FEBS J, 2005 Jan, 272(2), 327 - 40 Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity; Seydel U et al.; Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system . Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506) . Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition . The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups . We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A . To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties . We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively . In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-d-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed . These data allow a better understanding of endotoxic activity and its structural prerequisites. J Pediatr Hematol Oncol, 2005 Jan, 27(1), 37 - 38 Two Cases of Ralstonia pickettii Bacteremias in a Pediatric Oncology Unit Requiring Removal of the Port-A-Caths; Kismet E et al.; Ralstonia pickettii is an aerobic, gram-negative bacterium causing bacteremia following the use of contaminated saline vials, respiratory therapy solutions, skin disinfectants, blood culture mediums, and water supplies . It is rarely associated with human infections . The authors report two cases of R . pickettii bacteremia in patients with Port-A-Caths that could be treated only by removal of the ports. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 479 - 486 Brevundimonas mediterranea sp . nov., a non-stalked species from the Mediterranean Sea; Fritz I et al.; Six strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from the Mediterranean Sea . 16S rRNA gene sequence analysis indicated that the strains were affiliated within the alphaproteobacterial genus Brevundimonas, with Brevundimonas intermedia (99.4 %) and Brevundimonas vesicularis (99.2 %) as their closest relatives . This affiliation was supported by chemotaxonomic data (major polar lipids: phosphatidyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidyl glucopyranosyl diacylglycerol; major fatty acids: C(18 : 1), C(16 : 0), C(16 : 1), C(15 : 0), C(17 : 1)omega8c, 11-Me-C(18 : 1)omega5t) . The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strains from all recognized Brevundimonas species . The strains therefore represent a novel species, for which the name Brevundimonas mediterranea sp . nov . is proposed, with the type strain V4.BO.10(T) (=LMG 21911(T)=CIP 107934(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 423 - 6 Chryseobacterium formosense sp . nov., isolated from the rhizosphere of Lactuca sativa L . (garden lettuce); Young CC et al.; A yellow-pigmented bacterial strain (CC-H3-2(T)), isolated from the rhizosphere of Lactuca sativa L . (garden lettuce) in Taiwan, was investigated using a polyphasic taxonomic approach . The cells were Gram-negative, rod-shaped and non-spore-forming . Phylogenetic analyses using the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Chryseobacterium, with the highest sequence similarity to the type strains of Chryseobacterium indoltheticum (97.7 %), Chryseobacterium scophthalmum (97.5 %), Chryseobacterium joostei (97.2 %) and Chryseobacterium defluvii (97.2 %) . The major whole-cell fatty acids were iso-C(15 : 0) (52.2 %) and iso-C(17 : 0) 3-OH . DNA-DNA hybridization experiments revealed levels of only 27.4 % to C . scophthalmum, 27.1 % to C . indoltheticum, 14.1 % to C . joostei and 7.8 % to C . defluvii . DNA-DNA relatedness and biochemical and chemotaxonomic properties demonstrate that strain CC-H3-2 (T) represents a novel species, for which the name Chryseobacterium formosense sp . nov . is proposed . The type strain is CC-H3-2(T) (=CCUG 49271(T)=CIP 108367(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 309 - 313 Marinomonas ushuaiensis sp . nov., isolated from coastal sea water in Ushuaia, Argentina, sub-Antarctica; Prabagaran SR et al.; A Gram-negative, rod-shaped, psychrophilic, motile, non-spore-forming bacterium, strain U1(T), was isolated from Ushuaia located at the southernmost tip of Argentina . On the basis of 16S rRNA gene sequence similarity, strain U1(T) was found to be closely related to Marinomonas communis (DSM 5604(T)) and Marinomonas primoryensis (IAM 15010(T)) . At the DNA-DNA level, however, the values for similarity were 41 and 25 %, respectively . The major fatty acids present were iso-C(16 : 0), C(16 : 1)omega7c, iso-C(17 : 1) and C(18 : 1)omega7c and the G+C content of the DNA was 43.6 mol% . All of the above characteristics support the affiliation of strain U1(T) to the genus Marinomonas . Furthermore, on the basis of phenotypic features, chemotaxonomic characteristics and phylogenetic analysis of the 16S rRNA gene sequence, it appears that strain U1(T) is distinct from the four Marinomonas species with validly published names . Strain U1(T), therefore, represents a novel species, for which the name Marinomonas ushuaiensis sp . nov . is proposed . The type strain of M . ushuaiensis is U1(T) (=MTCC 6143(T)=DSM 15871(T)=JCM 12170(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 275 - 9 Marinomonas pontica sp . nov., isolated from the Black Sea; Ivanova EP et al.; A Gram-negative, polarly flagellated bacterium was isolated from a sea-water sample collected from the Karadag Natural Reserve of the Eastern Crimea and characterized to clarify its taxonomic position . 16S rRNA gene sequence-based phylogenetic analysis of this novel organism revealed Marinomonas vaga, Marinomonas communis, Marinomonas mediterranea, Marinomonas primoryensis and 'Marinomonas protea' as its closest relatives (similarity 95-97 %) . The G+C content of the DNA was 46.5 mol% . The organism grew between 4 and 33 degrees C, tolerated 10 % NaCl, was slightly alkaliphilic and was not able to degrade starch, gelatin, agar or Tween 80 . Phosphatidylethanolamine (53.4 %) and phosphatidylglycerol (46.6 %) were the predominant phospholipids . The major fatty acids were 16 : 0 (15.5 %), 16 : 1omega7 (26.7 %) and 18 : 1omega7 (47.1 %) . The phylogenetic, genetic and physiological properties of the organism placed it within a novel species, proposed as Marinomonas pontica sp . nov., the type strain of which is 46-16(T) (=LMG 22531(T)=KMM 3492(T)=UCM 11075(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 235 - 8 Salegentibacter mishustinae sp . nov., isolated from the sea urchin Strongylocentrotus intermedius; Nedashkovskaya OI et al.; A bacterial strain, designated KMM 6049(T), was isolated from the sea urchin Strongylocentrotus intermedius inhabiting the Sea of Japan . The bacterium studied was strictly aerobic, heterotrophic, yellow-pigmented, non-motile, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . 16S rRNA gene sequence analysis indicated that strain KMM 3524(T) was closely related to Salegentibacter holothuriorum and Salegentibacter salegens (sharing 97.7 and 98 % sequence similarity, respectively) . DNA-DNA relatedness levels between strains KMM 6049(T) and S . holothuriorum KMM 3524(T) and S . salegens DSM 5424(T) were 24 and 45 %, respectively, indicating that KMM 6049(T) belongs to a novel species of the genus Salegentibacter, for which the name Salegentibacter mishustinae sp . nov . is proposed . The type strain is KMM 6049(T) (=KCTC 12263(T)=LMG 22584(T)=NBRC 100592(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 231 - 4 Roseivirga ehrenbergii gen . nov., sp . nov., a novel marine bacterium of the phylum 'Bacteroidetes', isolated from the green alga Ulva fenestrata; Nedashkovskaya OI et al.; The taxonomic position of a novel marine bacterium isolated from the green alga Ulva fenestrata collected in the Sea of Japan was established . Cells of the strain studied, designated KMM 6017(T), were strictly aerobic, heterotrophic, pink-pigmented, non-motile by gliding, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . 16S rRNA gene sequence analysis indicated that the strain occupied a distinct lineage within the phylum 'Bacteroidetes' and formed a cluster with {Flexibacter} tractuosus and Reichenbachia agariperforans . The G+C content of the DNA of KMM 6017(T) was 40.2 mol% . The major respiratory quinone was MK-7 . The predominant fatty acids were i15 : 1, i15 : 0 and i17 : 0 3-OH (34.2, 24 and 7.7 %, respectively) . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium was assigned to the genus Roseivirga gen . nov., as Roseivirga ehrenbergii gen . nov., sp . nov . The type strain is KMM 6017(T) (=KCTC 12282(T)=LMG 22567(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 133 - 8 Chryseobacterium daecheongense sp . nov., isolated from freshwater lake sediment; Kim KK et al.; A novel nitrate-reducing bacterium, CPW406(T), was isolated from the sediment of a shallow, freshwater lake . The strain was a Gram-negative, non-motile, non-spore-forming rod, which formed yellow-pigmented colonies on nutrient agar and contained a polyamine pattern with sym-homospermidine as the major compound, MK-6 as the predominant menaquinone, 15 : 0 iso and 17 : 0 iso 3-OH as the major fatty acids and phosphatidylethanolamine and several unknown lipids in the polar lipid profile . The 16S rRNA gene sequence of strain CPW406(T) was found to be most similar to that of the type strain of Chryseobacterium defluvii (DSM 14219(T); 97.9 % similarity) . However, DNA-DNA relatedness data and its phenotypic properties showed that strain CPW406(T) could be distinguished from all known Chryseobacterium species and thus represented a novel species, for which the name Chryseobacterium daecheongense sp . nov . is proposed; the type strain is CPW406(T) (=DSM 15235(T)=KCTC 12088(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 41 - 7 Roseisalinus antarcticus gen . nov., sp . nov., a novel aerobic bacteriochlorophyll a-producing {alpha}-proteobacterium isolated from hypersaline Ekho Lake, Antarctica; Labrenz M et al.; A Gram-negative, aerobic to microaerophilic rod was isolated from 10 m depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica) . The strain was oxidase- and catalase-positive, metabolized a variety of carboxylic acids and sugars and produced lipase . Cells had an absolute requirement for artificial sea water, which could not be replaced by NaCl . A large in vivo absorption band at 870 nm indicated production of bacteriochlorophyll a . The predominant fatty acids of this organism were 16 : 0 and 18 : 1omega7c, with 3-OH 10 : 0, 16 : 1omega7c and 18 : 0 in lower amounts . The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine . Ubiquinone 10 was produced . The DNA G+C content was 67 mol% . 16S rRNA gene sequence comparisons indicated that the isolate represents a member of the Roseobacter clade within the alpha-Proteobacteria . The organism showed no particular relationship to any members of this clade but clustered on the periphery of the genera Jannaschia, Octadecabacter and 'Marinosulfonomonas' and the species Ruegeria gelatinovorans . Distinct morphological, physiological and genotypic differences to these previously described taxa supported the description of a new genus and a novel species, for which the name Roseisalinus antarcticus gen . nov., sp . nov . is proposed . The type strain is EL-88(T) (=DSM 11466(T)=CECT 7023(T)). FEMS Microbiol Rev, 2005 Jan, 29(1), 83 - 98 Enteropathogenic Escherichia coli: unravelling pathogenesis; Deborah Chen H et al.; Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to intestinal epithelial cells, causing diarrhoea . It constitutes a significant risk to human health and remains an important cause of infant mortality in developing countries . Although EPEC was the first E . coli strain to be implicated in human disease in the 1940s and 1950s, the mechanisms by which this pathogen induced diarrhoea remained a complete mystery throughout most of the 40 years since its description . It was only during the late 1980s that major advances were made in unravelling the mechanisms behind EPEC pathogenesis . Ever since, progress has been made at a stunning pace and there have been major breakthroughs in identifying the bacterial factors involved in attaching and effacing (A/E) lesion formation, host signal transduction pathways in response to EPEC infection and the genetic basis of EPEC pathogenesis . The rapid pace of discovery is a result of intensive research by investigators in this field and portends that EPEC will soon be among one of the most understood diarrhoea-causing infectious agents . This review aims to trace the progress of EPEC research since its existence was first reported by John Bray in 1945, highlighting the major findings that have revolutionised our understanding of EPEC pathogenesis. J Microbiol Methods, 2005 Mar, 60(3), 383 - 93 Isolation of Brucella abortus total RNA from B . abortus-infected murine RAW macrophages; Covert J et al.; Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages . However, the intracellular survival mechanisms used by Brucella are not fully understood . Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events . We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion . This method takes advantage of the B . abortus cell envelope 1that protects bacterial RNA and DNA . The cell envelope of B . abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B . abortus RNA . In our experiments, 4.5 mug of RNA was routinely isolated from 1 ml bacterial culture and 2-9 mug of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination . The method is rapid and uses inexpensive, commonly available reagents . Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis. BMC Bioinformatics . 2005 Jan 12;6(1):7 {Epub ahead of print} Evaluation of methods for predicting the topology of beta-barrel outer membrane proteins and a consensus prediction method; Bagos PG et al.; BACKGROUND: Prediction of the transmembrane strands and topology of beta-barrel outer membrane proteins is of interest in current bioinformatics research . Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist . The methods can be grossly divided to those based on Hidden Markov Models (HMMs), on Neural Networks (NNs) and on Support Vector Machines (SVMs) . In this work, we compare the different available methods for topology prediction of beta-barrel outer membrane proteins . We evaluate their performance on a non-redundant dataset of 20 beta-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution . Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method . RESULTS: We assess the statistical significance of the performance of each prediction scheme and conclude that Hidden Markov Model based methods, HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according to either the per-residue accuracy, the segments overlap measure (SOV) or the total number of proteins with correctly predicted topologies in the test set . Furthermore, we show that the available predictors perform better when only transmembrane beta-barrel domains are used for prediction, rather than the precursor full-length sequences, even though the HMM-based predictors are not influenced significantly . The consensus prediction method performs significantly better than each individual available predictor, since it increases the accuracy up to 4% regarding SOV and up to 15% in correctly predicted topologies . CONCLUSIONS: The consensus prediction method described in this work, optimizes the predicted topology with a dynamic programming algorithm and is implemented in a web-based application freely available to non-commercial users at http://bioinformatics.biol.uoa.gr/ConBBPRED. Crit Rev Microbiol, 2004, 30(4), 275 - 86 Autotransporter and two-partner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens; Newman CL et al.; A number of protein secretion mechanisms have been identified in gram-negative pathogens . Many of these secretion systems are dependent upon the Sec translocase for protein export from the cytoplasm into the periplasm and then utilize other mechanisms for transport from the periplasm through the outer membrane . In this article, we review secretion similarities between autotransporter and two-partner secretion systems, and we report similarities between the autotransporter secretion mechanism with that of intimin/invasins . Considering that many secreted proteins are virulence factors, a better understanding of their secretion mechanisms will aid in the development of disease treatments and new bacterial vaccines. J Biol Chem . 2005 Jan 10; {Epub ahead of print} Crystal structure of CD14 and its implications for lipopolysaccharide signaling; Kim JI et al.; Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome . The core receptors recognizing lipopolysaccharide are CD14, TLR4 and MD-2 . CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling . In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products . Here, we present the first crystal structure of CD14 to 2.5 A resolution . A large hydrophobic pocket was found on the N-terminal side of the horseshoe-like structure . Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide binding site . Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket . Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket and the multiplicity of grooves available for ligand binding. J Mol Biol, 2005 Feb 4, 345(5), 1185 - 97 Epub 2004 Dec 15. The Solution Structure of the C-terminal Domain of TonB and Interaction Studies with TonB Box Peptides; Sean Peacock R et al.; The TonB protein transduces energy from the proton gradient across the cytoplasmic membrane of Gram-negative bacteria to TonB-dependent outer membrane receptors . It is a critically important protein in iron uptake, and deletion of this protein is known to decrease virulence of bacteria in animal models . This system has been used for Trojan horse antibiotic delivery . Here, we describe the high-resolution solution structure of Escherichia coli TonB residues 103-239 (TonB-CTD) . TonB-CTD is monomeric with an unstructured N terminus (103-151) and a well structured C terminus (152-239) . The structure contains a four-stranded antiparallel beta-sheet packed against two alpha-helices and an extended strand in a configuration homologous to the C-terminal domain of the TolA protein . Chemical shift perturbations to the TonB-CTD (1)H-(15)N HSCQ spectrum titrated with TonB box peptides modeled from the E.coli FhuA, FepA and BtuB proteins were all equivalent, indicating that all three peptides bind to the same region of TonB . Isothermal titration calorimetry measurements demonstrate that TonB-CTD interacts with the FhuA-derived peptide with a K(D)=36(+/-7)muM . On the basis of chemical shift data, the position of Gln160, and comparison to the TolA gp3 N1 complex crystal structure, we propose that the TonB box binds to TonB-CTD along the beta3-strand. Lett Appl Microbiol, 2005, 40(2), 146 - 51 Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field; Berg J et al.; Abstract j . berg, a . tom-petersen and o . nybroe . 2004.Aims: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria . Methods and Results: Soil bacteria were isolated from untreated and Cu-amended field plots . Cu-amendment significantly increased the frequency of Cu-resistant isolates . A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics . More than 95% of the Cu-resistant isolates were Gram-negative . Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (>/=3) antibiotics than Cu-sensitive Gram-negative isolates . Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (>/=2) antibiotics than corresponding isolates from control plots . Significance and Impact of the Study: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria . Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance. Cardiovasc Res, 2005 Feb 1, 65(2), 317 - 27 Chlamydia pneumoniae infections in mouse models: relevance for atherosclerosis research; de Kruif MD et al.; Mouse models have been frequently used in the study of Chlamydia pneumoniae (also known as Chlamydophila pneumoniae) infections . This gram-negative obligate intracellular bacterium causes respiratory infections, followed by dissemination of the bacterium to various organs throughout the body, including cardiovascular tissues, supporting the current hypothesis of a relationship between C . pneumoniae and atherosclerosis . Recently, clinical trials evaluated the effect of antichlamydial antibiotics on secondary cardiovascular events . Although small studies showed some effect, the large WIZARD study did not confirm these results, and the role of antichlamydial antibiotics in prevention of secondary events was questioned . To address these issues, data obtained from mouse models were systematically reviewed here . C . pneumoniae infections showed atherogenic properties in mice that were reproducible and confirmed by different research groups . However, antibiotic therapy was of limited value in these mouse models . Antibiotic therapy effectively cleared the acute infection, but did not influence the atherogenic properties of C . pneumoniae unless the therapy was started early during the acute infection . The results summarized here may help to better understand the results of the clinical antibiotic trials. J Mol Evol, 2004 Oct, 59(4), 498 - 506 Molecular evolution of daphnia immunity genes: polymorphism in a gram-negative binding protein gene and an alpha-2-macroglobulin gene; Little TJ et al.; Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection . The genes of the arthropod immune system remain comparatively unstudied . We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity . For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation . For another gene, encoding a putative alpha-2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host-parasite arms race . We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution. J Biol Chem . 2005 Jan 4; {Epub ahead of print} Identification of a new membrane-associated protein which influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis; Sato K et al.; The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of the organisms . The organisms have developed several systems for transport across the inner and outer membranes . The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors . Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB and hagA on the chromosome . In this study, we isolated a P . gingivalis mutant (porT) which showed very weak activities of gingipains in the cell lysates and culture supernatants . Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells . Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm . The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum . These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured. Yi Chuan Xue Bao, 2004 Dec, 31(12), 1448 - 54 {Sequence of Escherichia coli O141 O-antigen gene cluster and analyses of its evolutionary history}; Kong QK et al.; Lipopolysaccharide (LPS) is one of the major components of the outer membrane of gram-negative bacteria . It is an amphipathic molecule compose of lipid A, a core oligosaccharide and an O-specific antigen . O-antigen, which is a repeat-unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface . The genes of O-antigen gene cluster are responsible for the synthesis of the O-antigen . The O-antigen gene cluster of E . coli O141 was sequenced and found to contain the genes rmlBDAC and manBC for the biosynthesis of nucleotide sugars dTDP-rhamnose and GDP-mannose, respectively, encoding genes for Ounit flippase (wzx), O-antigen polymerase (wzy) and potential transferase genes . The possible biosynthesis pathway for O-antigen of E . coli O141 was proposed . Two genes specific to E . coli O141 were identified . This work provides the basis for a sensitive test by PCR for the rapid detection of E . coli O141 . Phylogenetic trees for the rmlB, rmlD, rmlA, and rmlC genes and manB, manC genes were generated and the comparisons were made among different strains . We find that these genes are typical E . coli genes and might have been involved in recombination events between O-antigen gene clusters. Microbiology, 2005 Jan, 151(Pt 1), 259 - 68 Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA; Baumgarth B et al.; The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa . The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions . ExpG is a member of the MarR family of regulatory proteins . Analysis of target sequences of an ExpG(His)(6) fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs . Association and dissociation kinetics of the specific binding of ExpG(His)(6) to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM) . Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction. Eur J Pediatr Surg, 2004 Dec, 14(6), 418 - 21 {In Process Citation}; Ameh EA et al.; BACKGROUND: Fournier's gangrene is uncommon in childhood and little is known about the disease in this age group . METHOD: A retrospective review was carried out of neonates and infants treated for Fournier's gangrene over a period of 16 years . RESULTS: Twelve neonates and infants aged 5 days - 3 months (median 3 weeks) were treated in our hospital . The precipitating cause was omphalitis in 7 babies, strangulated inguinal hernia in 2 and in 3 babies there was no identifiable cause . Gram-negative bacteria were cultured in 3 patients, but in most the culture was sterile . Treatment consisted of debridement of devitalised tissue and administration of broad-spectrum antibiotics . Primary closure was achieved in 1 baby and secondary closure in 2 others . In 7 babies the wound contracted rapidly and healed . There was no mortality . CONCLUSION: Fournier's gangrene in neonates and infants in our environment is largely preventable . Early debridement and appropriate antibiotics give good results. East Afr Med J, 2004 Sep, 81(9), 474 - 9 Clinical and laboratory features of spontaneous bacterial peritonitis; Filik L et al.; BACKGROUND: Spontaneous Bacterial Peritonitis (SBP) is a complication of cirrhosis . The mortality rate is approximately 30-50% . SBP is defined as an ascitic fluid infection in the absence of any obvious intraabdominal infectious foci . While earlier reports of SBP emphasized high mortality rates, recently lower mortality rates have been reported . OBJECTIVE: To evaluate the clinical and laboratory features and prognostic indicators of SBP . DESIGN: Retrospective study . SETTING: Hacettepe University Hospitals . SUBJECTS: A total of 281 SBP episodes of 214 patients between 3rd march 1981 and 3rd August 1999, in Hacettepe University Hospital were evaluated . Statistical analysis was performed in the group of patients having chronic liver diseases . RESULTS: One hundred and forty nine of the patients 214 (69.6%) were males and 65(30.4%) were females . The mean age of all patients were 49.91+/-15.01 years (17 to 90 years) . All spontaneous ascites infection episodes were symptomatic . In all of the episodes, most common clinical features were as follows: icterus (54.5%), abdominal tenderness (54.5%), hepatic encephalopathy (50.7%), fatigue (46.7%), abdominal pain (44.4%) and fever (38.8%) . The culture of the ascitic fluid resulted in isolation of a bacteria in 25.4% of all episodes . The most frequently isolated microorganisms turned out to be gram negative enteric bacterias (76.2%) . Sixty seven patients in 179 cases with liver disease passed away (37.4%) . The use of cefotaxime and newer cephalosporins seemed to have less mortality (31.7%) as compared with that (42.2%) observed in patients treated with other antibiotic regimens . CONCLUSIONS: Of all the factors analysed in patients with chronic liver diseases, being Child-Pugh class C, having fatigue, hepatic encephalopathy, hypotension, higher peripheral blood leukocyte count (> or =12000/mm3), renal dysfunction (serum creatinine level > or = 2mg/dl), longer prothrombin time (INR > or = 2.5), lower ascites protein level (<Igr/ dl) and, liver disease for longer time, developing superinfection (an infection other than SBP starting during SBP treatment) were statistically correlated with a higher death rate (p<0.05). Invest Ophthalmol Vis Sci, 2005 Jan, 46(1), 114 - 20 Lipopolysaccharide-induced expression of intercellular adhesion molecule-1 and chemokines in cultured human corneal fibroblasts; Kumagai N et al.; PURPOSE: Invasion of bacteria into the corneal stroma induces the infiltration of leukocytes and subsequent corneal ulceration . The role of corneal fibroblasts in the detection of bacterial invasion into the stroma was investigated by examining the in vitro expression of the receptor complex for lipopolysaccharide (LPS), a common component of Gram-negative bacteria, as well as the possible effects of LPS on both the expression of adhesion molecules and the release of chemokines in cultured human corneal fibroblasts . METHODS: Expression of the LPS receptor complex, intercellular adhesion molecule (ICAM)-1, and the chemokines interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 was examined by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, or immunofluorescence analysis . RESULTS: Corneal fibroblasts were found to contain transcripts encoding toll-like receptor-4, CD14, and MD-2, all of which are components of the LPS receptor complex . The expression of ICAM-1 at the surface of corneal fibroblasts and the amount of ICAM-1 mRNA in the cells were both increased by LPS . Similarly, LPS increased both the release of IL-8 and MCP-1 by corneal fibroblasts as well as the amounts of the corresponding mRNAs in these cells . These various effects of LPS were potentiated by the presence of a low concentration of human serum . CONCLUSIONS: Corneal fibroblasts may play an important role in the defense system of the cornea by recognizing the presence of LPS and subsequently expressing adhesion molecules and chemokines that promote leukocyte infiltration. Nat Struct Mol Biol . 2004 Dec 26; {Epub ahead of print} Structural characterization of a type III secretion system filament protein in complex with its chaperone; Yip CK et al.; The type III secretion system (TTSS) mediates the specific translocation of bacterial proteins into the cytoplasm of eukaryotic cells, a process essential for the virulence of many Gram-negative pathogens . The enteropathogenic Escherichia coli TTSS protein EspA forms a hollow extracellular filament believed to be a molecular conduit for type III protein translocation . Structural analysis of EspA has been hampered by its polymeric nature . We show that EspA alone is sufficient to form filamentous structures in the absence of other pathogenicity island-encoded proteins . CesA is the recently proposed chaperone of EspA, and we demonstrate that CesA traps EspA in a monomeric state and inhibits its polymerization . Crystallographic analysis of the heterodimeric CesA-EspA complex at a resolution of 2.8 A reveals that EspA contains two long a-helices, which are involved in extensive coiled-coil interactions with CesA. Infect Immun, 2005 Jan, 73(1), 352 - 61 Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila; Akamine M et al.; The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice . Intracellular growth of L . pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages . There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice . Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L . pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages . Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L . pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain . There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria . An L . pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages . Treatment of L . pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production . Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L . pneumophila, although other TLRs may also contribute to innate immunity against this organism. Infect Immun, 2005 Jan, 73(1), 62 - 9 An immunoreactive 38-kilodalton protein of Ehrlichia canis shares structural homology and iron-binding capacity with the ferric ion-binding protein family; Doyle CK et al.; Ehrlichiae are tick-transmitted, gram-negative, obligately intracellular bacteria that live and replicate in cytoplasmic vacuoles, but little is known about iron acquisition mechanisms necessary for their survival . In this study, a genus-conserved immunoreactive ferric ion-binding protein (Fbp) of Ehrlichia canis was identified and its iron-binding capability was investigated . E . canis Fbp was homologous to a family of periplasmic Fbp's involved in iron acquisition and transport in gram-negative bacteria . E . canis Fbp had a molecular mass (38 kDa) consistent with those of Fbp's in other bacteria and exhibited substantial immunoreactivity in its native conformation . The predicted three-dimensional structure of E . canis Fbp demonstrated conservation of important Fbp family structural motifs: two domains linked with a polypeptide "hinge" region . Under iron-binding conditions, the recombinant Fbp exhibited an intense red color and an absorbance spectrum indicative of iron binding, and it bound Fe(III) but not Fe(II) . Fbp was observed primarily in the cytoplasm of the reticulate forms of E . canis and Ehrlichia chaffeensis but was notably found on extracellular morula fibers in morulae containing dense-cored organisms . Although expression of Fbp is regulated through an operon of three functionally linked genes in other gram-negative bacteria, the absence of an intact fbp operon in Ehrlichia spp . suggests that genes involved in ehrlichial iron acquisition have been subject to reductive evolution. J Periodontal Res, 2005 Feb, 40(1), 28 - 35 Chronic treatment with the glutamate receptor antagonist MK-801 alters periodontal disease susceptibility; Breivik T et al.; Breivik T, Gundersen Y, Osmundsen H, Opstad PK, Fonnum F . Chronic treatment with the glutamate receptor antagonist MK-801 alters periodontal disease susceptibility . J Periodont Res 2004; doi: 10.1111/j.1600-0765.2004.00765.x (c) Blackwell Munksgaard 2004Objective: Previous experiments in rats suggest that hypothalamic-pituitary-adrenal (HPA) axis over-responsiveness, which leads to increased secretion of immunoregulatory glucocorticoid hormones, increases periodontal disease susceptibility, whereas HPA axis under-responsiveness is associated with increased resistance to the disease . The present study was designed to investigate whether MK-801 (dizocilipine malate), an antagonist of the glutamate receptor N-methyl-d-aspartate (NMDA) in the brain, which has been found to play an important role in the regulation of the HPA axis, would influence the outcome of experimental ligature-induced periodontal disease in a rat model . Methods: Experimental periodontal disease was induced in periodontal disease susceptible and HPA axis high-responding Fischer 344 rats 2 days before chronic treatment with MK-801(1 mg/kg intraperitoneally) . The periodontal breakdown was assessed after the ligatures had been in place for 23 days . Following intraperitoneal Gram-negative bacterial lipopolysaccharide stimulation (Escherichia coli, 250 microg/kg), concentrations of glucocorticoid receptors (GRs) in the hippocampus, and levels of the cytokine tumour necrosis factor alpha (TNF-alpha), as well as the HPA axis-derived hormone corticosterone, were measured in serum . Results: Compared to vehicle-treated controls, MK-801-treated rats had significantly increased periodontal tissue destruction (p < 0.01) . MK-801-treated rats also showed significantly increased expression of GRs in the hippocampus (p < 0.05), elevated levels of corticosterone (p < 0.001) and reduced levels of TNF-alpha (p < 0.01) in serum 2 h after lipopolysaccharide stimulation . Conclusion: These findings may implicate glutamate receptor-dependent mechanisms in periodontal disease, and support the concept of a bidirectional immune-brain-immune regulatory network with importance for periodontal health and disease. Oral Microbiol Immunol, 2005 Feb, 20(1), 1 - 9 A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis; Yamane K et al.; Yamane K, Yamanaka T, Yamamoto N, Furukawa T, Fukushima H, Walker CB, Leung K-P . A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis . Oral Microbiol Immunol 2005: 20: 1-9 . (c) Blackwell Munksgaard, 2005 . Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions . We have isolated an exopolysaccharide-producing P . nigrescens, strain 22, from a chronic periodontitis lesion . The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate . The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis . To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established . The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes . Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 mug/mg), glucose (25.6 mug/mg), fructose (65.8 mug/mg), galactose (12.5 mug/mg), arabinose (6.2 mug/mg), xylose (3.2 mug/mg), rhamnose (6.1 mug/mg), and ribose (0.6 mug/mg) . Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3) . Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328 . The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328 . Exopolysaccharide alone failed to induce abscess formation in mice . Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors . The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated. J Physiol Pharmacol, 2004 Jul, 55 Suppl 2, 105 - 15 Association of the presence the Helicobacter pylori in the oral cavity and in the stomach; Czesnikiewicz-Guzik M et al.; Helicobacter pylori is a gram-negative, microaerophilic rod-shaped bacteria that lives beneath the gastric mucous layer, on the surface of epithelial cells . Stomach infection with this organism causes inflammation of the gastric mucosa, which can lead to gastritis, duodenal or gastric ulcer and even in rare cases to gastric carcinoma or MALT lymphoma . Approximately 50% of the world's population is believed to be infected with H . pylori . Most infections is probably acquired in childhood, but the exact route of transmission is unknown . It has been speculated that dental plaque might harbour Helicobacter pylori and, therefore, might be a source of gastric infection . In order to address this issue we studied the relationships between oral and gastric infections with H . pylori in 100 subjects . Methods: Gastric H . pylori infection was determined by (13)C-urea breath test (UBT) and the presence of the bacteria in oral cavity was monitored by the culture from the saliva and from dental plaque . Results: H . pylori was found in the stomach in 51% of studied individuals, while oral H . pylori was found in 54% (in saliva) and in 48.3% (in gingival pockets), the difference was not statistical significant (p=NS) . Interestingly, anti-Hp IgA was found in 84% of studied individuals . No relationship was found between the presence of the bacteria in the oral cavity and the H . pylori gastric infection . 54.9% of subjects with stomach infection showed concomitant presence of H . pylori in saliva . 52.3% of examined subjects with negative UBT-test revealed the presence of H . pylori in culture from the saliva . The X(2) value of relationship between UBT and culture H pylori in saliva was 0.029 (p=0.9) . Similarly, no relationship was found between the presence of H . pylori in the stomach and in the dental plaque (X2=0.6); p=0.4) . As expected, the presence of H . pylori in the dental plaque was significantly correlated with the presence of bacteria in the saliva (X2=18.4; p=0.0002) . We also compared the presence of H . pylori in the saliva of patients with and without teeth . The cultured H . pylori was found in 63.7% of patients without teeth and in 52.9% of patients with teeth . This indicates that the presence of teeth does not seem to affect the occurence of H . pylori in saliva . We conclude that oral activity contamination with of H . pylori occurs at similar degree to that in the stomach . However, there was no significant correlation between the occurence of H . pylori in the stomach and in the oral cavity indicating that other factors, like susceptibility to infection due to acid environment in the stomach may be the major factor in gastric infection with that bacteria, while oral cavity may serve only as transient food-related contamination without clear relation to gastric infection. Expert Rev Vaccines, 2004 Dec, 3(6), 681 - 691 Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors; Walcher P et al.; The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties . BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties . The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells . The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity . Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously . Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA . Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells . There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response . All can be used for subunit antigen to design new vaccine candidates with particle presentation technology . In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens . Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy . The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies. Arch Gynecol Obstet . 2004 Dec 17; {Epub ahead of print} Chemoprophylactic and bactericidal efficacy of 80 mg gentamicin in a single and once-daily dosing; Sifakis S et al.; OBJECTIVE: The objective was to examine the biodistribution, the chemoprophylactic, and the bactericide efficacy of 80-mg gentamicin single or once-daily dosing.STUDY DESIGN: Ninety-six patients who had had cesarean section or gynecological surgery received 80 mg gentamicin for chemoprophylaxis . A second group of 92 patients with Gram-negative infection received once-daily 80-mg gentamicin intramuscularly, combined with cefoxitin or ceforanide, for 5 days . Gentamicin serum and tissue concentration was determined 1 h after the first administration.RESULTS: The chemoprophylactic efficacy of gentamicin was 93.7% . The treatment efficacy was high in patients with chorioamnionitis and endometritis (92.9%), moderate in those with wound infection (69.5%), and less effective in those with septicemia (55.6%) . Twenty-six percent of patients continued with antibiotics for infection control . The mean serum level was 4.48+/-0.49 and 5.56+/-0.66 mug/ml in obstetrical and gynecological patients respectively (p>0.05) . Serum levels >4 mug/ml were achieved in 91% of patients.CONCLUSIONS: A single dose of 80 mg gentamicin offers chemoprophylaxis and achieves therapeutic serum-concentrations 1 h after administration . The 5-day combination of once-daily 80 mg gentamicin with a second-generation cephalosporin is effective in patients with chorioamnionitis and endometritis, but only moderately effective in those with wound infections and septicemia. APMIS, 2004 Oct, 112(10), 674 - 85 Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin; Belibasakis GN et al.; The cytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species and causes growth arrest and morphological alterations in mammalian cells . Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells . The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A . actinomycetemcomitans, and to evaluate the involvement of its Cdt . Human gingival fibroblasts and periodontal ligament cells were challenged with A . actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry . Cells exposed to an A . actinomycetemcomitans wild-type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls . The cellular enlargement occurred in both G1 and G2/M arrested cells . In contrast, cells exposed to an A . actinomycetemcomitans cdt-knockout mutant strain showed cell cycle phase distribution and size similar to the controls . In conclusion, A . actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt. Mol Plant Microbe Interact, 2004 Dec, 17(12), 1328 - 36 Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato; Kang L et al.; Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell . Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive . Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions . Pseudomonas syringae pv . tomato T1 causes an HR-like cell death on Nicotiana benthamiana . This rapid cell death was delayed significantly in plants inoculated with P . syringae pv . tomato expressing avrPto . In addition, P . syringae pv . tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines . Transient expression of avrPto in both N . benthamiana and tomato prf3 plants also was able to suppress nonhost HR . Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens . AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions . Experiments with avrPto mutants revealed several residues important for the suppression effects . AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N . benthamiana . These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion. Protein Expr Purif, 2005 Jan, 39(1), 27 - 34 Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp . 22b; Cieslinski H et al.; The gram-negative antarctic bacterium Pseudoalteromonas sp . 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase . The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized . The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues . The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose . Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp . 22b beta-galactosidase . The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity . The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0 . PNPG was its preferred substrate (58% higher activity than against ONPG) . The enzyme was particularly thermolabile, losing all activities within 10min at 50 degrees C . The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6h at 30 degrees C and during 28h at 15 degrees C . Because of its attributes, the recombinant Pseudoalteromonas sp . 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products. Biochemistry, 2004 Dec 21, 43(50), 15767 - 74 SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) analysis of the thermodynamics of synergistic anion binding by ferric-binding protein (FbpA), a bacterial transferrin; Roulhac PL et al.; SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes . Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion) . The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion . Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior . However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA . As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)) . The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol) . The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value . This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process. Microbiol Mol Biol Rev, 2004 Dec, 68(4), 771 - 95 Process of protein transport by the type III secretion system; Ghosh P; The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells . The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells . While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation . This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport . The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum . It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane . Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells . A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS . Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells . Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry. Microbiol Mol Biol Rev, 2004 Dec, 68(4), 692 - 744 Type V protein secretion pathway: the autotransporter story; Henderson IR et al.; Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides . To overcome this barrier, bacteria have developed several systems for protein secretion . The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins . Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria . Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis . However, most of the autotransporter proteins remain to be characterized . In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function. J Med Microbiol, 2004 Dec, 53(Pt 12), 1187 - 93 Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4; Haralambieva IH et al.; Chlamydophila pneumoniae, an obligately intracellular Gram-negative bacterium and a common causative agent of respiratory tract infections, has been implicated in the induction and progression of atherosclerosis and coronary artery disease . In this study, the signalling mechanism of C . pneumoniae in human fibroblasts, a prominent cell population in chronic inflammation and persistent infection, contributing to plaque formation, was investigated . C . pneumoniae elementary bodies were demonstrated to up-regulate the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in human fibroblasts . The effect was independent of the chlamydial lipopolysaccharide and was likely to be mediated by a heat-labile chlamydial protein . Furthermore, an anti-Toll-like receptor 4 (TLR4) antibody was shown to abolish C . pneumoniae-induced cell activation, whereas an anti-TLR2 antibody had no effect, indicating the role of TLR4 in p44/p42 MAPK activation . Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 and phosphodiesterase 4 (PDE 4) inhibitor Rolipram enhanced C . pneumoniae-induced MAPK phosphorylation and attenuated C . pneumoniae infectivity in vitro . Together the results indicate that C . pneumoniae triggers rapid TLR4-mediated p44/p42 MAPK activation in human fibroblasts and chemical enhancement of MAPK phosphorylation modulates in vitro infection at the molecular level. Immunol Lett, 2005 Jan 15, 96(1), 73 - 83 Polycationic lipids inhibit the pro-inflammatory response to LPS; Leon-Ponte M et al.; Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria . As such, it signals monocytes, macrophages and neutrophils to up-regulate phagocytic functions and to release pro-inflammatory cytokines . Despite the established role of CD14 as the main LPS receptor, the precise nature of the LPS signalling complex and its compartmentalization remain unknown . Interactions of LPS with other cell surface molecules such as TLR-4 and MD-2, and its subsequent internalization are required for LPS signalling . Here, we show that the polycationic lipid LipoFectaminetrade mark causes inhibition of the LPS-induced MAPK activation and lack of pro-inflammatory cytokine production, despite proper localization of CD14 within lipid rafts and massive LPS internalization . The ability of LipoFectaminetrade mark to inhibit LPS induced pro-inflammatory responses may be due to uncoupling of CD14 from TLR-4/MD-2 in the LPS signalling complex of mouse macrophages/microglial cells, as suggested by inhibition of LPS-induced concomitant internalization of these surface molecules . Thus, LipoFectaminetrade mark may be a useful tool to dissect the molecular interactions leading to LPS signalling, and identifies a potential therapeutic strategy for LPS clearance. Comb Chem High Throughput Screen, 2004 Dec, 7(8), 733 - 47 Anti-endotoxin agents . 2 . Pilot high-throughput screening for novel lipopolysaccharide-recognizing motifs in small molecules; Wood SJ et al.; Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are an integral part of the outer leaflet of the outer-membrane of Gram-negative bacteria . Lipopolysaccharides play a pivotal role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient, worldwide . The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis . We have earlier shown that the pharmacophore necessary for small molecules to bind LPS is simple, comprising of two protonatable cationic functions separated by about 15 A, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin . In this report, we employ high-throughput screening methods, using a novel fluorescent probe displacement method . Searches in three-dimensional structure databases yielded about approximately 4000 commercially available small molecules, each possessing two cationic functions spaced approximately 15 A apart . Approximately 400 such compounds have been screened in an effort to validate the method by which high-affinity endotoxin binders can be identified . We show that the IC50 values that are obtained from the fluorescence-based primary screen are correlated both to the enthalpy of binding, as measured by isothermal titration calorimetry, as well as to biological potency in vitro assays . By performing rapid toxicity screens in tandem with the bioassays, lead compounds of interest can be easily identified for further systematic structural modifications and SAR studies. J Biol Chem . 2004 Dec 2; {Epub ahead of print} Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts; Tefsen B et al.; The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown . Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins . Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released . We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane . The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP) . We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes . In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts . Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane. Biotechnol Prog, 2004 Nov-Dec, 20(6), 1757 - 65 Effect of process variables on supercritical fluid disruption of Ralstonia eutropha cells for poly(R-hydroxybutyrate) recovery; Khosravi-Darani K et al.; This research focuses on the disruption of the gram-negative bacterium Ralstonia eutropha cells by supercritical CO2 for poly(R-hydroxybutyrate) (PHB) recovery . The variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical CO2 pressure, have been studied . Effect of this disruption technique on PHB molecular mass was also investigated . PHB recovery was examined using a combination of this method and chemical pretreatments . For salt pretreatment, the cells were exposed to 140 mM NaCl and heat (60 degrees C, 1 h) . The cells were also exposed to 0.2-0.8% (w/w) NaOH to examine the effect of alkaline pretreatment . Bacterial cells treated in growth phase exhibited less resistance to disruption than nutrient-limited cells in the stationary phase . It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells . Pretreatment with a minimum of 0.4% (w/w) NaOH was necessary to enable complete disruption with two times pressure release . Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock . The proposed method is economic and comparable with other recovery methods in terms of the percentage of PHB recovery and energy consumption, while it is environmentally more benign. Ambio, 2004 Nov, 33(7), 436 - 47 Effects on the structure of Arctic ecosystems in the short- and long-term perspectives; Callaghan TV et al.; Species individualistic responses to warming and increased UV-B radiation are moderated by the responses of neighbors within communities, and trophic interactions within ecosystems . All of these responses lead to changes in ecosystem structure . Experimental manipulation of environmental factors expected to change at high latitudes showed that summer warming of tundra vegetation has generally led to smaller changes than fertilizer addition . Some of the factors manipulated have strong effects on the structure of Arctic ecosystems but the effects vary regionally, with the greatest response of plant and invertebrate communities being observed at the coldest locations . Arctic invertebrate communities are very likely to respond rapidly to warming whereas microbial biomass and nutrient stocks are more stable . Experimentally enhanced UV-B radiation altered the community composition of gram-negative bacteria and fungi, but not that of plants . Increased plant productivity due to warmer summers may dominate food-web dynamics . Trophic interactions of tundra and sub-Arctic forest plant-based food webs are centered on a few dominant animal species which often have cyclic population fluctuations that lead to extremely high peak abundances in some years . Population cycles of small rodents and insect defoliators such as the autumn moth affect the structure and diversity of tundra and forest-tundra vegetation and the viability of a number of specialist predators and parasites . Ice crusting in warmer winters is likely to reduce the accessibility of plant food to lemmings, while deep snow may protect them from snow-surface predators . In Fennoscandia, there is evidence already for a pronounced shift in small rodent community structure and dynamics that have resulted in a decline of predators that specialize in feeding on small rodents . Climate is also likely to alter the role of insect pests in the birch forest system: warmer winters may increase survival of eggs and expand the range of the insects . Insects that harass reindeer in the summer are also likely to become more widespread, abundant and active during warmer summers while refuges for reindeer/caribou on glaciers and late snow patches will probably disappear. Dermatol Ther, 2004, 17(6), 499 - 504 Treatment and prevention of rickettsial and ehrlichial infections; Maender JL et al.; Rickettsial and ehrlichial infections are both carried by arthropod vectors . Both Rickettsia and Ehrlichia are small intracellular gram-negative coccobacilli . Clinical manifestations of Rickettsia range from spotted fevers to various forms of typhus . Human ehrlichiosis can present as monocytic ehrlichiosis or granulocytic anaplasmosis . Prevention is by avoidance of the responsible vectors . Therapy is usually with doxycycline, but chloramphenicol can also be used. Acta Microbiol Immunol Hung, 2004, 51(3), 351 - 8 The antimotility action of a trifluoromethyl ketone on some gram-negative bacteria; Spengler G et al.; The inhibition of bacterial motility was studied by a trifluoro methyl ketone derivative on two Escherichia coli strains (wild strain having a proton pump system and the proton pump-deficient mutant strain) and two Helicobacter pylori strains (clarithromycin susceptible and clarithromycin resistant) . Evidence is presented of the inhibitory action of 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18) on the proton motive forces of the two bacterial strains by affecting the action of biological motor and proton efflux in the membranes . The swimming, the forward motion was more sensitive than the vibration or tumbling to the inhibition . We suppose that the inhibiton of bacterial motility is related to the virulence of bacteria: consequently the pathogenicity can be reduced in the presence of TF18. Photochem Photobiol Sci, 2004 Nov-Dec, 3(11-12), 992 - 8 Epub 2004 Nov-Dec. Photodynamic activity of cationic and non-charged Zn(ii) tetrapyridinoporphyrazine derivatives: biological consequences in human erythrocytes and Escherichia coli; Dupouy EA et al.; The photodynamic activity of a cationic Zn(ii) tetramethyltetrapyridinoporphyrazinium salt (ZnPc ) was compared with that of a non-charged Zn(ii) tetrapyridinoporphyrazine (ZnPc ), both in vitro using human red blood (HRB) cells and a typical Gram-negative bacterium Escherichia coli . Absorption and fluorescence spectroscopic studies were analyzed in different media . Fluorescence quantum yields ({small phi}(F)) of 0.35 for ZnPc and 0.30 for ZnPc were calculated in N,N-dimethylformamide (DMF) . The singlet molecular oxygen, O(2)((1){capital Delta}(g)), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of {capital Phi}({capital Delta})= 0.56 for ZnPc and 0.50 for ZnPc . In biological medium, the photodynamic effect was first evaluated in HRB cells . Both phthalocyanines produce similar photohemolysis of HRB cells, reaching values >90% of lysis after 5 min of irradiation with visible light . The photodynamic effect is accompanied by an increase in the membrane fluidity of HRB cells . However, these studies on E . coli cells showed that the cationic ZnPc produces a higher photoinactivation of Gram-negative bacteria than ZnPc . Also, these results were established by stopped of growth curves for E . coli . Therefore the studies show that cationic ZnPc is an efficient phototherapeutic agent with potential applications in tumor cell and Gram-negative bacteria inactivation by photodynamic therapy. Histopathology, 2004 Dec, 45(6), 633 - 7 Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization; Hudelist G et al.; AIMS: The presence of laminated, calcified extracellular debris known as psammoma bodies is a well-known histomorphological feature of ovarian adenocarcinomas and other human malignancies . Biomineralization has recently been found to be associated with a group of extremely small Gram-negative bacteria capable of precipitating calcium salts . The aim of the present study was to evaluate a possible pathogenic link between the development of psammoma bodies and nanobacteria infection . MATERIAL AND RESULTS: Immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyse nanobacterial protein and gene expression in eight psammona body-containing adenocarcinomas and in 10 malignant ovarian tumours without signs of biomineralization . Nanobacterial proteins were detected in eight out of eight (100%) psammoma-positive tumour samples . Conversely, none of the 10 psammoma-negative tissues (0%) was positive for nanobacterial antigens . Furthermore, nanobacterial mRNA was detectable in all of the four tissues (100%) that contained psammoma bodies, but was absent in all 10 ovarian cystadenocarcinomas (0%) that were psammoma negative . CONCLUSIONS: We found a 100% concordance between the expression of nanobacteria and the presence of psammoma bodies in malignant ovarian tumours . Several lines of evidence suggest the involvement of these organisms in the process of biomineralization . We therefore conclude that nanobacterial infection of malignant ovarian tissue contributes to mechanisms leading to the formation of calcified deposits known as psammoma bodies. Genes Immun . 2004 Nov 25; {Epub ahead of print} The TLR4 +896 polymorphism is not associated with lipopolysaccharide hypo-responsiveness in leukocytes; Imahara SD et al.; Toll-like receptor 4 (TLR-4) is required for detection of Gram negative bacterial infections by binding lipopolysaccharide (LPS) and for the initiation of inflammatory signaling . Recent studies have demonstrated that a nonsynonymous single-nucleotide polymorphism (Asp299Gly, A+896G) is associated with decreased endotoxin responsiveness and poor outcomes from sepsis . We show that human carriers of this polymorphism show no deficit in LPS induced peripheral blood mononuclear cell (PBMC) mitogen-activated protein kinase (MAPK) activity, no reduction in sensitivity to endotoxin, and variable differences in whole-blood inflammatory cytokine production . These results indicate that this mutation is not a primary determinant of human endotoxin sensitivity.Genes and Immunity advance online publication, 25 November 2004; doi:10.1038/sj.gene.6364147. Clin Chim Acta, 2005 Jan, 351(1-2), 17 - 29 Clinical laboratory differentiation of infectious versus non-infectious systemic inflammatory response syndrome; Mitaka C; OBJECTIVE: To evaluate the accuracy of C-reactive protein (CRP), procalcitonin (PCT), neopterin, and endotoxin in the differential diagnosis of sepsis and non-infectious systemic inflammatory response syndrome (SIRS) . METHODS: A Medline database and references from identified articles were used to perform a literature search relating to the differential diagnosis of sepsis versus non-infectious SIRS . RESULTS: CRP, PCT, and neopterin are released both in sepsis and in non-infectious inflammatory disease . CRP and PCT are equally effective, although not perfect, in differentiating between sepsis and non-infectious SIRS . However, CRP and PCT have different kinetics and profiles . The kinetics of CRP is slower than that of PCT, and CRP levels may not further increase during more severe stages of sepsis . On the contrary, PCT rises in proportion to the severity of sepsis and reaches its highest levels in septic shock . PCT tends to be higher in nonsurvivor than in survivor . Therefore, PCT demonstrated a closer correlation with the severity of sepsis and outcome than CRP . Unlike CRP and PCT, neopterin is increased in viral infection as well as bacterial infection, and neopterin is also a useful indicator of sepsis . Endotoxemia was detected in no more than half of patients with Gram-negative bacteremia, and Gram-negative bacteremia was detected in half of patients with endotoxemia . CONCLUSIONS: The diagnostic capacity of PCT is superior to that of CRP due to the close correlation between PCT levels and the severity of sepsis and outcome . Neopterin is very useful in the diagnosis of viral infection . The endotoxin assay in combination with CRP, PCT, or neopterin may help as a diagnostic marker for Gram-negative bacterial infection. J Biol Chem . 2004 Nov 23; {Epub ahead of print} NF-kappa B-and C/EBP beta-driven interleukin-1beta gene expression and PAK1-mediated caspase 1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide (LPS)-stimulated macrophages; Basak C et al.; Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer and gastric carcinoma . Interleukin 1 beta (IL-1 beta) is one of the potent proinflammatory cytokines elicited by H . pylori infection . We have dissected the signaling pathways leading to H . pylori LPS-induced IL-1 beta secretion . Both the NF- kappa B and the C/EBP beta binding elements of the IL-1 beta promoter drive LPS-induced IL-1 beta gene expression . NF-kappa B activation requires the classical TLR4-initiated signaling cascade leading to IB phosphorylaiton as well as PI3-K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBP beta activation requires PI3-K/Akt/p38 mitogen activated protein (MAP) kinase signaling . We observed a direct interaction between activated p38 MAP kinase and C/EBP beta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBP beta . Most importantly, we observed a role of Rac1/PAK1 signaling in activation of caspase 1 which is necessary for maturation of pro- IL-1 beta . H . pylori LPS induced direct interaction between PAK1 and caspase 1 which was inhibited in cells transfected with dominant-negative Rac1 . PAK1 immunoprecipitated from lysates of H . pylori LPS-challenged cells was able to phosphorylate recombinant caspase 1, but not its S376A mutant . LPS-induced caspase 1 activation was abrogated in cells transfected with caspase 1(S376A) . These results suggested a role of PAK1-induced phosphorylation of caspase 1 at S376 in activation of caspase 1 . Our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase 1 phosphorylation, is crucial for caspase 1 activation . These studies also provide detailed insight into the regulation of IL-1 beta gene expression by H . pylori LPS, and are particularly important in the light of the observations that IL-1 beta gene polymorphisms are associated with increased risk of H . pylori-associated gastric cancer. Klin Mikrobiol Infekc Lek, 2004 Oct, 10(5), 207 - 13 {Bartonelloses.}; Medkova Z; Bartonellae belong to less known causal agents of many human diseases . They are gram-negative bacteria growing slowly on culture media enriched with hemin or bovine serum . The genus Bartonella, which currently involves more than 15 species, is present worldwide . Bartonellae live in natural foci in dependence on the occurrence of natural host (rodents, felines, canidae, human) and insect vector (flea, tick, louse) . By reservoir animals they usually cause permanent intraerythrocytic bacteraemia without system inflammation symptoms . A classical example of a human disease is cat scratch disease (CSD) caused by Bartonella henselae and characterised by regional lymphagoitis and lymphadenitis . Increasing interest is being devoted to the ability of Bartonella sp . (e.i . B . quintana) to cause the opportune infections with diverse clinical manifestation: bacillary angiomatosis, specific liver and spleen vasculitis (peliosis hepatis, splenis), endocarditis and others . The issue of Bartonella infections is relatively new and its importance is still growing with increasing knowledge in this field. Infect Immun, 2004 Dec, 72(12), 7315 - 7 In vitro model of Bartonella henselae-induced angiogenesis; Kirby JE; Bartonella henselae is a gram-negative pathogen that causes angiogenesis . Here, I establish in vitro models to study Bartonella-induced blood vessel formation . I found that B . henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix. Infect Immun, 2004 Dec, 72(12), 7231 - 9 Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia trachomatis infection; Gervassi A et al.; Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans . Both genital and ocular C . trachomatis infections are associated with tissue inflammation and pathology . Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines . To determine the potential contribution of DC to the inflammatory process, human DC were infected with C . trachomatis serovar E or L2 . Both C . trachomatis serovars were found to infect and replicate in DC . Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules . Furthermore, chlamydial infection induced the secretion of interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-alpha) . The mechanisms involved in Chlamydia-induced IL-1beta and IL-18 secretion differed from those of the other cytokines . Chlamydia-induced IL-1beta and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia-induced activation of caspase-1 in infected host cells . In contrast, TNF-alpha and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia-induced cytokine secretion in DC . Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia-induced IL-1beta, IL-6, and TNF-alpha secretion . The data herein demonstrate that DC can be infected by human C . trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC . Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections. Infect Immun, 2004 Dec, 72(12), 7202 - 11 Toll-like receptor 9 can be expressed at the cell surface of distinct populations of tonsils and human peripheral blood mononuclear cells; Eaton-Bassiri A et al.; Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9) . However, the nature and cellular localization of TLR9 in primary human cells remain controversial . Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface . The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed . Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells . Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10% . In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression . Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization . Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9. Am J Gastroenterol, 2004 Nov, 99(11), 2147 - 9 Novosphingobium aromaticivorans: a potential initiator of primary biliary cirrhosis; Kaplan MM; Primary biliary cirrhosis (PBC) is characterized by a T-cell-mediated destruction of bile duct epithelial cells that line the small intrahepatic bile ducts . The targets of activated T-lymphocytes are the dihydrolipoamide acetyltransferase components of the 2 oxo acid dehydrogenases, enzyme complexes that are important in oxidative energy metabolism . Pyruvate dehydrogenase is the best known of these . Its dihydrolipoamide acetyltransferase component is referred to as PDC-E2 . A major question in understanding the pathogenesis of PBC is why PBC patients lose their tolerance to antigens that are found in virtually every cell in the body . A possible cause is molecular mimicry between microbial agents and self-antigens . Infection with or exposure to a microorganism whose PDC-E2 bears close homology with human PDC-E2 could act as an immunological trigger that initiates the development of PBC . Emerging data suggest that there is a microorganism that may initiate the onset of PBC . Novosphingobium aromaticivorans is a gram negative strictly aerobic bacteria that is found worldwide in soil, water, and coastal plain sediments . Its PDC-E2-like proteins have a higher degree of homology with the immunodominant region of human PDC-E2 than any microorganism thus far studied (100-1,000 times greater than that of Escherichia coli) . In addition, N . aromaticivorans can metabolize xenobiotics that are similar to the chemical compounds that react with sera from PBC patients . Some of these xenobiotics are immunologically related to lipoic acid, the cofactor that is at the active center of PDC-E2 . Thus, N . aromaticivorans can theoretically break down self-tolerance in two ways: by molecular mimicry due to subclinical infection and by the metabolism of xenobiotics that are present in the environment . In an initial study, investigators found that antibodies against N . aromaticivorans were found in 77 of 77 PBC patients from Milan, Italy, who had antibodies to PDC-E2 and that the titers to N . aromaticivorans proteins were similar to those to human PDC-E2 . The report in this issue of The American Journal of Gastroenterology confirms these earlier findings and demonstrates that exposure to N . aromaticivorans occurs in genetically different PBC patients from other regions . Thirteen of 14 Icelandic PBC patients who were AMA positive reacted against at least one of the 2 oxo acid dehydrogenase-E2 complexes . These observations provide additional evidence that exposure to N . aromaticivorans may trigger the development of PBC. Clin Exp Pharmacol Physiol, 2004 Oct, 31(10), 723 - 31 Role of endogenous macrophage inflammatory protein-2 in regulating fever induced by bacterial endotoxin in normal and immunosuppressed rats; Minano FJ et al.; During myelosuppressive chemotherapy, Gram-negative bacterial infection with consequent exposure to lipopolysaccharide (LPS) is one of the most important causes of persistent fever . The classical model of the pathogenesis of fever suggests that pro-inflammatory cytokines, produced by leucocytes in the bloodstream in response to exogenous pyrogens such as bacterial LPS, represent the distal mediators of the febrile response . Neutrophils are the first effectors cells and the most prominent leucocyte population involved in acute bacterial infection . Macrophage inflammatory protein (MIP)-2 plays a crucial role in influencing early cell trafficking and neutrophil activation during pathophysiological processes and serves the same chemotactic function as human interleukin-8 . In the present study, we investigated the role of MIP-2 in the development of a febrile response induced by LPS in immunocompetent and leukopenic rats . Intraperitoneal injection of LPS in leukopenic rats elicited a biphasic febrile response of rapid onset, the magnitude and duration of which were significantly greater than in immunocompetent animals . The febrile responses to LPS were accompanied by a pronounced induction of serum MIP-2 levels at 1, 2 and 4 h compared with their respective controls . In both normal and leukopenic rats, neutralization of endogenous MIP-2 bioactivity by systemic administration of antirat MIP-2 antibody caused a significant attenuation of the early phase of LPS fever . However, in contrast with normal rats, the second phase of fever was unimpaired by anti-MIP-2 in leukopenic rats . These findings suggest that circulating MIP-2 is involved in the generation of the early phase of LPS fever that contributes to the maintenance of the later phase of fever in immunocompetent, but not leukopenic, rats . Our data support a regulatory role for endogenous MIP-2 in initiating the fever responses to LPS . Furthermore, these results provide evidence that different cellular and humoral mechanisms are implicated in the development of a febrile response triggered by Gram-negative bacterial infections in leukopenic hosts. Drug Des Discov, 2003, 18(4), 109 - 16 Conformational analysis of globomycin with a signal peptidase II inhibitory activity using molecular dynamics simulation; Kiho T et al.; Globomycin (1), a 19-membered cyclic depsipeptide, exhibited an antibiotic activity against gram-negative bacteria by inhibiting signal peptidase II in the cytoplasmic membrane . Although only one conformation of 1 was observed for the crystal structure, it was revealed by 1H NMR spectroscopic analysis that 1 exists as a mixture of two rotational isomers in solution (CDCl3 and CD3OD) . A conformational analysis of 1 was, therefore, performed by high-temperature molecular dynamics simulation in combination with 1H NMR analysis to elucidate the conformations in solution . The relative ratio of the major and minor isomers present, which differs depending on the solvent, was then derived from their relative energy differences obtained in the conformational analysis . The difference in the relative ratios corresponded with that calculated from the 1H NMR analysis . Finally, the predicted conformations in solution were compared with that of the X-ray crystal structure to find local and global differences that characterize these conformations. Mol Plant Microbe Interact, 2004 Nov, 17(11), 1259 - 68 NopB, a soybean cultivar-specificity protein from Sinorhizobium fredii USDA257, is a type III secreted protein; Lorio JC et al.; The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes . Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.) . This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction . Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium . We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257 . The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NoIB) . Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB . Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops . A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography . Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages . Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter . Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB . We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2003, 14(2), 133 - 41 {Comparison of a rapid antigen test and cultures for diagnosis of meningitis in children}; Hashikita G et al.; Fourteen pediatric patients diagnosed as bacterial meningitis between August 1997 and April 2002 were enrolled in this study . Both rapid antigen detection test, Slidex Meningite 5 Kit (Biomerieux) and culture were performed using cerebrospinal fluids (CSF) . H . influenzae was isolated from 11 samples and was the most frequently isolated bacteria, followed by S . pneumoniae from 4 samples and enteric bacteriae from 2 samples . Five out of six samples with positive result by culture were also positive by the rapid antigen test . Gram-negative rod was identified in smear specimens of CSF from all these 5 samples . Significance of the rapid antigen test should be recognized under drug resistance of those bacteriae are increasing. J Infect Dis, 2004 Dec 15, 190(12), 2121 - 8 Epub 2004 Dec 15. Virulence factor profiles and phylogenetic background of Escherichia coli isolates from veterans with bacteremia and uninfected control subjects; Sannes MR et al.; BACKGROUND: Escherichia coli is the most common cause of gram-negative bloodstream infections, causing an estimated 40,000 deaths from sepsis each year in the United States . The present study sought to determine specifically which virulence factors (VFs) and phylogenetic groups of E . coli are epidemiologically associated with bacteremia . METHODS: E . coli isolates from 63 veterans with bacteremia and rectal isolates from 71 matched uninfected control subjects were compared both for phylogenetic group and for the presence of VFs and O antigens . RESULTS: Bacteremia isolates exhibited a significantly greater prevalence of most VFs studied . In multivariate logistic regression analysis, ompT (outer membrane protein T) was the strongest VF predictor of bacteremia (P<.001) . Despite the concentration of most individual VFs within group B2, bacteremia and rectal isolates differed little by phylogenetic distribution, a finding explained by the greater prevalence of VFs among bacteremia isolates than rectal isolates within groups B2 and D . CONCLUSIONS: Although phylogenetic group partially corresponds with virulence potential in E . coli bacteremia, VFs are more-powerful predictors of pathogenic potential . Bacteremia isolates exhibit an arsenal of VFs that distinguishes them from rectal isolates from uninfected hosts, which makes these differences attractive potential targets in vaccine or drug development. Neoplasia, 2004 Sep-Oct, 6(5), 423 - 31 Chemotherapy-induced and/or radiation therapy-induced oral mucositis--complicating the treatment of cancer; Naidu MU et al.; The term mucositis is coined to describe the adverse effects of radiation and chemotherapy treatments . Mucositis is one of the most common adverse reactions encountered in radiation therapy for head and neck cancers, as well as in chemotherapy, in particular with drugs affecting DNA synthesis (S-phase-specific agents such as fluorouracil, methotrexate, and cytarabine) . Mucositis may limit the patient's ability to tolerate chemotherapy or radiation therapy, and nutritional status is compromised . It may drastically affect cancer treatment as well as the patient's quality of life . The incidence and severity of mucositis will vary from patient to patient . It will also vary from treatment to treatment . It is estimated that there is 40% incidence of mucositis in patients treated with standard chemotherapy and this will not only increase with the number of treatment cycles but also with previous episodes . Similarly, patients who undergo bone marrow transplantation and who receive high doses of chemotherapy have a 76% chance of getting mucositis . Patients receiving radiation, in particular to head and neck cancers, have a 30% to 60% chance . The exact pathophysiology of development is not known, but it is thought to be divided into direct and indirect mucositis . Chemotherapy and/or radiation therapy will interfere with the normal turnover of epithelial, cells leading to mucosal injury; subsequently, it can also occur due to indirect invasion of Gram-negative bacteria and fungal species because most of the cancer drugs will cause changes in blood counts . With the advancement in cytology, a more precise mechanism has been established . With this understanding, we can select and target particular mediators responsible for the mucositis . Risk factors such as age, nutritional status, type of malignancy, and oral care during treatment will play important roles in the development of mucositis . Many treatment options are available to prevent and treat this condition, but none of them can completely prevent or treat mucositis . More and more pathological methods are being developed to understand this condition so that better therapeutic regimens can be selected . Emphasis also should be made in assessing the patient's psychologic condition, particular depressive disorders . This is important because treatment with antidepressants will not only contribute in lifting depression but also reduces pain somatization . Although mucositis is rarely life-threatening, it will interfere with treatment of cancer to a great extent. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 235 - 57 Protein secretion through autotransporter and two-partner pathways; Jacob-Dubuisson F et al.; Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity . Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains . As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter . In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters . The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria . Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane . Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface . A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter . In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules . In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 181 - 206 Type III protein secretion mechanism in mammalian and plant pathogens; He SY et al.; The type III protein secretion system (TTSS) is a complex organelle in the envelope of many Gram-negative bacteria; it delivers potentially hundreds of structurally diverse bacterial virulence proteins into plant and animal cells to modulate host cellular functions . Recent studies have revealed several basic features of this secretion system, including assembly of needle/pilus-like secretion structures, formation of putative translocation pores in the host membrane, recognition of N-terminal/5' mRNA-based secretion signals, and requirement of small chaperone proteins for optimal delivery and/or expression of effector proteins . Although most of our knowledge about the TTSS is derived from studies of mammalian pathogenic bacteria, similar and unique features are learned from studies of plant pathogenic bacteria . Here, we summarize the most salient aspects of the TTSS, with special emphasis on recent findings. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 163 - 79 The underlying mechanisms of type II protein secretion; Filloux A; The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the peptidoglycan-containing periplasm . Whereas the envelope forms an essential barrier against harmful substances, it is nevertheless a compartment of intense traffic for large proteins such as enzymes and toxins . Numerous studies dealing with the molecular mechanism of protein secretion have revealed that Gram-negative bacteria evolved different strategies to achieve this process . Among them, the type II secretion mechanism is part of a two-step process . Exoproteins following this pathway are synthesized as signal peptide-containing precursors . After cleavage of the signal peptide, the mature exoproteins are released into the periplasm, where they fold . The type II machinery, also known as the secreton, is responsible for the translocation of the periplasmic intermediates across the OM . The type II system is broadly conserved in Gram-negative bacteria and involves a set of 12-16 different proteins named GspC-M, GspAB, GspN, GspO, and GspS . The type II secretion system is highly reminiscent of the type IV piliation assembly system . Based on findings about the subcellular localisation of the Gsp components, protein-protein interactions between Gsps and their multimerisation status, structural data and electron microscopy observation, it could be proposed a working model that strikingly runs both systems in parallel. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 149 - 61 Type I secretion in gram-negative bacteria; Delepelaire P; In gram-negative bacteria, type I secretion is carried out by a translocator made up of three proteins that span the cell envelope . One of these proteins is a specific outer membrane protein (OMP) and the other two are cytoplasmic membrane proteins: an ATP-binding cassette (ABC) and the so-called membrane fusion or adaptor protein (MFP) . Type I secretion is sec-independent and bypasses the periplasm . This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal . This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex . This report will mainly deal will recent data concerning the structure and assembly of the secretion complex as well as the effects and role of substrate folding on secretion by this pathway. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 121 - 34 Quality control in the bacterial periplasm; Duguay AR et al.; Studies of the mechanisms that Gram-negative bacteria use to sense and respond to stress have led to a greater understanding of protein folding in both cytoplasmic and extracytoplasmic locations . In response to stressful conditions, bacteria induce a variety of stress response systems, examples of which are the sigma(E) and Cpx systems in Escherichia coli . Induction of these stress response systems results in upregulation of several gene targets that have been shown to be important for protein folding under normal conditions . Here we review the identification of stress response systems and their corresponding gene targets in E . coli . In addition, we discuss the apparent redundancy of the folding factors in the periplasm, and we consider the potential importance of the functional overlap that exists. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 5 - 16 Translocation of bacterial proteins-an overview; Holland IB; Recent progress in the understanding of the nature of the extraordinary variety of protein translocation systems, mainly in Gram negative bacteria, is reviewed . This takes us from the insertion of proteins into the inner membrane via the sophisticated Sec apparatus, the lethal injection of Type III proteins into host cells and on to the beautiful machine that assembles the flagellum . Attempts are made to establish some order, some common principles that might explain the variety and the complexity of some systems . The fundamentals considered are the nature of different transport signals, the nature of translocons (a wide variety of inner membrane types, outer membrane translocons are more conserved), the process of docking to translocons, the role of chaperones and the folding of transported proteins, the energetics of translocation, and prospects for future advances. Mar Biotechnol (NY), 2004 Jul-Aug, 6(4), 335 - 46 Epub 2004 Jun 23. Isolation and characterization of a fucoidan-degrading marine bacterial strain and its fucoidanase; Sakai T et al.; A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory . The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36% . The cells of the strain were rod-shaped (2.0 microm long x 1.0 microm wide), and each cell was motile by means of one polar flagellum . Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae . It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme . The enzyme was purified with 3500-fold purity at 12.0% yield . Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30 degrees to 35 degrees C . The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2263 - 7 Saccharibacter floricola gen . nov., sp . nov., a novel osmophilic acetic acid bacterium isolated from pollen; Jojima Y et al.; Three Gram-negative, aerobic, rod-shaped bacterial strains were isolated, from the pollen of Japanese flowers, as producers of xylitol; these strains were subjected to a polyphasic taxonomic study . Phylogenetic analyses of the 16S rRNA gene sequences demonstrated that these three isolates formed a new cluster within a group of acetic acid bacteria in the alpha-Proteobacteria . The characteristics of the three isolates were as follows: (i) their predominant quinone was Q-10; (ii) their cellular fatty acid profile contained major amounts of 2-hydroxy acids and an unsaturated straight-chain acid (C(18 : 1)omega7c); and (iii) their DNA G+C contents were in the range 51.9-52.3 mol%, which is around the lower limit of the reported range for the genera of acetic acid bacteria . The negligible or very weak productivity of acetic acid from ethanol and the osmophilic growth properties distinguished these strains from other acetic acid bacteria . The unique phylogenetic and phenotypic characteristics suggest that the three isolates should be classified within a novel genus and species with the proposed name Saccharibacter floricola gen . nov., sp . nov . The type strain is strain S-877(T) (=AJ 13480(T)=JCM 12116(T)=DSM 15669(T)). Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2223 - 30 Proposals of Curvibacter gracilis gen . nov., sp . nov . and Herbaspirillum putei sp . nov . for bacterial strains isolated from well water and reclassification of {Pseudomonas} huttiensis, {Pseudomonas} lanceolata, {Aquaspirillum} delicatum and {Aquaspirillum} autotrophicum as Herbaspirillum huttiense comb . nov., Curvibacter lanceolatus comb . nov., Curvibacter delicatus comb . nov . and Herbaspirillum autotrophicum comb . nov; Ding L et al.; Two strains of curved bacteria, 7-1(T) and 7-2(T), isolated from well water, were phylogenetically examined to determine their taxonomic position . Strain 7-1(T) is a Gram-negative, slightly curved rod . Analysis of the 16S rRNA gene sequence showed that strain 7-1(T) formed a cluster with {Aquaspirillum} delicatum and {Pseudomonas} lanceolata . It has some similar characteristics to {A.} delicatum and {P.} lanceolata, but has sufficient distance to separate it from other genera . DNA-DNA hybridization analysis, as well as chemotaxonomic and morphological studies, demonstrated that strain 7-1(T), {A.} delicatum and {P.} lanceolata belong to a new genus, Curvibacter gen . nov . Strain 7-1(T) (=IAM 15033(T)=ATCC BAA-807(T)) is classified as the type strain of Curvibacter gracilis gen . nov., sp . nov., and {A.} delicatum and {P.} lanceolata are classified as Curvibacter delicatus comb . nov . and Curvibacter lanceolatus comb . nov., respectively . Strain 7-2(T) is a Gram-negative spirillum . Phylogenetic study based on the 16S rRNA gene sequences showed that it formed a cluster with the members of the genus Herbaspirillum, {Pseudomonas} huttiensis and {Aquaspirillum} autotrophicum . The classification is therefore proposed of strain 7-2(T) (=IAM 15032(T)=ATCC BAA-806(T)) as the type strain of Herbaspirillum putei sp . nov., and {P.} huttiensis and {A.} autotrophicum are transferred to the genus Herbaspirillum as Herbaspirillum huttiense comb . nov . and Herbaspirillum autotrophicum comb . nov., respectively. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2185 - 90 Sphingomonas oligophenolica sp . nov., a halo- and organo-sensitive oligotrophic bacterium from paddy soil that degrades phenolic acids at low concentrations; Ohta H et al.; The taxonomic position of a halo- and organo-sensitive, oligotrophic soil bacterium, strain S213(T), was investigated . Cells were Gram-negative, non-motile, strictly aerobic, yellow-pigmented rods of short to medium length on diluted nutrient broth . When 0.1-0.4 % (w/v) NaCl was added to diluted media composed of peptone and meat extract, growth was inhibited with increasing NaCl concentration and the cells became long aberrant forms . When 6 mM CaCl(2) was added, the cells grew quite normally and aberrant cells were no longer found at 0.1-0.5 % (w/v) NaCl . Chemotaxonomically, strain S213(T) contains chemical markers that indicate its assignment to the Sphingomonadaceae: the presence of ubiquinone Q-10 as the predominant respiratory quinone, C(18 : 1) and C(16 : 0) as major fatty acids, C(14 : 0) 2-OH as the major 2-hydroxy fatty acid and sphingoglycolipids . 16S rRNA gene sequence analysis indicated that strain S213(T) belongs to the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas mali IFO 15500(T) (98.3 %), Sphingomonas pruni IFO 15498(T) (98.0 %), Sphingomonas asaccharolytica IFO 15499(T) (97.9 %) and Sphingomonas echinoides DSM 1805(T) (97.8 %) . The results of DNA-DNA hybridization experiments and its phenotypic characteristics clearly distinguished the strain from its nearest neighbours and demonstrate that strain S213(T) represents a novel Sphingomonas species, for which the name Sphingomonas oligophenolica sp . nov . is proposed . The type strain is S213(T) (=JCM 12082(T)=CIP 107926(T)). Int J Syst Evol