bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET
Authors: Veal‐Carr Wendy L. , Stibitz Scott
Published: January 2005

Bordetella pertussis, the etiologic agent of whooping cough, causes disease by employing an array of virulence factors controlled by the BvgA–BvgS two‐component signal transduction system. Regulation by this system has been extensively characterized in vitro, where bvg‐activated genes are repressed in a process known as phenotypic modulation. Differential regulation of these genes by the response regulator BvgA results in promoters that are activated early, middle, or late after being released from modulation. However, the in vivo environmental signal and regulation pattern has not been described. In order to investigate BvgAS‐mediated regulation of B. pertussis virulence factors in vivo using the mouse aerosol challenge model, we have adapted the recombinase‐based in vivo technology (RIVET) system for use in B. pertussis. We have demonstrated that these strains show resolution during in vitro growth under non‐modulating conditions. In addition, we have demonstrated that modulating strains by growth on media containing MgSO4 does not affect virulence in the mouse aerosol challenge model. We have therefore used the RIVET system to reveal the time‐course of gene expression in vivo for selected B. pertussis virulence factors (cya, fha, prn and ptx). Our data indicate that this method can be effectively used to monitor and compare in vivo and in vitro gene expression in B. pertussis, and that temporal regulation patterns previously observed in vitro are mirrored in vivo.
Population density‐dependent regulation of exopolysaccharide formation in the hyperthermophilic bacterium Thermotoga maritima
Authors: Johnson Matthew R. , Montero Clemente I. , Conners Shannon B. , Shockley Keith R. , Bridger Stephanie L. , Kelly Robert M.
Published: January 2005

Co‐cultivation of the hyperthermophiles Thermotoga maritima and Methanococcus jannaschii resulted in fivefold higher T. maritima cell densities when compared with monoculture as well as concomitant formation of exopolysaccharide and flocculation of heterotroph‐methanogen cellular aggregates. Transcriptional analysis of T. maritima cells from these aggregates using a whole genome cDNA microarray revealed the induction of a putative exopolysaccharide synthesis pathway, regulated by intracellular levels of cyclic diguanosine 3′,5′‐(cyclic)phosphate (cyclic di‐GMP) and mediated by the action of several GGDEF proteins, including a putative diguanylate cyclase (TM1163) and a putative phosphodiesterase (TM1184). Transcriptional analysis also showed that TM0504, which encodes a polypeptide containing a motif common to known peptide‐signalling molecules in mesophilic bacteria, was strongly upregulated in the co‐culture. Indeed, when a synthetically produced peptide based on TM0504 was dosed into the culture at ecologically relevant levels, the production of exopolysaccharide was induced at significantly lower cell densities than was observed in cultures lacking added peptide. In addition to identifying a pathway for polysaccharide formation in T. maritima, these results point to the existence of peptide‐based quorum sensing in hyperthermophilic bacteria and indicate that cellular communication should be considered as a component of the microbial ecology within hydrothermal habitats.
Recruitment of penicillin‐binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates
Authors: Pinho Mariana G. , Errington Jeff
Published: January 2005

Staphylococcus aureus penicillin‐binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d‐cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by binding to its substrate, which is localized at that place. In methicillin‐resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.
Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells
Authors: Capecchi Barbara , Adu‐Bobie Jeannette , Di Marcello Federica , Ciucchi Laura , Masignani Vega , Taddei Annarita , Rappuoli Rino , Pizza Mariagrazia , Aricò Beatrice
Published: January 2005

Neisseria  meningitidis  is  a  human  pathogen,  which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood–brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N‐terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.
The Neurospora crassa gene responsible for the cut and ovc phenotypes encodes a protein of the haloacid dehalogenase family
Authors: Youssar Loubna , Schmidhauser Thomas J. , Avalos Javier
Published: January 2005

Light stimulation of carotenogenesis in Neurospora crassa, mediated by the White Collar proteins, is enhanced in some regulatory mutants, such as vivid and ovc. The gene responsible for the vivid mutation has been identified, but not the one responsible for the ovc phenotype. The ovc mutant is sensitive to high osmotic conditions and allelic with another mutant, cut, also osmosensitive but not affected in carotenogenesis. A phenotypic characterization of both strains is presented. Light induction of mRNA levels of the carotenoid genes al‐1 and al‐2, the regulatory gene wc‐1 or the conidiation‐specific gene con‐10 is not significantly changed in the ovc mutant when compared with the wild type. We have identified the gene affected in the ovc mutant by complementation of osmosensitivity with a cosmid library. This gene, which we call cut‐1, codes for an enzyme of the haloacid dehalogenase family, which includes different classes of phosphatases. cut‐1 is able to restore the wild‐type phenotype of the ovc and cut strains, confirming that they are affected in the same gene. DNA sequence analysis identified a point mutation in the cut mutant, leading to a truncated protein. The ovc mutant represents a deletion encompassing the entire gene and surrounding sequences. The cut‐1 promoter contains putative regulatory elements involved in osmotic or thermal stress. We show that cut‐1 transcription is low in illuminated or dark‐grown cultures, and is induced by high osmotic conditions or by heat shock.
Degradation of the HilC and HilD regulator proteins by ATP‐dependent Lon protease leads to downregulation of Salmonella pathogenicity island 1 gene expression
Authors: Takaya Akiko , Kubota Yohsuke , Isogai Emiko , Yamamoto Tomoko
Published: January 2005

Salmonella pathogenicity island 1 (SPI1) enables infecting Salmonella to cross the small intestinal barrier and to escape phagocytosis by inducing apoptosis. Several environmental signals and transcriptional regulators modulate the expression of hilA, which encodes a protein playing a central role in the regulatory hierarchy of SPI1 gene expression. We have previously shown that Lon, a stress‐induced ATP‐dependent protease, is a negative regulator of hilA, suggesting that it targets factors required for activating hilA expression. To elucidate the mechanisms by which Lon protease negatively regulates SPI1 transcription, we looked for its substrate proteins. We found that HilC and HilD, which are positive regulators of hilA expression, accumulate in Lon‐depleted cells, and that the enhancement of SPI1 expression that occurs in a lon‐disrupted mutant is not observed in the lon hilC hilD triple null mutant. Furthermore, we demonstrated that the half‐lives of HilC and HilD are, respectively, about 12 times and three times longer in the Lon‐depleted mutant, than in the Lon+ cells, suggesting that Lon targets both of HilC and HilD. In view of these findings, we suggest that the regulation of SPI1 expression is negatively controlled through degradation of the HilC and HilD transcriptional regulators by Lon.
Contact with host cells induces a DNA repair system in pathogenic Neisseriae
Authors: Morelle Sandrine , Carbonnelle Etienne , Matic Ivan , Nassif Xavier
Published: January 2005

DNA repair systems play a major role in maintaining the integrity of bacterial genomes. Neisseria meningitidis, a human pathogen capable of colonizing the human nasopharynx, possesses numerous DNA repair genes but lacks inducible DNA repair systems such as the SOS response, present in most bacteria species. We recently identified a set of genes upregulated by contact with host cells. An open reading frame having high homology with the small subunit of Escherichia coli exonuclease VII (xseB) belongs to this regulon. The increased sensitivity of a mutant in this coding sequence to UV irradiation, alkylating agent and nalidixic acid demonstrates the participation of this gene in meningococcal DNA repair. In addition, the upregulation of the transcription of this open reading frame upon interaction of N. meningitidis with host cells increased not only the bacterial ability to repair its DNA but also the rate of phase variation by frameshifting. Together these data demonstrate that N. meningitidis possesses an inducible DNA repair system that might be used by the bacteria to adapt to its niches when it is colonizing a new host.
PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability
Authors: Roosen Johnny , Engelen Kristof , Marchal Kathleen , Mathys Janick , Griffioen Gerard , Cameroni Elisabetta , Thevelein Johan M. , De Virgilio Claudio , De Moor Bart , Winderickx Joris
Published: January 2005

In the yeast Saccharomyces cerevisiae, PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read‐outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome‐wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress‐responsive element (STRE)‐ and post‐diauxic shift (PDS)‐driven gene expression, Sch9 appears to exert additional positive control on the Rim15‐effector Gis1 to regulate PDS‐driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)‐gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9‐controlled signalling route. This mechanism allows fine‐tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.
Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds
Authors: Draskovic Irena , Dubnau David
Published: January 2005

ComEC is a putative channel protein for DNA uptake in Bacillus subtilis and other genetically transformable bacteria. Membrane topology studies suggest a model of ComEC as a multispanning membrane protein with seven transmembrane segments (TMSs), and possibly with one laterally inserted amphipathic helix. We show that ComEC contains an intramolecular disulphide bond in its N‐terminal extracellular loop (between the residues C131 and C172), which is required for the stability of the protein, and is probably introduced by BdbDC, a pair of competence‐induced oxidoreductase proteins. By in vitro cross‐linking using native cysteine residues we show that ComEC forms an oligomer. The oligomerization surface includes a transmembrane segment, TMS‐G, near the cytoplasmic C‐terminus of ComEC.
The σ54‐dependent transcriptional activator SfnR regulates the expression of the Pseudomonas putida sfnFG operon responsible for dimethyl sulphone utilization
Authors: Endoh Takayuki , Habe Hiroshi , Nojiri Hideaki , Yamane Hisakazu , Omori Toshio
Published: January 2005

Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO2), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a σ54‐dependent transcriptional regulator is essential for DMSO2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO2‐utilization‐defective phenotype) using mini‐Tn5, which contains two outward‐facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO2. The DMSO2‐positive mutant carried a mini‐Tn5 insertion in the intergenic region between two opposite‐facing operons, sfnAB and sfnFG. Both sfnA and sfnB products were similar to acyl‐CoA dehydrogenase family proteins, whereas sfnF and sfnG encoded a putative NADH‐dependent FMN reductase (SfnF) and an FMNH2‐dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH2‐dependent DMSO2 monooxygenase that converts DMSO2 to MSA. It was revealed that the expression of the sfnFG operon is directly activated by the binding of SfnR at its upstream region. Site‐directed mutagenesis of the SfnR binding sequences allowed us to define a potential recognition sequence for SfnR. These results provided insight into regulation of sulphate starvation‐induced genes in bacteria.
Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein
Authors: Lara Beatriz , Rico Ana Isabel , Petruzzelli Sabrina , Santona Antonella , Dumas Jacques , Biton Jacques , Vicente Miguel , Mingorance Jesús , Massidda Orietta
Published: January 2005

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide‐dependent manner, forming long corkscrew‐like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.
The Legionella IcmS–IcmW protein complex is important for Dot/Icm‐mediated protein translocation
Authors: Ninio Shira , Zuckman‐Cholon Deborah M. , Cambronne Eric D. , Roy Craig R.
Published: January 2005

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two‐hybrid screen. It was determined that the IcmS–IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS–IcmW complex being involved in the recognition and Dot/Icm‐dependent translocation of substrate proteins during Legionella infection of host cells.
The svpA‐srtB locus of Listeria monocytogenes: Fur‐mediated iron regulation and effect on virulence
Authors: Newton Salete M. C. , Klebba Phillip E. , Raynaud Catherine , Shao Yi , Jiang Xiaoxu , Dubail Iharilalao , Archer Crystal , Frehel Claude , Charbit Alain
Published: January 2005

In Listeria monocytogenes the promoter region of the svpA‐srtB locus contains a well‐conserved Fur box. We characterized the iron‐regulation of this locus: real‐time polymerase chain reaction analyses and anti‐SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80‐fold and 10‐fold respectively), when initiated by either the addition of the iron chelator 2,2′‐bipyridyl to BHI media, or by growth in iron‐restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA‐srtB promoter in Escherichia coli (37‐fold) and in L. monocytogenes (two‐ to threefold) when the bacteria were grown in iron‐deficient conditions. A Δfur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA‐srtB promoter. Cellular fractionation data revealed that in iron‐rich media wild‐type SvpA was exclusively secreted to the culture supernatant. However, both the Δfur derivative and wild‐type L. monocytogenes grown in iron‐deficient media anchored a fraction of the SvpA proteins (∼5%) to peptidoglycan, and produced a lower‐molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA‐srtB locus (through Fur‐mediated regulation), and attachment of SvpA to the cell wall (through SrtB‐mediated covalent linkage). SvpA bears homology to IsdC, a haemin‐binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site‐directed deletions of four structural genes and the promoter of the svpA‐srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA‐srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA‐srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.
Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon‐associated degradation
Authors: Losada Liliana C. , Hutcheson Steven W.
Published: January 2005

The hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon‐associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS‐dependent effector secretion. In this study we report that TTSS‐dependent effectors are subject to the proteolytic degradation that appears to be rate‐limiting to secretion. The half‐lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS‐dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones  appears  to  be  protection  of  effectors  from Lon‐associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon‐targeting and chaperone‐binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.
Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide
Authors: Wong Sandy M. , Akerley Brian J.
Published: January 2005

In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence‐associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in‐frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS‐PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.
A master regulator for biofilm formation by Bacillus subtilis
Authors: Kearns Daniel B. , Chu Frances , Branda Steven S. , Kolter Roberto , Losick Richard
Published: January 2005

Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR‐mediated  repression.  We  further  propose  that  SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.
Mechanism of lipid‐body formation in prokaryotes: how bacteria fatten up
Authors: Wältermann Marc , Hinz Andreas , Robenek Horst , Troyer David , Reichelt Rudolf , Malkus Ursula , Galla Hans‐Joachim , Kalscheuer Rainer , Stöveken Tim , Von Landenberg Philipp , Steinbüchel Alexander
Published: January 2005

Neutral lipid accumulation is frequently observed in some Gram‐negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation  of  wax  ester‐  and  triacylglycerol  (TAG)‐bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid‐body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid‐bodies starts with the docking of wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid‐body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane‐associated enzyme. SLDs conglomerated subsequently to membrane‐bound lipid‐prebodies which are then released into the cytoplasm. The formation of matured lipid‐bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half‐unit membrane of phospholipids.
Mig‐14 is an inner membrane‐associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection
Authors: Brodsky Igor E. , Ghori Nafisa , Falkow Stanley , Monack Denise
Published: January 2005

Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid‐like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig‐14 and virK genes of Salmonella enterica are important in bacterial resistance to anti‐microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon‐gamma (IFN‐γ) is crucial to controlling replication of mig‐14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig‐14 contributes to long‐term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig‐14 and virK contribute to the survival of Salmonella in macrophages treated with IFN‐γ and are necessary for resistance to cathelin‐related anti‐microbial peptide (CRAMP), an anti‐microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig‐14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig‐14 is an inner membrane‐associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig‐14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig‐14 in anti‐microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.
Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O‐acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus
Authors: Bera Agnieszka , Herbert Silvia , Jakob Andreas , Vollmer Waldemar , Götz Friedrich
Published: January 2005

Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild‐type strain was O‐acetylated at C6‐OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan‐specific O‐acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.
MicroMeeting: Who's talking to whom? Epithelial–bacterial pathogen interactions
Authors: Aldridge Phillip D. , Gray Michael A. , Hirst Barry H. , Khan C. M. Anjam
Published: January 2005

Our perception that host–bacterial interactions lead to disease comes from rare, unsuccessful interactions resulting in the development of detectable symptoms. In contrast, the majority of host–bacterial interactions go unnoticed as the host and bacteria perceive each other to be no threat. In July 2004, a focused international symposium on epithelial–bacterial pathogen interactions was held in Newcastle upon Tyne (UK). The symposium concentrated on recent advances in our understanding of bacterial interactions at respiratory and gastrointestinal mucosal epithelial layers. For the host these epithelial tissues represent a first line of defence against invading bacterial pathogens. Through the discovery that the innate immune system plays a pivotal role during host–bacterial interactions, it has become clear that epithelia are being utilized by the host to monitor or communicate with both pathogenic and commensal bacteria. Interest in understanding the bacterial perspective of these interactions has lead researchers to realize that the bacteria utilize the same factors associated with disease to establish successful long‐term interactions. Here we discuss several common themes and concepts that emerged from recent studies that have allowed physiologists and microbiologists to interact at a common interface similar to their counterparts – epithelia and bacterial pathogens. These studies highlight the need for further multidisciplinary studies into how the host differentiates between pathogenic and commensal bacteria.
STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *
Authors: ISHIMODA TADASHI
Published: January 2005

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti‐chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea‐polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ‐nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi, 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.
MITOTIC ACTIVITY AND CELL PROLIFERATION IN PRIMARY INDUCTION OF NEWT EMBRYO
Authors: SUZUKI AKIO , KUWABARA KAZUSHI
Published: January 2005

Mitotic activity and cell proliferation of newt (Triturus pyrrhogaster) embryo were examined with special reference to primary induction.

Mitotic activity of gastrula ectoderm gradually decreases during gastrulation. The ectoderm, which is isolated from mid‐gastrula (stage 12b) and cultured in vitro, also shows gradual decrease in mitotic activity during cultivation and the mitotic activity steeply decreases after 48 hr.

The ectoderm cultured with heterologous inductor (GPL‐extract) shows a temporal suppression in mitotic activity. The ectoderm of the whole gastrula also shows a regional suppression where it is in contact with the chorda‐mesoderm.

The number of the ectodermal cells increases about 2 times after 24 hr culture and to more than 3 times after 48 hr culture. Accordingly it is certain that the majority of the ectodermal cells divides at least one time in the course of 48 hr.

Histological examination of the ectoderm cultured together with the inductor reveals that differentiation of undifferentiated ectoderm to neural tissues is accomplished at least within 48 hr after cultivation with the inductor.

The present examination shows the possibility that the mitotic activity of the ectoderm may be temporarily suppressed by the inductor and that it then decreases along with neural cell differentiation after recovery of the activity.

The results also suggest that the determination of undifferentiated ectoderm to neural tissues occurs before the second cell division after the contact with the inductor and the events occurring during the first cell cycle after activating by the inducing stimulus are critical for the primary induction.
STUDIES ON DROSOPHILA EMBRYONIC CELLS IN VITRO I. CHARACTERISTICS OF CELL TYPES IN CULTURE *
Authors: KURODA Y.
Published: January 2005

Embryonic cells from Drosophila melanogaster were cultured in medium K‐17 supplemented with 0.1 mg/ml of fetuin and 15% fetal bovine serum, and the behavior of embryonic cells, the characteristics of some cell types and the maintenance of the phenotypic properties of the cells were examined. Embryos at the stage with a saclike midgut (12 hr eggs) were found to be the best materials for cultivation. Muscle cells, epithelial cells, fibroblastic cells and small cells were observed as distinct migrating cell types. Muscle cells pulsated synchrónously when two or more cells made contact with each other and this pulsation continued for more than six weeks under the culture conditions employed. Epithelial cells matured with increase in size and in the number of cytoplasmic granules. Fibroblastic cells had conspicuous motile activity. Small cells were peculiar for their size of about 3 μm diameter and formed groups of about forty to fifty cells. Their characteristic shape and size suggested that they were imaginal disc cells. Cellular spheres, which consisted of epithelial cells and fibroblastic cells, and nerve fibers of nerve cells were also observed. Some mitotic figures, suggesting the occurrence of cell division, were observed in cultures of epithelial cells.
INITIATION OF CLEAVAGE IN FISH EGGS BY INJECTION OF FLAGELLA OR MICROTUBULES OF SEA URCHIN SPERMATOZOA
Authors: OHTA TADAYUKI , IWAMATSU TAKASHI
Published: January 2005

Flagella and their microtubules obtained from sea urchin (Hemicentrotus pulcherrimus) spermatozoa were injected into unfertilized eggs of the medaka (Oryzias latipes) with a micropipette. Upon activation, some of the eggs began the first cleavage with three or more irregular blastomeres, and developed to the morula stage. It is suggested that sperm flagellar microstubule material is one of the cleavage initiation substances.
CHANGES IN SUSCEPTIBILITY OF CHICK EMBRYONIC BLOOD CELLS TO NEWCASTLE DISEASE VIRUS
Authors: TAHARA YUTAKA , HYODO NOBUYUKI
Published: January 2005

To analyse the age‐dependent changes in susceptibility of chick embryonic cells to viral infection, observations were made on the blood cells after the inoculation of Newcastle disease virus. A lethal dose of Sato strain of Newcastle disease virus was introduced into chick embryos via injection of inoculum into the blood vessel and allantoic sac. Observations of blood cells by immunofluorescent technique revealed two types of cells, susceptible and resistant to the virus infection. Erythroblasts of both primitive and definitive lines, embryonic thromboblasts and thrombocytes were of the former type and mid‐ and late‐polychromatic erythrocytes and mature ones were of the latter. Erythroblasts gradually decrease in their viral susceptibility as they differentiate into polychromatic erythrocytes and finally become resistant to the virus at the mid‐polychromatic erythrocyte stage or later. Thromboblasts, on the other hand, retain high susceptibility to the virus throughout the course of their differentiation to mature thrombocytes. The change in the viral susceptibility of erythroid cells with differentiation is discussed in relation to the structural and functional alterations during the cell specialization.
PRESENCE OF 1‐METHYLADENINE IN SEA URCHIN GONAD AND ITS RELATION TO OOCYTE MATURATION
Authors: KANATANI HARUO
Published: January 2005

A water extract of sea urchin ovary was found to induce maturation of starfish oocytes in vitro. The presence of the active substance was demonstrated in ovaries of the sea urchins, Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus, and the sand dollars, Clypeaster japonicus and Peronella japonica. The active substance was also contained in the testes of these echinoids. That the content of this substance increases during the reproductive season was demonstrated with Anthocidaris gonads. The active substance present in ovary or testis of the sea urchins was successfully extracted with 85% ethanol and purified with gel‐filtrations on Sephadex G‐15 columns after washing with chloroform and ether. The purified active substances were the same and were identified as 1‐1‐methyladenine by thin layer chromatography. 1‐Methyladenine was found to be effective in inducing oocyte maturation in Anthocidaris crassispina in vitro. Therefore, 1‐methyladenine seems to play an important role in oocyte maturation in echinoids as well as in asteroids.
FORMATION OF ASCOSPORE DELIMITING MEMBRANE IN SORDARIA HUMANA
Authors: SAKAI AIKO
Published: January 2005

The origin and the formation of the ascospore delimiting membrane in Sordaria humana were studied by electron microscopy. The complete delimiting membrane consists of a double unit membrane system. It originates from tubular elements of 70–100 nm diameter which are formed with a thin membrane 5–6 nm thick and filled with electron translucent materials. The process of development of the delimiting membrane is as follows: Tubular elements first aggregate, and then become arranged in a plane. Soon afterwards, they fuse laterally and become transformed into a delimiting membrane. Spore delimitation starts from the distal end of the centriolar plaque and develops towards the opposite side of the nucleus, preceding the formation of the membrane system.
ULTRASTRUCTURAL STUDY OF THE PROCESS OF ROSETTE FORMATION BY DISSOCIATED NEURAL RETINAL CELLS IN STATIONARY CULTURE
Authors: FUJISAWA HAJIME
Published: January 2005

Well orientated two‐dimensional rosettes were produced in stationary cultures of dissociated neural retinal cells of 5 1/2‐day‐old chick embryos. Observations by electron microscopy were made on aggregates at the different steps in the process leading to rosette formation. Though all of the dissociated neural retinal cells showed a clear morphological polarity, primary cell‐to‐cell adhesion occurred at random with respect to the cellular polarity. A special junction of the zonula adhaerens type was found at the contact area between the “mitochondria‐containing portions” or their neighboring regions of both adjoining cells. During stationary culturing, the contact area between the cells gradually increased. Where the “mitochondria‐containing portions” of adjoining cells were brought into contact with each other, junctions of zonula adhaerens type were formed between them. In this way, a radial arrangement of all the cells within an aggregate was established, leading to the formation of the fundamental structures of rosettes.
DNA CONTENTS IN PURKINJE CELLS AND INNER GRANULE NEURONS IN THE DEVELOPING RAT CEREBELLUM
Authors: FUJITA SETSUYA , HATTORI TAKANORI , FUKUDA MASARU , KITAMURA TADAHISA
Published: January 2005

Cytophotometric studies revealed that the content of Feulgen DNA in Purkinje cells in the developing rat cerebellum remains at the diploid level throughout the postnatal life. No evidence was found to suggest resumption of DNA synthesis in the neurons. This finding is in accord with autoradiographic observations that neuroblasts once differentiated from matrix cells do not synthesize DNA nor divide again.
LOCATION AND ULTRASTRUCTURE OF PRIMORDIAL GERM CELLS (PGCs) IN AMBYSTOMA MEXICANUM
Authors: IKENISHI KOHJI , NIEUWKOOP PIETER D.
Published: January 2005

The location and ultrastructure of the primordial germ cells (PGCs) were studied in Ambystoma mexicanum larvae of stages 23 to 47.

PGCs were found in the spaces between the endodermal cell mass and the lateral plate mesoderm at stages 23 to 35. Some of the PGCs at stage 35, and most of them at stages 40 and 42, were located near the Wolffian duct. At stages 46 and 47 all the PGCs were situated in the genital ridges. Cilia, which have hitherto never been reported in PGCs, were occasionally seen in PGCs of Ambystoma from stage 23 till stage 46.

No “germinal plasm” was found in the PGCs prior to stage 40. Specific structures or “nuage material”, corresponding to the germinal granules or their derivatives in Xenopus, were first recognized in the vicinity of the nucleus at stage 40. Between stages 40 and 46, the amount of “nuage material” markedly increased. It was finally localized mainly in “intermitochondrial spaces”. A possible transfer of material from the nucleus to the cytoplasm or vice versa through nuclear pores was first noticed at stage 40, the material concerned being quite similar in ultrastructure to the “nuage material”.
TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS
Authors: REMINGTON J. A. , FLICKINGER R. A.
Published: January 2005

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC‐rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.
ACETYLCHOLINE AND ACETYLCHOLINESTERASE ACTIVITY IN EARLY EMBRYOS OF THE MEDAKA ORYZIAS LATIPES, A TELEOST
Authors: FLUCK RICHARD A.
Published: January 2005

Acetylcholinesterase (AChE) activity is present in homogenates of medaka embryos during cleavage and epiboly. The levels of AChE activity change little during this period of development and are similar in embryos grown at either 15°C or 25°C. The specific activity of AChE in cells isolated from blastulae is 0.06 mmoles substrate hydrolyzed/min/g protein, a value comparable to that of chicken myoblasts and myotubes in vitro. Acetylcholinesterase activity was detected cytochemically in all cells dissociated from blastulae and gastrulae. In deep blastomeres AChE activity is present nearly throughout the cytoplasm; it is absent from peripheral regions of the cytoplasm which are involved in circus, or limnicolor, movements. Acetylcholine‐like activity in extracts of the embryos was assayed using the clam heart ventricle bioassay. The active principle, which caused a decrease in both the frequency and magnitude of ventricular contractions, was inactivated by heating at pH 10 and by incubation at pH 7.0 with commercial AChE. The effect of the active principle on the clam heart was blocked by mytolon chloride, a drug which specifically blocks the effect of acetylcholine on the clam heart. The active principle migrated on Whatman #1 chromatography paper with the same Rf as authentic acetylcholine in three solvent systems. The amount of acetylcholine in blastulae is about 4 picomoles/embryo.
DEFORMATION MOVEMENT INDUCED BY DIVALENT IONOPHORE A23187 IN THE TUBIFEX EGG
Authors: SHIMIZU TAKASHI
Published: January 2005

Deformation movement (groove‐forming activity of the equatorial surface) was induced in the spherical Tubifex egg by treatment with ionophore A23187 (5–10 μg/ml). The degree of induced movement was maximal when the treatment was commenced at the beginning of the round stages, i.e., immediately after disappearance of the grooves formed in the preceding normal deformation movement; it gradually decreased as the onset of the treatment was delayed. This induction was independent of either external Ca2+ or Mg2+. Ultrastructural examination of eggs undergoing the induced movement revealed the presence of organized microfilaments in the cortical layer of the grooving regions. It is assumed that the absence of contractile activity during the round stages of the Tubifex egg is attributed basically to the low availability of intracellularly stored Ca2+. The results also suggest that the distribution and/or the amount of the contractile elements change during these stages.