Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1281 - 3 Epub 2004 Jun 22. Crystallization and preliminary X-ray analysis of a C-terminal TonB fragment from Escherichia coli; Koedding J et al.; The TonB protein located in the cell wall of Gram-negative bacteria mediates the proton motive force from the cytoplasmic membrane to specific outer membrane transporters . A C-terminal fragment of TonB from Escherichia coli consisting of amino-acid residues 147-239 (TonB-92) has been purified and crystallized . Crystals grew in space group P2(1) to dimensions of about 1.0 x 0.12 x 0.12 mm . A native data set has been obtained to 1.09 A resolution. Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9618 - 23 Epub 2004 Jun 21. The integral membrane enzyme PagP alternates between two dynamically distinct states; Hwang PM et al.; PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria . A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP beta barrel . The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located . To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity . Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T) . The kinetics and thermodynamics of the interchange have been investigated by (1)H-(15)N NMR spectroscopy, with Delta H = -10.7 kcal/mol and Delta S = -37.5 cal/mol.K for the R--> T transition . A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel . In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible . Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis . The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP. Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9780 - 5 Epub 2004 Jun 21. Microarray analysis of transposition targets in Escherichia coli: the impact of transcription; Manna D et al.; Transposable elements have influenced the genetic and physical composition of all modern organisms . Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function . Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction . Replicated copies are excised from the chromosome and packaged into virus particles . Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion . To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA . Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency . A moderate preference was observed for genes near the origin compared to terminus of replication . Large biases were found at hot and cold spots, which often include several consecutive genes . Efficient transcription of genes had a strong negative influence on transposition . Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection. MedGenMed . 2004 Mar 09;6(1):10. Causative agents of liver abscess in Thai hepatitis B carriers; Wiwanitkit V et al.; Liver abscess and hepatitis B virus (HBV) infection are two significant tropical gastrointestinal disorders . The concurrence between these two disorders yields poor prognosis, which then often leads to the need for intensive care . The aim of this study was to investigate the causal pathogens of liver abscess in HBV carriers . This retrospective case review was conducted on 35 Thai hospitalized HBV carriers who had diagnosis of liver abscess . A high rate of amoebic liver abscess in this series (37.1%) was demonstrated; Gram-negative aerobes were the major abscess pathogens . The causative pathogens of HBV carriers were similar to those in the overall patient population with abscess . The treatment plan for liver abscess in the general population can also be applied to HBV carriers. Vet Immunol Immunopathol, 2004 Aug, 100(3-4), 179 - 86 Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila; Entrican G et al.; Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia . Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms . Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Ikappa-Balpha, the endogenous inhibitor of NF-kappaB . However, Ikappa-Balpha is not upregulated in response to bacterial lipopolysaccharide (LPS) . The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14 . Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies. Eur J Biochem, 2004 Jul, 271(13), 2691 - 704 A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region; Leone S et al.; Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons . A novel lipo-oligosaccharide from P . stutzeri OX1 was isolated and characterized . For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration . The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis . Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH . The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups . The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy . These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A . All sugars are D-pyranoses and alpha-linked, if not stated otherwise . Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain . This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P . sutzeri OX1 to a hydrocarbon-containing environment. Urol Res, 2004 Jun, 32(3), 190 - 5 Epub 2004 Feb 06. Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population; Khullar M et al.; The aim of this study was to detect, isolate and characterize the nanobacteria from human renal stones from a north Indian population, and to determine their role in biomineralization . Renal stones retrieved from the kidneys of 65 patients were processed and subjected to mammalian cell culture conditions . The isolated bacteria were examined using scanning (SEM) and transmission electron microscopy (TEM) . They were characterized for the presence of DNA, proteins and antigenicity . The role of these bacteria in biomineralization was studied by using the (14)C-oxalate based calcium oxalate monohydrate (COM) crystallization assay . We observed the presence of apatite forming, ultrafilterable gram negative, coccoid microorganisms in 62% of the renal stones . SEM studies revealed 60-200 nm sized organisms with a distinct cell wall and a capsule . TEM images showed needle like apatite structures both within and surrounding them . They were heat sensitive, showed antibiotic resistance and accelerated COM crystallization . A potent signal corresponding to the presence of DNA was observed in demineralized nanobacterial cells by flow cytometry . The protein profile showed the presence of several peptide bands of which those of 18 kDa and 39kDa were prominent . Apatite forming nanosized bacteria are present in human renal stones and may play a role in the pathophysiology of renal stone formation by facilitating crystallization and biomineralization . However, further studies are required to establish the exact mechanism by which nanobacteria are involved in the causation of renal stones. J Bacteriol, 2004 Jul, 186(13), 4168 - 76 Sinorhizobium meliloti sulfotransferase that modifies lipopolysaccharide; Cronan GE et al.; Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of a plant structure called the nodule . Symbiosis between S . meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor . S . meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity . Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R . A . Cedergren, J . Lee, K . L . Ross, and R . I . Hollingsworth, Biochemistry 34:4467-4477, 1995) . We utilized the genomic sequence of S . meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity . We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase . Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro . However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S . meliloti that can compensate for the loss of LpsS . The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa . These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions. J Occup Environ Hyg, 2004 Feb, 1(2), 62 - 8 Microbial air quality in offices at municipal landfills; Lis DO et al.; The purpose of this study was to evaluate the influence of two municipal landfills on the microbiological air quality in offices on landfill sites . The evaluation was based on the concentration levels of airborne bacteria and fungi and the identification of isolated strains . Air samples were collected with a six-stage Andersen impactor . The concentrations of bacterial aerosol ranged from 1.0 x 10(3) to 7.2 x 10(4) colony forming units (CFU)/m(3) indoors, and from 7.0 x 10 to 4.0 x 10(4) CFU/m(3) outdoors . The corresponding fungal aerosol ranges were from 2.3 x 10(2) to 7.3 x 10(3) CFU/m(3) indoors and from 2.0 x 10(2) to 1.2 x 10(4) CFU/m(3) outdoors . The concentration levels were affected by the season of the year . The study showed that both indoor and outdoor air were heavily contaminated with bacteria and fungi . The proximity of the unpaved transport route and the weighing of refuse loads contributed to the increase of bacterial and fungal aerosol concentrations significantly . The air in the offices was characterized not only by elevated concentrations of bacteria and fungi but also by high frequencies of gram-negative bacteria, along with fungal species characteristic of landfills . The quantitative and qualitative changes in the composition of the bacterial and fungal aerosol posed a possible health risk to office workers at municipal waste landfill sites. Inhal Toxicol, 2004 Apr, 16(4), 217 - 29 The measurement and health impact of endotoxin contamination in organic dusts from multiple sources: focus on the cotton industry; Lane SR et al.; Endotoxin is derived from Gram-negative bacterial membranes, and its inflammatory effects following inhalation are well characterized . The significance of this fact becomes apparent when the wide-ranging environments containing high levels of this microbial product are considered . Endotoxin is present in numerous industrial environments, especially where organic fibers are processed . Microbial contamination of these fibers mainly occurs at the agricultural stage . Materials such as flax and hemp are affected in this way, but the most important product in this context is cotton, from which chronic dust inhalation causes the disease byssinosis . Despite the fact that endotoxin constitutes a significant threat to public health, there are currently no occupational exposure limits for this toxicant . This communication describes the toxicology of endotoxin, and its role in inhalation-induced disease, focusing on measurement of airborne endotoxin in the occupational and domestic environments using the Limulus amebocyte lysate (LAL) enzyme assay . Following the success of the LAL assay for measuring endotoxin in dusts, our laboratory has examined its application to aqueous washes from cotton fibers . Reproducibility of the results was high, and data are presented displaying levels of endotoxin contamination in fibers from different cotton producing countries . Hence, worldwide comparison of industrial endotoxin concentrations can be readily made using this test . It would be highly desirable if the performance of the LAL assay facilitated introduction of industrial endotoxin safety limits, and in spite of minor surmountable shortcomings, the test is accurate, reliable, and well field-tested, so its continued widespread use may achieve this goal. Inhal Toxicol, 2004 Jun, 16(6-7), 461 - 71 Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains; Elder AC et al.; Epidemiological studies associate morbidity and mortality with exposure to particulate air pollution in elderly individuals with existing cardiopulmonary disease . These associations led to the hypothesis that inhaled particles can exert adverse effects outside of the lung, particularly on the cardiovascular system . We tested this hypothesis by examining the pulmonary and peripheral effects of inhaled ultrafine carbon particles in old rats that were injected with endotoxin (lipopolysaccharide, LPS) to model systemic gram-negative bacterial infection . Fischer 344 rats (23 mo) and spontaneously hypertensive (SH) rats (11-14 mo) were injected with LPS (2 mg/kg, i.p.) immediately before being exposed to inhaled ultrafine carbon particles for 6 h (150 microg/m(3), CMD = 36 nm) . Controls were injected with sterile saline or were sham exposed . Twenty-four hours after LPS injection, bronchoalveolar lavage (BAL) fluid, cells, and blood were obtained to assess endpoints of inflammation, oxidant stress, coagulability, and the acute-phase response . LPS did not cause an influx of neutrophils (PMNs) into the alveolar space, but did increase the number and percentage of circulating PMNs and the concentration of plasma fibrinogen in both rat strains . Inhaled ultrafine particles did not induce lung inflammation in either rat strain . In both strains, ultrafine particles (UFP) were found to decrease the number of blood PMNs, increase the intracellular oxidation of a fluorescent dye (DCFD) in blood PMNs, and affect plasma thrombin-anti-thrombin (TAT) complex and fibrinogen levels . UFP were also found to interact with ip LPS with respect to plasma TAT complex levels and blood PMN DCFD oxidation . Differences between the two rat strains were also found for TAT complex levels, BAL cell reactive oxygen species release, and DCFD oxidation in both BAL macrophages and blood PMNs . These results suggest that inhaled ultrafine carbon particles inhaled at concentrations mimicking high episodic increases in urban air can exert extrapulmonary effects in old rats and that they can change the systemic response to an inflammatory stimulus. Mol Membr Biol, 2004 May-Jun, 21(3), 151 - 61 The simulation approach to bacterial outer membrane proteins; Bond PJ et al.; The outer membrane of Gram-negative bacteria serves as a protective barrier against the external environment but is rendered selectively permeable to nutrients and waste by proteins called porins . Other outer membrane proteins (OMPs) provide the membrane with a variety of other functions including active transport, catalysis, pathogenesis and signal transduction . A relatively small number of crystal or NMR structures of these proteins are known, and it is therefore essential that the maximum possible information be extracted . In this respect, computational techniques enable extrapolation from time- and space-averaged static structures to dynamic, physiological events . Electrostatics approaches have been used to investigate the structures of porins . The stochastic simulation of ion trajectories through these channels has been possible with Brownian dynamics, which treats the membrane and solvent approximately, enabling the prediction of conduction properties . Molecular dynamics has also been applied, enabling fully atomistic descriptions of 'virtual outer membranes' . This has provided atomic resolution descriptions of solute permeation through porins . It has also yielded insights into the dynamics of gating in active transporters and ion channels, as well as providing clues to catalytic mechanisms in outer membrane enzymes . Additionally, simulations are beginning to reveal the common features of interactions between membrane proteins and lipids, with biological implications for OMP folding, stability and mechanism . Future prospects include the simulation of longer, larger and more complex outer membrane systems, with more accurate descriptions of inter-atomic forces. Vestn Khir Im I I Grek, 2004, 163(2), 12 - 7 {Septic shock}; Grinev MV et al.; The problem of sepsis and septic shock (SSH) is known to be very actual due to peak-shaped growth of the number of such patients and unsatisfactory results of treatment . The occurrence of SSH is different--from 10% in the structure of infectious complications after polytrauma to 50% in patients with gram-negative sepsis in patients with burn disease . An analysis of treatment of 165 patients with SSH developed against the background of peritonitis has revealed high level of lethality (64%) . In the pathogenesis of SSH an important role is played by immune disorders resulting in the development of generalized inflammation and polyorganic insufficiency . Treatment of patients with SSH must be necessarily supplemented with immuno-correcting techniques: use of medicines of nonspecific anti-cytokine action (pentoxyphillin) and regulators of the immune response (ronkoleukin, blood perfusion through the donor porcine spleen) . The timely performed immunomodulating therapy allowed lethality of patients with SSH to be reduced to 32%. Arch Surg, 2004 Jun, 139(6), 652 - 4; discussion 655 Endotoxin has an indirect vasodilatory effect on isolated human skeletal muscle arterioles; Campbell M et al.; BACKGROUND: Septic shock and its effects are a major cause of mortality in the intensive care environment . The exact effect and mechanism of endotoxin has yet to be fully described . With a better understanding of this process, better clinical tools could be developed to treat these patients . HYPOTHESIS: Endotoxin has no direct effect on human skeletal muscle microvasculature and requires the release of an endothelial-derived factor to produce the vasodilation seen in gram-negative sepsis . DESIGN: Benchtop research using an isolated arteriole model with controlled exposure to endotoxin . SETTING: University medical center . METHODS: First-order arterioles (approximately 150- micro m diameter) were isolated from human cremasteric muscles and pressurized to physiologic levels before exposure to an endotoxin-rich effluent with and without an upstream conduit vessel (superficial epigastric vein) . The vasodilatory effect was measured with videomicroscopy and compared with control samples . MAIN OUTCOME MEASURES: Mean vessel diameter and percentage of loss in tone . RESULTS: When compared with controls, the isolated arteriole had no significant response when exposed to endotoxin alone (3.5% change in basal tone) . When the endotoxin was allowed to pass over an upstream conduit vessel, the arteriole showed marked dilation (14.2% loss of basal tone) . CONCLUSIONS: This study demonstrates that endotoxin has no direct vasodilatory effect on human skeletal muscle arterioles, but it is the release of an endothelial factor from the upstream conduit vessels that produces the loss of tone in the microvasculature . Further research is ongoing to characterize the factors involved (nuclear factor-kappaB, tumor necrosis factor alpha, and interleuklin 6) for possible clinical intervention (antioxidants, cyclosporine, and nitric oxdide synthase inhibitors). Res Vet Sci, 2004 Oct, 77(2), 93 - 100 Q fever (coxiellosis): epidemiology and pathogenesis; Woldehiwet Z; Q fever is a widespread zoonosis caused by the Gram-negative bacterium Coxiella burnetii . Aborting domestic ruminants are the main sources of human infection but the reservoir of infection is extremely wide . In humans, Q fever may occur as acute pneumonia, hepatitis or flu-like illness or may take a severe chronic form, characterized by endocarditis, chronic hepatitis and chronic fatigue syndrome . In animals, the main clinical manifestation is late abortion . Infection with C . burnetii can be diagnosed using cultural, serological and genetic methods but because the organism is potentially dangerous and requires specialized skills only specialist laboratories are capable of undertaking diagnostic tests . This paper provides a brief overview of the epidemiology and pathogenesis of Q fever (coxiellosis). Gut, 2004 Jul, 53(7), 987 - 92 Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis; Franchimont D et al.; BACKGROUND AND AIMS: Elicitation of an innate immune response to bacterial products is mediated through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) and the NODs . The recently characterised Asp299Gly polymorphism in the lipopolysaccharide (LPS) receptor TLR4 is associated with impaired LPS signalling and increased susceptibility to Gram negative infections . We sought to determine whether this polymorphism was associated with Crohn's disease (CD) and/or ulcerative colitis (UC) . METHODS: Allele frequencies of the TLR4 Asp299Gly polymorphism and the three NOD2/CARD15 polymorphisms (Arg702Trp, Gly908Arg, and Leu1007fsinsC) were assessed in two independent cohorts of CD patients (cohort 1, n = 334; cohort 2, n = 114), in 163 UC patients, and in 140 controls . A transmission disequilibrium test (TDT) was then performed on 318 inflammatory bowel disease (IBD) trios . RESULTS: The allele frequency of the TLR4 Asp299Gly polymorphism was significantly higher in CD (cohort 1: 11% v 5%, odds ratio (OR) 2.31 (95% confidence interval (CI) 1.28-4.17), p = 0.004; and cohort 2: 12% v 5%, OR 2.45 (95% CI 1.24-4.81), p = 0.007) and UC patients (10% v 5%, OR 2.05 (95% CI 1.07-3.93), p = 0.027) compared with the control population . A TDT on 318 IBD trios demonstrated preferential transmission of the TLR4 Asp299Gly polymorphism from heterozygous parents to affected children (T/U: 68/34, p = 0.01) . Carrying polymorphisms in both TLR4 and NOD2 was associated with a genotype relative risk (RR) of 4.7 compared with a RR of 2.6 and 2.5 for TLR4 and NOD2 variants separately . CONCLUSION: We have reported on a novel association of the TLR4 Asp299Gly polymorphism with both CD and UC . This finding further supports the genetic influence of PRRs in triggering IBD. Apoptosis, 2004 Jul, 9(4), 467 - 74 LPS-induced apoptosis is dependent upon mitochondrial dysfunction; Kuwabara T et al.; Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN) . Apoptosis occurs in a manner that is independent of bacterial virulence proteins . In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells . LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases . While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential . Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP . These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells . Harefuah, 2004 May, 143(5), 364 - 7, 390 {Q fever pericarditis}; Edelstein S et al.; Q fever is a zoonotic disease caused by Coxiella burnetii--an obligate, gram negative, intracellular bacteria . The term Q (Query) was first used because at the time the disease was named its etiology was unknown . Q fever is divided into acute and chronic infections characterized by different evolution, serological profiles and treatment . Pericarditis, as a manifestation of Q fever is rare and difficult to diagnose . This is due to the following: firstly, the clinical presentation of acute Q fever is pleomorphic, nonspecific and self limited, and secondly, the diagnosis relies on the physician's interest and the presence of a reliable diagnostic laboratory . The objective of that review is to increase the physician's awareness of the clinical presentation of Q fever, to discuss the importance of the diagnosis and laboratory tests and to guide the physician as to when to provide treatment and the relevant patient population to be treated. Annu Rev Biochem, 2004, 73, 107 - 46 Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins; Tugarinov V et al.; Recent developments in NMR spectroscopy, which include new experiments that increase the lifetimes of NMR signals or that precisely define the orientation of internuclear bond vectors with respect to a common molecular frame, have significantly increased the size of proteins for which quantitative structural and dynamic information can be obtained . These experiments have, in turn, benefited from new labeling strategies that continue to drive the field . The utility of the new methodology is illustrated by considering applications to malate synthase G, a 723 residue enzyme, which is the largest single polypeptide chain for which chemical shift assignments have been obtained to date . New experiments developed specifically to address the complexity and low sensitivity of spectra recorded on this protein are presented . A discussion of the chemical information that is readily available from studies of systems in the 100 kDa mol wt range is included . Prospects for membrane protein structure determination are discussed briefly in the context of an application to an Escherichia coli enzyme, PagP, localized to the outer membrane of gram-negative bacteria. Proteomics, 2004 May, 4(5), 1280 - 92 Proteome analysis of Madrid E strain of Rickettsia prowazekii; Chao CC et al.; Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus . The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol . Consequently, it is listed as a select agent and warrants more research to understand its pathogenesis . Although the genomic DNA sequence of strain Madrid E has been completed, the actual expression of the individual protein has not been investigated . In order to provide a global view of the expressed protein profile, the whole cell lysate of purified rickettsia (Madrid E strain) was reduced, alkylated, and digested with trypsin . The total digest was characterized by a two-dimensional liquid chromatography mass spectrometry system and analyzed with a modified version of the ProteomeX workstation . A total of 252 proteins out of 834 predicted protein-coding genes were identified, 238 proteins were identified by the detection of at least two unique peptides . Only 14 proteins were identified by the detection of one unique peptide in all three separate analyses . Among the 238 proteins identified by multiple unique peptides, 230 proteins were found in at least two of three separate analyses . The reproducible and convenient methodology and the information described here have provided a foundation for future proteome study of various R . prowazekii strains with different virulence. Chembiochem, 2004 Apr 2, 5(4), 491 - 9 Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars; Jose J et al.; Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups . In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed . For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay . Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway . Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions . The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS) . The activity of whole cells displaying SDH at the surface was determined in an optical test . Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose . The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells). J Biol Chem, 2004 Aug 20, 279(34), 35709 - 18 Epub 2004 Jun 07. Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter; Clarke BR et al.; The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis . This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria . Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule . The polymannan O antigens have nonreducing terminal methyl groups . The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD(O8) enzyme, and this modification terminates polymerization . Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation . The bifunctional WbdD(O9a) catalyzes both reactions, but only the kinase activity controls chain length . Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export . An E . coli wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane. Appl Environ Microbiol, 2004 Jun, 70(6), 3687 - 94 Identification of differential gene expression in bacteria associated with coral black band disease by using RNA-arbitrarily primed PCR; Frias-Lopez J et al.; RNA-arbitrarily primed PCR techniques have been applied for the first time to identify differential gene expression in black band disease (BBD), a virulent coral infection that affects reef ecosystems worldwide . The gene activity for the BBD mat on infected surfaces of the brain coral Diploria strigosa was compared with that for portions of the BBD mat that were removed from the coral and suspended nearby in the seawater column . The results obtained indicate that three genes (DD 95-2, DD 95-4, and DD 99-9) were up-regulated in the BBD bacterial mat on the coral surface compared to the transcript base levels observed in the BBD mat suspended in seawater . Clone DD 95-4 has homology with known amino acid ABC transporter systems in bacteria, while clone DD 99-9 exhibits homology with chlorophyll A apoprotein A1 in cyanobacteria . This protein is essential in the final conformation of photosystem I P700 . DD 95-2, the only gene that was fully repressed in the BBD mat samples suspended in seawater, exhibited homology with the AraC-type DNA binding domain-containing proteins . These transcriptional activators coordinate the expression of genes essential for virulence in many species of gram-negative bacteria. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Jun, 16(6), 358 - 60 {Anti-endotoxin core glycolipid antibody: the preparation of immune serum of E . Coli J5}; Xu XN et al.; OBJECTIVE: To prepare high titer anti-endotoxin core glycolipid (J5) antibody (CGL) for the treatment of Gram-negative bacteremia and septic shock . METHODS: Nontoxic bacterial vaccine (50 x 10(12)U/L) against E.Coli O111:B4 mutant strain J5 was prepared . J5 bacterial vaccine was injected into rabbits through ear marginal vein (saline as control preparation), one time pre three days, totally five times . Injected doses were as following: 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml, and 0.8 ml . One week after fifth injection, blood samples from heart were collected and immune serum was isolated . Indirect clotting test was used to determine the titer of antibody and cross reaction . RESULTS: Among 12 immunized rabbits, titers of antibody against E . Coli J5 were exceeding 1:1 024 in 6 rabbits, and they had cross reaction with various kinds of Gram-negative bacterial endotoxins . CONCLUSION: The titer of anti-endotoxin core glycolipid (E.Coli J5) antibody prepared by us appears to be high, and it can combine with various kinds of Gram-negative bacterial endotoxins. Scand J Immunol, 2004 Jun, 59(6), 553 - 8 Anti-inflammatory cytokines induce lipopolysaccharide tolerance in human monocytes without modifying toll-like receptor 4 membrane expression; Moreno C et al.; Toll-like receptor 4 (TLR4) participates in innate immunity by detecting lipopolysaccharides (LPS) of Gram-negative bacterial cell walls . TLR4 macrophage expression in mice is modulated by LPS . This fact constitutes, at least partially, the molecular basis for LPS tolerance . Very recently, the effect of interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, has been described on TLR4 membrane expression of human monocytes . IFN-gamma up-regulates TLR4 expression and antagonizes the LPS-induced TLR4 down-regulation . These data prompted us to study the expression of membrane TLR4 in human mono- cytes in which LPS tolerance was induced by LPS and by anti-inflammatory cytokines {interleukin-10 (IL-10) and transforming growth factor beta1 (TGFbeta1)} . Data concerning this latter model, and more specifically, the effect of anti-inflammatory cytokines over TLR4 expression, are not available at present . We show here that membrane TLR4 expression in human monocytes falls after LPS exposure . The effect was prolonged for 12 h, but then expression returned to normal levels . The incubation of human monocytes with IL-10, TGFbeta1 or a mixture of both induces no alterations in membrane TLR4 expression . However, these cytokines are able to substitute the tolerizing LPS exposure in order to induce LPS tolerance . Our data help to achieve a better understanding of the way cytokines control the cellular expression of TLR. J Biol Chem, 2004 Aug 13, 279(33), 34833 - 9 Epub 2004 Jun 04. Solution structure of Cox11, a novel type of beta-immunoglobulin-like fold involved in CuB site formation of cytochrome c oxidase; Banci L et al.; Cytochrome c oxidase assembly process involves many accessory proteins including Cox11, which is a copper-binding protein required for Cu incorporation into the Cu(B) site of cytochrome c oxidase . In a genome wide search, a number of Cox11 homologs are found in all of the eukaryotes with complete genomes and in several Gram-negative bacteria . All of them possess a highly homologous soluble domain and contain an N-terminal fragment that anchors the protein to the membrane . An anchor-free construct of 164 amino acids was obtained from Sinorhizobium meliloti, and the first structure of this class of proteins is reported here . The apoform has an immunoglobulin-like fold with a novel type of beta-strand organization . The copper binding motif composed of two highly conserved cysteines is located on one side of the beta-barrel structure . The apoprotein is monomeric in the presence of dithiothreitol, whereas it dimerizes in the absence of the reductant . When copper(I) binds, NMR and extended x-ray absorption fine structure (EXAFS) data indicate a dimeric protein state with two thiolates bridging two copper(I) ions . The present results advance the knowledge on the poorly understood molecular aspects of cytochrome c oxidase assembly. J Bacteriol, 2004 Jun, 186(12), 3903 - 10 Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens; Shouldice SR et al.; We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A . M . haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever . The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host . The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion . The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion . Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm. J Bacteriol, 2004 Jun, 186(12), 3814 - 25 Regulation of hypercompetence in Legionella pneumophila; Sexton JA et al.; Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable . Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake . We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction . For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37 degrees C . However, it was previously reported that microaerophilic growth at 37 degrees C allows L . pneumophila serogroup 1 strain AA100 to be naturally transformed . Here we report that another L . pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions . Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30 degrees C . In contrast to Lp02, AA100 is only minimally transformable at 30 degrees C, indicating that Lp02 is hypercompetent under these conditions . To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake . Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L . pneumophila . This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable. J Bacteriol, 2004 Jun, 186(12), 3712 - 20 The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures; Soderberg MA et al.; The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite . The PilD prepilin peptidase of L . pneumophila promotes type IV pilus biogenesis and type II protein secretion . Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence . Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37 degrees C . Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L . pneumophila is grown at 30, 25, and 17 degrees C . Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures . Whereas strains specifically lacking type IV pili were not defective for low-temperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L . pneumophila to form colonies at 25, 17, and 12 degrees C . Indeed, the lsp mutants were completely unable to grow at 12 degrees C . The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene . Interestingly, the lsp mutants displayed improved growth at 25 degrees C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth . Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25 degrees C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s) . In summary, these data document, for the first time, that L . pneumophila replicates at temperatures below 20 degrees C and that a bacterial type II protein secretion system facilitates growth at low temperatures. Proteomics, 2004 Jun, 4(6), 1597 - 613 Fine-tuning the prediction of sequences cleaved by signal peptidase II: a curated set of proven and predicted lipoproteins of Escherichia coli K-12; Gonnet P et al.; A curated set of 81 proven and 44 predicted lipoproteins of Escherichia coli K-12 was defined with the combined use of a literature survey, a variety of predictive tools and human expertise . The well-documented Gram-negative proteome of E . coli K-12 was chosen to assess how the different approaches complement each other and to ensure a stable definition of a consistent set of lipoproteins . The results of detailed analysis of such proteins at the level of a single proteome are presented, corroborated and rationalized. J Fr Ophtalmol, 2004 Apr, 27(4), 420 - 3 {Rules and regulations concerning contact lens-related infection}; Feys J; Contact lens-related infectious keratitis is a potentially sight-threatening complication . Bacterial keratitis, mostly due to Gram-negative bacteria, is associated with poor lens hygiene, overnight wear, and contaminated lens care solutions . Contamination of the lens storage case may cause fungal keratitis . Acanthamoeba infection is related to the use of tap water or swimming while wearing soft lenses . Viruses are of less concern among contact lens wearers . Possible transmission of Creutzfeldt-Jakob disease by multi-patient trial lenses must be taken in account . To minimize these risk factors, regulations are applied at various levels: CE marking of contact lenses and care products as they are medical devices; contact lens fitting only by health care professionals; distribution of contact lenses by opticians and lens care solutions by opticians and pharmacists; hygienic management of trial lenses following official recommendations . Contact lens-related keratitis must be reported to health care Authorities. Chem Phys Lipids, 2004 Jul, 130(2), 83 - 98 The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes; McIntosh TJ; This review details how bilayer structural/elastic properties impact three distinct areas of biological significance . First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition . The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage . Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains ("rafts") enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer . This is likely due to the large area compressibility modulus of SM:cholesterol bilayers . Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side . We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol's strong interaction with saturated chains. Biosci Biotechnol Biochem, 2004 May, 68(5), 1146 - 8 Isolation of an exopolysaccharide-producing bacterium, Sphingomonas sp . CS101, which forms an unusual type of sphingan; Seo EJ et al.; An exopolysaccharide-producing Gram negative bacterium was isolated and determined to be a Sphingomonas sp . (CS101) . A sugar composition analysis of an exopolysaccharide indicated that the Sphingomonas sp . CS101 secreted an exopolysaccharide composed of glucose, mannose, fucose, and rhamnose in the ratio of 2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is an unusual type of sphingan family . The mean molecular weight of the exopolysaccharide was determined to be 4.2x10(5) Da by size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis . An exopolysaccharide was produced up to 17 g/l (pH 7; 30 degrees C) with the optimal medium condition over 4 days of cultivation. Shock, 2004 Jun, 21(6), 566 - 71 Increased lymphoid tissue apoptosis in baboons with bacteremic shock; Efron PA et al.; The molecular mechanisms of immune cell apoptosis during sepsis remain unclear . Two young adult baboons (Papio sp.) received a lethal dose of live Escherichia coli and were sacrificed at either 16 (for animal welfare concerns) or 24 h post-septic shock . An additional baboon, which received no bacteria, served as a control . Necropsy was performed immediately with subsequent immunohistochemical staining of lymphoid tissue . Immunohistologic analysis of tissues from the septic baboons revealed marked systemic lymphocyte apoptosis occurring in all lymphoid tissues examined . Focally, pyknotic and karyorrhectic lymphocytes demonstrated activation of a mitochondrial-dependent cell death pathway (active caspase 9 and apoptosis-inducing factor) . Other regions demonstrated apoptotic lymphocytes with activation of a death receptor-dependent cell pathway (Fas ligand) . Thus, we have demonstrated for the first time in primates that overwhelming gram-negative bacteremia produces an early and profound lymphocyte death that occurs through multiple cell death pathways . Bacteremic shock in the baboon may be an appropriate model for studying experimental therapies aimed at blocking lymphocyte apoptosis because their response appears comparable to humans dying from sepsis. Can J Microbiol, 2003 Dec, 49(12), 733 - 40 Controlled induction of the RpoS regulon in Escherichia coli, using an RpoS-expressing plasmid; Chen G et al.; RpoS, an alternative sigma factor produced by many gram-negative bacteria, primarily controls genes that are expressed in stationary phase in response to nutrient deprivation . To test the idea that induction of RpoS in the exponential phase, when RpoS is not normally expressed, increases RpoS-dependent gene expression, we constructed a plasmid carrying the rpoS gene under the control of an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible T7lac promoter . Northern and Western analyses revealed that levels of RpoS mRNA and protein, respectively, increased in response to the inducer IPTG . Assays of changes in RpoS-dependent functions (catalase activity and glycogen accumulation), confirmed that induced RpoS was functional in exponential phase and was sufficient for the expression of RpoS-dependent functions . Controlled expression of RpoS and RpoS-dependent genes by plasmid-encoded rpoS may thus offer a useful tool for the study of RpoS-dependent gene expression. Invest Ophthalmol Vis Sci, 2004 Jun, 45(6), 1871 - 8 Expression of toll-like receptor 4 and its associated lipopolysaccharide receptor complex by resident antigen-presenting cells in the human uvea; Chang JH et al.; PURPOSE: To investigate the in vivo expression of toll-like receptor 4 (TLR4) and its associated lipopolysaccharide (LPS) receptor complex in the human eye . METHODS: Normal human ocular tissues were evaluated for in vivo TLR4, MD-2, and CD14 mRNA and protein expression by RT-PCR and immunohistochemistry, respectively . The distribution patterns and phenotypes of the cells expressing these proteins were further characterized by confocal microscopy and double-label immunofluorescence studies . RESULTS: Normal human uvea, retina, sclera, and conjunctiva constitutively expressed TLR4, MD-2, and CD14 mRNA . The protein expression of these molecules was restricted, however, to resident antigen-presenting cells (APCs) in the normal human uvea, consisting mainly of HLA-DR(+) dendritic cells (DCs) . These APCs endowed with the complete LPS receptor complex appeared to be strategically positioned in perivascular and subepithelial locations for surveying blood-borne or intraocular LPS . In contrast, other cell types of the normal human cornea, conjunctiva, retina, and sclera did not express TLR4/MD-2 protein in vivo as detectable by immunohistochemistry . CONCLUSIONS: The present study demonstrates for the first time that resident APCs in the normal human uvea express TLR4 and its associated LPS receptor complex . This has significant implications for the understanding of normal ocular immunity as well as unraveling the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis (AAU). Med Pr, 2004, 55(1), 31 - 40 {Occupational bio hazards: current issues}; Dutkiewicz J; Over the last decade, there was noted a large advancement of knowledge on living organisms and their products posing a potential occupational risk . Novel risk factors, often new to science, were identified, the role and significance of already known factors better comprehended, and occupational groups endangered by biological hazards more thoroughly recognized . Novel viruses and prions, emerging in different parts of the world, may pose a particular threat to health and life of health care workers, agriculture workers and veterinarians . A new coronavirus (SCoV) that evoked a rapid outbreak of disease described as severe acute respiratory syndrome (SARS) in the first half of 2003 may serve as an example . The disease was particularly common among health care workers . Previously discovered zoonotic viruses, Nipah virus in pigs and Hendra virus in horses, may be a cause of fatal encephalitis in animal farmers . Hantaviruses (Puumala, Hantaan, Sin Nombre and others) infecting field rodents may be a cause of hemorrhagic fever with renal syndrome (HFRS) or pulmonary syndrome (HPS) in farmers and laboratory workers . Prions responsible for inducing a zoonotic variant of Creutzfeldt-Jakob disease (vCJD) are considered to be a potential cause of work-related infections in agricultural and health care workers, however, this assumption has not as yet been supported by any conclusive evidence . In many countries, blood-borne occupational infections with hepatitis C virus (HCV) is the major epidemiological problem among health care workers, mostly because no vaccine against this virus has been produced to date . Vaccinations effectively restricted the number of occupational infections with hepatitis B virus (HBV), and work-related infections with human immunodeficiency virus (HIV) causing acquired immunodeficiency syndrome (AIDS) are very rare . Hazardous bioserosols, occurring in many work environments, pose an occupational health hazard of particular importance . Many new biological factors present in organic dusts that may induce work-related allergic and immunotoxic diseases among farmers and workers of the agricultural and wood industries have been identified . Droplet aerosols, which are generated from water, oils, oil-water emulsions and other liquids in various work environments, may contain infectious agents (Legionella spp.) as well as allergic and/or toxic agents . It has been shown that allergens and endotoxins produced by Gram-negative bacteria occurring in oil mist from metalworking fluids may cause occupational respiratory diseases in workers of the metallurgic industry. Clin Infect Dis, 2004 Jun 1, 38(11), 1579 - 84 Epub 2004 May 07. Clinical significance of Roseomonas species isolated from catheter and blood samples: analysis of 36 cases in patients with cancer; De I et al.; This report analyzes 36 cases of bacteremia or catheter-related infection caused by Roseomonas species, a group of pink, slimy, waterborne, gram-negative coccobacilli . The causative species included the newly described Roseomonas mucosa (22 cases {61%}) and Roseomonas gilardii subspecies rosea (8 cases {22%}) and known species R . gilardii subspecies gilardii (5 cases {14%}) and Roseomonas genomospecies 4 (1 case {3%}) . Twenty-nine (81%) of the cases were symptomatic, with fever being the most common symptom (in 27 {75%} of the cases) . Twenty (56%) of the infections were monomicrobic . Six cases (17%) involved persistent catheter colonization, and 5 of these cases required removal of the catheter to clear the infection . All infections resolved, most with empirical antibiotic treatment . A summary of the antibiotic susceptibility pattern of these strains and other reported series show that Roseomonas species are consistently susceptible to amikacin and imipenem and frequently susceptible to ciprofloxacin and ticarcillin, but essentially nonsusceptible to ceftazidime and cefepime . This result may guide future therapy for infections due to Roseomonas species. J Biol Chem, 2004 Jul 30, 279(31), 32116 - 24 Epub 2004 May 20. AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex; Tikhonova EB et al.; Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes . These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm . In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli . We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification . All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC . We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex . However, the formation of the AcrAB-TolC complex does not require an input of energy. Infect Immun, 2004 Jun, 72(6), 3350 - 8 Suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by Bordetella pertussis and identification of an immunodominant lipoprotein; Carbonetti NH et al.; Pertussis toxin (PT), a virulence factor secreted by Bordetella pertussis, contributes to respiratory tract infection and disease caused by this pathogen . By comparing a wild-type (WT) B . pertussis strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (DeltaPT), we recently found that the lack of PT confers a significant defect in respiratory tract colonization in mice after intranasal inoculation . In this study, we analyzed serum antibody responses in mice infected with the WT or DeltaPT strain and found that infection with the DeltaPT strain elicited greater responses to several B . pertussis antigens than did infection with the WT, despite the lower colonization level achieved by the DeltaPT strain . The same enhanced antibody response was observed after infection with a strain expressing an enzymatically inactive PT; but this response was not observed after infection with B . pertussis mutant strains lacking filamentous hemagglutinin or adenylate cyclase toxin, nor when purified PT was administered with the DeltaPT inoculum, indicating a specific role for PT activity in this immunosuppressive effect . In particular, there were consistent strong serum antibody responses to one or more low-molecular-weight antigens after infection with the DeltaPT strain . These antigens were Bvg independent, membrane localized, and also expressed by the closely related pathogens Bordetella parapertussis and Bordetella bronchiseptica . Two-dimensional gel electrophoresis and mass spectrometry were used to identify one of the immunodominant low-molecular-weight antigens as a protein with significant sequence homology to peptidoglycan-associated lipoprotein in several other gram-negative bacterial species . However, a serum antibody response to this protein alone did not protect mice against respiratory tract infection by B . pertussis. Infect Immun, 2004 Jun, 72(6), 3260 - 6 Lipopolysaccharide protects primary B lymphocytes from apoptosis by preventing mitochondrial dysfunction and bax translocation to mitochondria; Souvannavong V et al.; Mature B lymphocytes undergo apoptosis when they are cultured in the absence of survival factors . Gram-negative bacterial lipopolysaccharide (LPS) prevents this spontaneous apoptosis . This study aimed to better define the signaling pathway(s) involved in the antiapoptotic activity of this endotoxin . We report here that, in addition to its effects on spontaneous apoptosis, LPS protects B cells from apoptosis induced by the broad-spectrum protein kinase inhibitor staurosporine . LPS increased cell viability and concomitantly maintained the mitochondrial transmembrane potential (DeltaPsim) and high glutathione levels . Moreover, LPS inhibited cytosolic cytochrome c release and decreased caspase-9 activation . Unlike staurosporine, LPS induced the retention of Bax, a proapoptotic protein of the Bcl-2 family, in the cytosol by preventing its translocation to mitochondria . These results suggest that Bax relocalization from the cytosol to the mitochondria is an important step of mature B-cell apoptosis and that the antiapoptotic activity of LPS occurs upstream of mitochondrial events. Infect Immun, 2004 Jun, 72(6), 3252 - 9 Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: safety, immunogenicity, and efficacy; Rossi G et al.; Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer . The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits . The present study describes the therapeutic vaccination of experimentally H . pylori-infected beagle dogs, an animal model that reproduces several aspects of the human infection with H . pylori . The vaccine consisted of three recombinant H . pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 microg each) with alum and administered intramuscularly either weekly or monthly . No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens . Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule . In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H . pylori colonization and the related gastric pathology. J Mol Microbiol Biotechnol, 2003, 6(3-4), 164 - 73 Analysis of fruE, a novel developmental gene of Myxococcus xanthus; Akiyama T et al.; Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation . In the present study, a TnV insertion developmental mutation, Omega773, of M . xanthus was analyzed . The TnV Omega773 insertion was found to be located within a novel developmental gene, fruE . The FruE protein is composed of 140 amino acid residues and bears an N-terminal signal peptide . The amino acid sequence of FruE shared no significant similarity with any other known protein in the databases . The fruE mutant displayed a development-delayed phenotype . The formation of tightly aggregated mounds in the fruE mutant was slower than that in the wild-type strain . The initiation of spore production in the fruE mutant was delayed by 12 h in comparison to the wild-type strain, and the process of spore formation was more asynchronous than that of the wild-type strain . The transcription initiation sites of the fruE gene were located 81 bp (P1) and 57 bp (P2) upstream of the fruE initiation codon . Although both promoters were active during vegetative growth and development, the P1 promoter was more active during development and the P2 promoter was more active during vegetative growth . The expression of the fruE gene increased to a peak at 6 h poststarvation and then decreased . The decrease in fruE expression was not observed in the D and E signal mutants . J Comput Aided Mol Des, 2004 Jan, 18(1), 1 - 11 Theoretical study of selective methylation in the synthesis of azithromycin; Duran D et al.; Azithromycin is a 15-membered macrolide antibiotic which is active in vitro against clinically important gram-negative bacteria . In this study, the selectivity of the methylation mechanism was analyzed computationally on the 2'-OCbz-3'-NMeCbz derivative of azithromycin in vacuum and in DMF . We have shown that the methylation of the hydroxy group on C-6 is energetically unfavorable compared to the other hydroxy groups in vacuum; the softness values further showed that the C-6 anion is not reactive towards CH3I in the methylation mechanism . To understand the effect of the solvent on the methylation process, detailed molecular dynamics simulations were performed in DMF using the anions at the C-4", C-6, C-11 and C-12 positions . We find the conformations of the anions not to be affected by the presence of the solvent . The radial distribution functions of the solvent molecules around the O- of the anions demonstrate that DMF molecules cluster around the C-6 anion . The relative strength of the anion-solvent interactions reveal that the solvent molecules provide the largest stabilization to the C-6 anion and prevent the methylation at this position . The latter descriptor was found to be an important factor in explaining the experimentally observed selectivity towards the methylation of the C-4", C-6, C-11 and C-12 anions. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 877 - 83 Prevotella shahii sp . nov . and Prevotella salivae sp . nov., isolated from the human oral cavity; Sakamoto M et al.; Two bacterial strains, EHS11(T) and EPSA11(T), which were isolated from the human oral cavity, were characterized in terms of phenotypic and biochemical characteristics, cellular fatty acid profiles and phylogenetic position based on 16S rRNA gene sequence analysis . 16S rRNA gene sequence analysis showed that each of the isolates belonged to a novel species of the genus Prevotella . Strain EHS11(T) was related to Prevotella loescheii (about 95 % similarity), whereas strain EPSA11(T) was related to Prevotella oris (about 94 % similarity) . Both strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods . The cellular fatty acid composition of strain EPSA11(T) was very similar to that of P . oris JCM 8540(T) . On the other hand, the cellular fatty acid composition of strain EHS11(T) was significantly different from those of other Prevotella species . The predominant fatty acids in strain EHS11(T) are C(18 : 1)omega9c, C(16 : 0) and C(16 : 0) 3-OH, whereas other Prevotella species, except for P . loescheii JCM 8530(T), possess anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(18 : 1)omega9c . The predominant fatty acids in P . loescheii JCM 8530(T) are anteiso-C(15 : 0), C(16 : 0) and C(18 : 1)omega9c . DNA-DNA hybridization experiments revealed a genomic distinction of strains EHS11(T) and EPSA11(T) from P . loescheii JCM 8530(T) and P . oris JCM 8540(T) . On the basis of these data, two novel Prevotella species are proposed: Prevotella shahii sp . nov . and Prevotella salivae sp . nov . The type strains of P . shahii and P . salivae are EHS11(T) (=JCM 12083(T)=DSM 15611(T)) and EPSA11(T) (=JCM 12084(T)=DSM 15606(T)), respectively. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 847 - 50 Pseudomonas lutea sp . nov., a novel phosphate-solubilizing bacterium isolated from the rhizosphere of grasses; Peix A et al.; A phosphate-solubilizing bacterial strain designated OK2(T) was isolated from rhizospheric soil of grasses growing spontaneously in a soil from Spain . Cells of the strain were Gram-negative, strictly aerobic, rod-shaped and motile . Phylogenetic analysis of the 16S rRNA gene indicated that this bacterium belongs to the gamma-subclass of Proteobacteria within the genus Pseudomonas and that the closest related species is Pseudomonas graminis . The strain produced catalase but not oxidase . Cellulose, casein, starch, gelatin and urea were not hydrolysed . Aesculin was hydrolysed . Growth was observed with many carbohydrates as carbon sources . The main non-polar fatty acids detected were hexadecenoic acid (16 : 1), hexadecanoic acid (16 : 0) and octadecenoic acid (18 : 1) . The hydroxy fatty acids detected were 3-hydroxydecanoic acid (3-OH 10 : 0), 3-hydroxydodecanoic acid (3-OH 12 : 0) and 2-hydroxydodecanoic acid (2-OH 12 : 0) . The G+C DNA content determined was 59.3 mol% . DNA-DNA hybridization showed 48.7 % relatedness between strain OK2(T) and P . graminis DSM 11363(T) and 26.2 % with respect to Pseudomonas rhizosphaerae LMG 21640(T) . Therefore, these results indicate that strain OK2(T) (=LMG 21974(T)=CECT 5822(T)) belongs to a novel species of the genus Pseudomonas, and the name Pseudomonas lutea sp . nov . is proposed. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 699 - 703 Legionella drancourtii sp . nov., a strictly intracellular amoebal pathogen; La Scola B et al.; A Legionella-like amoebal pathogen (LLAP), formerly named LLAP12(T), was characterized on the basis of microscopic appearance, staining characteristics, growth in Acanthamoeba polyphaga at different temperatures, DNA G+C content, serological cross-reactivity and 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analysis . LLAP12(T) was found to be a motile, Gram-negative bacterium that grew within cytoplasmic vacuoles in infected amoebae . The infecting bacteria induced lysis of their amoebal hosts and time taken to do so was dependent on incubation temperature . Recovery of LLAP12(T) from amoebae onto axenic media could not be achieved . Phylogenetic analysis of LLAP12(T), based on 16S rRNA and mip gene sequence analysis, indicated that it lay within the radiation of the Legionellaceae and that it clustered specifically with Legionella lytica and Legionella rowbothamii . The divergence observed between LLAP12(T) and these two species was of a degree equal to, or greater than, that observed between other members of the family . In support of this delineation, LLAP12(T) was found not to cross-react serologically with any other Legionella species . The mip and 16S rRNA gene sequence-based analyses also indicated that LLAP12(T) was related very closely to two other previously identified LLAP isolates, LLAP4 and LLAP11 . Taken together, these results support the proposal of LLAP12(T) as the type strain of Legionella drancourtii sp . nov. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 693 - 7 Agarivorans albus gen . nov., sp . nov., a gamma-proteobacterium isolated from marine animals; Kurahashi M et al.; Six bacterial strains were isolated from healthy marine organisms that were collected from the coast of the Kanto area in Japan . Phylogenetic analysis based on 16S rDNA sequence similarity showed that the six isolates formed a separate cluster in the gamma-Proteobacteria and were related to the genera Alteromonas and Glaciecola (<91.6 % similarity) . The isolates were related closely to each other (DNA-DNA reassociation values of 74-93 %) . The isolates had a polar flagellum and were Gram-negative, mesophilic, strictly aerobic rods that required salt for growth . Distinct phenotypic features of this group included the ability to hydrolyse agar and white pigmentation of colonies . The DNA G+C content of the isolates was 48-50 mol% . The major quinone was Q-8 . Phenotypic characteristics of the isolates differed from those of members of the genera Alteromonas and GLACIECOLA: The name Agarivorans albus gen . nov., sp . nov . is proposed for the six isolates; the type strain is MKT 106(T) (=IAM 14998(T)=LMG 21761(T)). Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 669 - 73 Reinekea marinisedimentorum gen . nov., sp . nov., a novel gammaproteobacterium from marine coastal sediments; Romanenko LA et al.; A Gram-negative, oxidase- and catalase-positive, rod-shaped bacterium, designated strain KMM 3655(T), was isolated from a coastal marine sediment sample . The novel bacterium required sodium ions for growth and grew between 0.5 and 5 % NaCl and at 4-37 degrees C, but not at 40 degrees C . It reduced nitrate, formed acids from glucose under aerobic and anaerobic conditions, utilized a limited spectrum of organic substrates and did not produce gelatinase, caseinase, amylase or chitinase . The major isoprenoid quinone was Q8 . Polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid . Fatty acid analysis of strain KMM 3655(T) revealed C(16 : 0), C(16 : 1)omega7c and C(18 : 1)omega7c as predominant components . The G+C content of the DNA was 51.1 mol% . Phylogenetic analysis of the 16S rDNA sequence placed the new isolate within the gamma-Proteobacteria as a separate deep branch, with about 90 % sequence similarity to representatives of the genus Oceanospirillum and other remotely related genera . Combined phylogenetic and physiological data show that the new marine sediment isolate, KMM 3655(T), represents a novel genus and species, for which the name Reinekea marinisedimentorum gen . nov., sp . nov . is proposed . The type strain is KMM 3655(T) (=DSM 15388(T)). Environ Microbiol, 2004 Jun, 6(6), 611 - 21 Lentisphaera araneosa gen . nov., sp . nov, a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae; Cho JC et al.; Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155(T) and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format . When cultured in low-nutrient seawater media, these strains copiously produced Alcian Blue-stainable viscous TEP . Growing cells were attached to each other by the TEP in a three dimensional network . Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans . The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community . In phylogenetic analyses, the strains consistently formed a new phylum-level lineage within the domain Bacteria, together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats . Five putative subgroups were delineated within this phylum-level lineage, including a marine group and an anaerobic group . The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere-shaped bacteria . The DNA G+C content of strain HTCC2155(T) was 48.3 mol% and the genome size was 2.9 mb . It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155(T) = ATCC BAA-859(T) = KCTC 12141(T)) gen . nov., sp . nov., the cultured marine representative of the Lentisphaerae phyl . nov., and the phylum be divided into two novel orders named the Lentisphaerales ord . nov . and the Victivallales ord . nov. Mol Plant Microbe Interact, 2004 May, 17(5), 447 - 55 The HopPtoF locus of Pseudomonas syringae pv . tomato DC3000 encodes a type III chaperone and a cognate effector; Shan L et al.; Type III secretion systems are highly conserved among gram-negative plant and animal pathogenic bacteria . Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells . Analysis of the whole genome sequence of Pseudomonas syringae pv . tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P . syringae pv . phaseolicola . The HopPtoF locus harbors two genes, ShcF(Pto) and HopF(Pto), that are preceded by a single hrp box promoter . We present evidence here to show that ShcF(Pto) and HopF(Pto) encode a type III chaperone and a cognate effector, respectively . ShcF(Pto) interacts with and stabilizes the HopF(Pto) protein in the bacterial cell . Translation of HopF(Pto) starts at a rare initiation codon ATA that limits the synthesis of the HopF(Pto) protein to a low level in bacterial cells. Nucleic Acids Res, 2004 May 11, 32(8), 2566 - 77 Print 2004. Predicting transmembrane beta-barrels in proteomes; Bigelow HR et al.; Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence . One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far . Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs . The method carefully attempts to avoid over-fitting the sparse experimental data . While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different . In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score . The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86% . Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy) . This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria . We found over 164 previously uncharacterized TMB proteins at high confidence . Database searches did not implicate any of these proteins with membranes . We challenge that the vast majority of our 164 predictions will eventually be verified experimentally . All proteome predictions and the PROFtmb prediction method are available at services/PROFtmb/. Cell, 2004 May 14, 117(4), 483 - 94 Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease; Wilken C et al.; Gram-negative bacteria respond to misfolded proteins in the cell envelope with the sigmaE-driven expression of periplasmic proteases/chaperones . Activation of sigmaE is controlled by a proteolytic cascade that is initiated by the DegS protease . DegS senses misfolded protein in the periplasm, undergoes autoactivation, and cleaves the antisigma factor RseA . Here, we present the crystal structures of three distinct states of DegS from E . coli . DegS alone exists in a catalytically inactive form . Binding of stress-signaling peptides to its PDZ domain induces a series of conformational changes that activates protease function . Backsoaking of crystals containing the DegS-activator complex revealed the presence of an active/inactive hybrid structure and demonstrated the reversibility of activation . Taken together, the structural data illustrate in molecular detail how DegS acts as a periplasmic stress sensor . Our results suggest a novel regulatory role for PDZ domains and unveil a novel mechanism of reversible protease activation. J Am Chem Soc, 2004 May 19, 126(19), 6005 - 16 Spectroscopic description of the two nitrosyl-iron complexes responsible for fur inhibition by nitric oxide; D'Autreaux B et al.; Ferric uptake regulation protein (Fur) is a global regulator, ubiquitous in Gram negative bacteria, that acts as a transcriptional repressor when it binds ferrous ion . Fur is involved in responses to several types of stress related to iron metabolism, such as stress induced by nitric oxide (NO) generated by macrophages against bacterial invasion . NO was recently shown to react with Fe(2+) ions in FeFur (iron substituted Fur protein) leading to an Fur bound iron-nitrosyl complex, unable to bind DNA, and characterized by a g = 2.03 EPR signal, associated with an S = (1)/(2) ground state . This electronic configuration could arise from either a mononitrosyl-iron {Fe(NO)}(7) or a dinitrosyl-iron {Fe(NO)(2)}(9) complex . The use of several spectroscopic tools such as EPR, ENDOR, FTIR, Mossbauer, and UV-visible spectroscopies as well as mass spectrometry analysis was necessary to characterize the iron-nitrosyl species in Fur . Furthermore, changes of C132 and C137 into glycines by site directed mutagenesis reveal that neither of the two cysteines is required for the formation of the g = 2.03 signal . Altogether, we found that two species are responsible for Fur inhibition in NO stress conditions: the major species, S(1/2), is an {Fe(NO)(2)}(9) (S = (1)/(2)) complex without bound thiolate and the minor species is probably a diamagnetic {Fe(NO)(2)}(8) (S = 0) complex . This is the first characterization of these physiologically relevant species potentially linking iron metabolism and the response to NO toxicity in bacteria. FEMS Microbiol Lett, 2004 May 15, 234(2), 333 - 41 NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins; Joshi SG et al.; Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever . In human infections, the primary target of R . rickettsii infection is vascular endothelium . Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R . rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3 . To understand upstream signaling mechanisms, we have determined the effect of NF-kappaB blockade on the status of different Bcl-2 (B-cell lymphoma 2) proteins in this study . Quantitative analysis following TUNEL and Hoechst staining confirmed that infection of endothelial cells with R . rickettsii for 6 h in the presence of a specific NF-kappaB inhibitor, MG132, resulted in induction of apoptosis . Infection-induced apoptosis of EC was associated with decreased level of Bid and accumulation of Bad, while cytosolic level of Bax remained relatively unchanged . Further, the cellular levels of apoptosis antagonist Bcl-2 were found to be down-regulated and apoptogenic mitochondrial proteins Smac and cytochrome c were released into cytoplasm . These results implicate an important regulatory role for NF-kappaB in controlling the intracellular levels and/or localization of pro- as well as anti-apoptotic proteins of Bcl-2 family, the intricate balance of which is a critical determinant of downstream signaling mechanisms governing cell fate during intracellular infection . Comb Chem High Throughput Screen, 2004 May, 7(3), 239 - 49 Anti-endotoxin agents . 1 . Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents; Wood SJ et al.; Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria . Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient . Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time . We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy . Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds . In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A . The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS . The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range . The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED(50)values. Biochemistry, 2004 May 18, 43(19), 5811 - 9 Kinetics and mechanism of iron(III) complexation by ferric binding protein: the role of phosphate; Gabricevic M et al.; Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp) . This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane . A synergistic anion is required for tight Fe(3+) sequestration by Fbp . Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro . Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5 . The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product . The first three steps exhibit half-lives ranging from ca . 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability . The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate . Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate . A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles . These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site. Microbiology, 2004 May, 150(Pt 5), 1475 - 83 Molecular and functional characterization of type I signal peptidase from Legionella pneumophila; Lammertyn E et al.; Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia . Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated . As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L . pneumophila was of particular interest . This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane . This paper reports the cloning and the transcriptional analysis of the L . pneumophila lepB gene encoding the type I signal peptidase (SPase) . Reverse transcription PCR experiments showed clear lepB expression when L . pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L . pneumophila . In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e . a LepA homologue and a ribonuclease III homologue . SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant . Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens . Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition . Finally it was shown by in vivo experiments that L . pneumophila LepB is a target for (5S,6S)-6-{(R)-acetoxyethyl}-penem-3-carboxylate, a specific inhibitor of type I SPases. Biomacromolecules, 2004 May-Jun, 5(3), 903 - 13 Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles; Joralemon MJ et al.; Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate) . The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator . Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles . SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers . The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU) . Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E . coli) via TEM imaging. Appl Environ Microbiol, 2004 May, 70(5), 2741 - 7 Dissimilatory arsenate reduction with sulfide as electron donor: experiments with mono lake water and Isolation of strain MLMS-1, a chemoautotrophic arsenate respirer; Hoeft SE et al.; Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors . Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction . We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water . Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide . No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide . The rate of arsenate reduction in lake water was dependent on the amount of available arsenate . We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution . The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite . Chemoautotrophy was confirmed by the incorporation of H(14)CO(3)(-) into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products . Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the delta-Proteobacteria, located near sulfate reducers like Desulfobulbus sp . (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake . However, strain MLMS-1 does not grow with sulfate as its electron acceptor. J Biomed Mater Res, 2004 Jun 1, 69A(3), 561 - 6 Titanium implants enhance pulmonary nitric oxide production and lung injury in rats exposed to endotoxin; Yang RS et al.; An increase in levels of elemental Ti in the blood and lung of rats with a Ti alloy implant has been demonstrated . However, the pathophysiological role of the elevated elemental Ti level in the circulation remains unclear . Rats were implanted with Ti alloy discs for 4 weeks . The levels of elemental Ti in the blood and lung were especially increased compared with other tissues . The Ti alloy implant enhanced lung injury related to endotoxin from Gram-negative bacteria (lipopolysaccharide, LPS), which was characterized by lung edema and other histological changes such as recruitment of neutrophils, interstitial edema, and alveolar hemorrhage in the lung . In the presence of endotoxin, an increase of nitrite production was shown in the plasma and bronchoalveolar lavage fluid of rats implanted with a Ti alloy . Moreover, the Ti alloy implant further enhanced the induction of inducible nitric oxide (NO) synthase (iNOS) protein expression induced by LPS in the lung . These endotoxin-related responses in the presence or absence of the Ti alloy implant could be inhibited by aminoguanidine (an iNOS inhibitor) . These results provide the first experimental evidence that circulating Ti released from Ti alloy implants has an ability to affect pulmonary iNOS protein expression, and enhance the pathogenesis of acute lung injury during endotoxemia . Adv Dent Res, 2003 Dec, 17, 95 - 9 Genetic and molecular characterization of a dental pathogen using genome-wide approaches; Actis LA et al.; Actinobacillus actinomycetemcomitans causes periodontitis, a costly chronic infection that affects a large number of patients . The pathogenesis of this dental infection is a multifactorial process that results in a serious degenerative disease of the periodontium . Although significant progress has been achieved after the identification of this Gram-negative bacterium as the etiological agent of this infection, much remains to be done to understand in detail the bacterial factors and host-pathogen interactions involved in the pathogenesis of this disease . Classic research approaches have resulted in the identification of important virulence factors and cellular processes, although they have provided a rather narrow picture of some of the steps of this complex process . In contrast, a much wider picture could be obtained with the application of tools such as bioinformatics and genomics . These tools will provide global information regarding the differential expression of genes encoding factors and processes that lead to the pathogenesis of this disease . Furthermore, comparative genomics has the potential of helping us to understand the emergence and evolution of this human pathogen . This genome-wide approach should provide a more complete picture of the pathogenesis process of this disease, and will facilitate the development of efficient diagnostic, preventive, and therapeutic measures for this disease. J Endotoxin Res, 2004, 10(2), 97 - 106 rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21; Horwitz AH et al.; rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin . rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23 . Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus . To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids . A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI) . These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure) . Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities . In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21 . In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease . We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI. J Endotoxin Res, 2004, 10(2), 85 - 95 A new method for removing endotoxin from plasma using hemocompatible affinity chromatography technology, applicable for extracorporeal treatment of septic patients; Amoureux MC et al.; The pathogenesis of sepsis begins with the proliferation of micro-organisms at a site of infection, followed by invasion of the bloodstream and other organs . Gram-negative bacteria account for a large part of sepsis cases . The structural component of Gram-negative bacteria, endotoxin or lipopolysaccharide (LPS), induces the synthesis and release of endogenous mediators of sepsis . A growing number of investigations of the molecular mechanisms occurring in sepsis, point to endotoxin as a central mediator leading to multi-organ failure and death . In numerous clinical trials, attempts to target molecules downstream of endotoxin have been made, but have not been associated with improved survival . We describe an affinity-based system for the selective removal of endotoxin from plasma . The small-scale device, a 1.5 ml cartridge, contains beads that bind endotoxin with high specificity and efficiency . In addition, evidence is presented that this device does not affect plasma hemostasis, nor does it activate the complement system . Taken together, these results represent a proof of principle for endotoxin removal from plasma, which may be of clinical value to treat sepsis by extracorporeal circulation of the blood through a scaled-up version of this endotoxin-removing device. Curr Issues Mol Biol, 2004 Jul, 6(2), 111 - 24 Type V protein secretion: simplicity gone awry? Desvaux M, Parham NJ, Henderson IR. Since its discovery in the late 1980's, the family of secreted proteins termed the autotransporters has been expanding continuously to become the largest group of secreted proteins in Gram-negative bacteria . The type V secretion pathway, which includes the autotransporters (type Va) together with the two-partner secretion system (type Vb) and the Oca family (type Vc), can be defined by secreted proteins that are (i) translocated across the outer membrane via a transmembrane pore formed by a beta-barrel and (ii) contain all the information required for translocation through the cell envelope . In the light of new discoveries and controversies in this research field, the secretion process of autotransporters, or the type Va secretion system, will be discussed here and placed in the context of the more general field of bacterial protein translocation. Eur J Immunol, 2004 May, 34(5), 1441 - 50 Inhibition of hepatic transcriptional induction of lipopolysaccharide-binding protein by transforming-growth-factor beta 1; Hallatschek W et al.; LPS-binding protein (LBP) is an acute-phase protein with the ability to bind and transfer LPS of Gram-negative bacteria, as well as cell wall compounds of other pathogenic bacteria . This soluble pattern-recognition molecule is present in high concentrations in serum and represents an important defense mechanism of the host . Regulation of the hepatic acute-phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host organism . We show here that TGF-beta 1, in a dose-dependent fashion, is able to inhibit LBP transcript accumulation and LBP protein synthesis induced by IL-6, IL-1 beta and dexamethasone in hepatoma cell lines . These data were confirmed employing primary human hepatocytes, where TGF-beta 1 also inhibited LBP protein synthesis . We identified and analyzed several Smad-binding sites (Smads are major regulatory elements of TGF-beta 1) within the LBP promoter, and found that one of them was active . We furthermore identified an AP-1-binding site clearly conferring inhibitory effects of TGF-beta 1 towards LBP promoter activity, shown by gel shift and promoter mutagenesis experiments . Further elucidating the mechanism of transcriptional regulation of proteins involved in innate immune responses may potentially help to develop novel intervention strategies for the acute-phase response, sepsis, and septic shock. FEMS Microbiol Lett, 2004 May 1, 234(1), 81 - 6 Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression; Garcia Gonzalez O et al.; The complete amino acid and nucleotide sequence of a secreted metalloprotease produced by Actinobacillus pleuropneumoniae serotype 1 is reported . A clone showing proteolytic activity in cell-free culture media was selected from a genomic library of A . pleuropneumoniae serotype 1 in pUC 19 . The sequence obtained contained an open reading frame encoding a protein with 869 amino acids . This protein was identified as a zinc neutral-metalloprotease belonging to the aminopeptidase family, with a predicted molecular weight of approximately 101 kDa . This sequence showed high homology with other predicted or sequenced aminopeptidases reported for different Gram-negative bacteria . Expression of the protease was observed in lung tissue from pigs that died of porcine pleuropneumonia suggesting a role in pathogenesis. OMICS, 2004 Spring, 8(1), 43 - 55 Genomic insights into gene regulation of Desulfovibrio vulgaris Hildenborough; Hemme CL et al.; Traditional laboratory studies of the sulfate-reducing bacteria have focused primarily on the biochemistry of the organisms . As genomic sequences of sulfate-reducing species have become available, insights have been gained into the metabolic and regulatory networks of these organisms . A computational analysis is reported of the transcriptional regulatory networks of Desulfovibrio vulgaris Hildenborough, the first mesophilic gram-negative sulfate-reducing bacterium for which a genome sequence is available . A set of conserved DNA motifs were derived from libraries of potential promoter regions of putative D . vulgaris regulons with the AlignACE program suite . Although one motif showed similarity to the Escherichia coli GlpR binding site, most of the motifs returned were apparently unique . A number of expected orthologs for regulatory proteins have not yet been recognized in D . vulgaris. Trends Immunol, 2004 Feb, 25(2), 53 - 5 Meet the relatives: a family of BPI- and LBP-related proteins; Bingle CD et al.; Until recently, two key members of the innate immune response to Gram negative bacteria, bactericidal permeability-increasing protein (BPI) and lipopolysaccharide (LPS)-binding protein, have been considered to be members of a small family of lipid-binding proteins that also contains cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) . A recent paper has characterised three related proteins that are expressed in the mouth, nose and upper airways . Taken together with other recent data, it is clear that a large family of such proteins exists and these additional members might also function in the innate immune response. Biotechnol Lett, 2004 Mar, 26(5), 379 - 83 Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate; Morono Y et al.; Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell . However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell . Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells . We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining . We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell. J Mass Spectrom, 2004 Apr, 39(4), 378 - 83 Determination of fatty acid positions in native lipid A by positive and negative electrospray ionization mass spectrometry; Sforza S et al.; Lipid A is the endotoxic principle of the lipopolysaccharide fraction from Gram-negative bacteria . It is involved in the elicitation of cytokine production that leads to massive inflammation and to septic shock as a lethal consequence . For this reason, the structural elucidation of lipopolysaccharides from toxic Gram-negative bacteria is an important and complicated task, mainly owing to its natural heterogeneity . Here, a new methodology to infer the distribution of the primary and secondary acyl residues is described, based on electrospray ionization mass spectrometry (ESI-MS) of intact lipid A under high cone voltage in order to achieve in-source fragmentation . Under these conditions, acyl fragmentation is induced and a different regioselective cleavage of secondary fatty acids is observed in positive and negative ESI-MS, allowing the rapid identification of the lipid A structure . Infect Immun, 2004 May, 72(5), 3042 - 7 Presence of pili on the surface of Francisella tularensis; Gil H et al.; Francisella tularensis is a highly infectious gram-negative bacterium with potential for use as a bioweapon . Analysis of the F . tularensis live vaccine strain (LVS) ultrastructure by electron microscopy revealed the presence of long, thin fibers, similar in appearance to type 4 pili . The highly virulent F . tularensis Schu S4 strain was found to contain type 4 pilus genes, and we confirmed that these genes are present and expressed in the LVS. Infect Immun, 2004 May, 72(5), 2837 - 42 pagP is required for resistance to antibody-mediated complement lysis during Bordetella bronchiseptica respiratory infection; Pilione MR et al.; To efficiently colonize and persist in the lower respiratory tract, bacteria must survive multiple host immune mechanisms . Bordetella bronchiseptica is a gram-negative respiratory pathogen that naturally infects mice and persists in the lower respiratory tract for up to 49 days postinoculation . In this work, we examined the effect of mutation of the pagP gene on the persistence of B . bronchiseptica in the lower respiratory tract of mice . The pagP gene encodes a palmitoyl transferase that is responsible for the addition of a palmitoyl group to the lipid A region of B . bronchiseptica lipopolysaccharide . Data presented here confirm that a B . bronchiseptica deltapagP mutant demonstrates defective persistence in the lower respiratory tract of wild-type mice . We hypothesized that the defective persistence of the B . bronchiseptica deltapagP mutant was due to an increased susceptibility of this mutant to a host immune response . In vivo data indicate that both B cells and the complement component C3 are required for the reduced bacterial numbers of the deltapagP mutant on day 14 postinoculation . In addition, an in vitro complement killing assay demonstrated that B . bronchiseptica exhibits pagP-dependent resistance to antibody-mediated complement killing at low concentrations of immune serum . Taken together, these results suggest that pagP is required for B . bronchiseptica to resist antibody-mediated complement lysis during respiratory infection. Biol Chem, 2004 Feb, 385(2), 137 - 43 The periplasmic E . coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization; Schlapschy M et al.; The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria . Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins . Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution . Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions . Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found . A first data set was collected to 2.60 A resolution. Hokkaido Igaku Zasshi, 2004 Mar, 79(2), 111 - 5 {What is Helicobacter pylori?: associated diseases in human}; Sugiyama T et al.; Helicobacter pylori (H . pylori) is a spiral gram-negative bacterium, characterized by positive urease, catalase and oxidase activity . The complete resolution of the full genome has elucidated the biological characteristics, the pathogenicity, and the bacterial evolution . The infection is acquired in childhood by oral-oral transmission in developed countries, probably an intra-familiar transmission . The infection has an association with chronic histological gastritis, peptic ulcer, gastric cancer and MALT lymphoma in the stomach . All patients with H . pylori infection have histological gastritis and most of them remain asymptomatic for life . Only a minority of the infected individuals occurs ulceration or gastric cancer . Cure of the infection prevents recurrence of peptic ulcer without persistent treatments against ulceration . Concerning gastric cancer, WHO concluded in 1994 that H . pylori is a definite carcinogen in humans on the basis of epidemiological studies . Evidences that the infection leads to the development of gastric cancer have been accumulated in animal models as well as in vitro experimental studies . Such clinical diversities are caused by variations of H . pylori pathogenicity, host susceptibility, environmental factors including foods and those interactions . At present, an eradication treatment of H . pylori, combined with a proton-pump inhibitor and two antibiotics (clarithromycin and amoxicillin) is restrictedly approved in the patients with gastric or duodenal ulcer by Japanese health care system. Genet Mol Res, 2004 Mar 31, 3(1), 181 - 94 Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification; Carepo MS et al.; Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems . The complete genome sequence of C . violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use . We examined the biotechnological potential of C . violaceum for environmental detoxification . Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail . Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins. Genet Mol Res, 2004 Mar 31, 3(1), 148 - 61 Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity; Brito CF et al.; Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil . Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals . Although rare, infections with C . violaceum are characterized by rapid dissemination and high mortality . With the complete genome sequence of C . violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed . Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged . The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis . We described a number of genes involved in infectious processes, such as host cell adhesion, "contact-dependent secretion" of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins . We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response. Genet Mol Res, 2004 Mar 31, 3(1), 92 - 101 Chemotaxis and flagellar genes of Chromobacterium violaceum; Pereira M et al.; The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism . This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C . violaceum . C . violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome . We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C . violaceum . A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins . The chemoreceptors found in C . violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C . violaceum genome . Also, the C . violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system . Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C . violaceum genome. Protein Sci, 2004 May, 13(5), 1402 - 6 Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions; Yu CS et al.; Gram-negative bacteria have five major subcellular localization sites: the cytoplasm, the periplasm, the inner membrane, the outer membrane, and the extracellular space . The subcellular location of a protein can provide valuable information about its function . With the rapid increase of