fe. Added manganous ion competes for binding to the iron site and can restore DNA‐binding activity to the metal‐dependent form of PerR, presumably generating PerR:Zn,Mn. The DNA‐binding activity of PerR:Zn,Fe is eliminated by exposure to H2O2 whereas PerR:Zn,Mn is comparatively resistant. DNA‐binding activity can be restored by a thiol‐reducing agent, suggesting that redox‐active cysteines are involved in peroxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H2O2. In contrast, in cells grown with iron supplementation, a PerR‐repressed gene is completely derepressed by H2O2. These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.
The incompatibility between the PlcR‐ and AtxA‐controlled regulons may have selected a nonsense mutation in Bacillus anthracis
Authors: Mignot Tâm , Mock Michèle , Robichon Denis , Landier Annie , Lereclus Didier , Fouet Agnès
Published: March 2003

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non‐specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR+ strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA‐controlled virulence regulon. We propose that the PlcR‐controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.