Microorganisms

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2291 - 5
Nocardiopsis metallicus sp . nov., a metal-leaching actinomycete isolated from an alkaline slag dump; Schippers A et al.; A taxonomic study was carried out on a metal-mobilizing, alkaliphilic bacterium from an alkaline slag dump, strain KBS6(T) . The strain produced substrate and aerial mycelia . Growth occurred in the pH range 7.0-10.5, with an optimum at pH 8.5 . A salt concentration of up to 10% was tolerated, and various organic substrates were used for growth . The results of a 16S rDNA sequence comparison revealed that strain KBS6(T) belongs to the genus Nocardiopsis . DNA-DNA hybridization with the two closest relatives, Nocardiopsis exhalans and Nocardiopsis prasina, gave similarity values of 18.2 and 44.1%, respectively, which indicated that strain KBS6(T) represents a novel species of the genus Nocardiopsis . This is consistent with the morphological, physiological and chemotaxonomic data . Because of the ability of this micro-organism to solubilize metals, the name Nocardiopsis metallicus sp . nov . is proposed for strain KBS6(T) (= DSM 44598(T) = NRRL B-24159(T)), this being the type strain.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2035 - 41
Emended description of Rickettsia felis (Bouyer et al . 2001), a temperature-dependent cultured bacterium; La Scola B et al.; On the basis of phenotypic data obtained on the strain Marseille-URRWFXCal2(T), isolated from the cat flea Ctenocephalides felis, the description of Rickettsia felis (Bouyer et al., 2001) is emended and Marseille-URRWFXCal2(T) is proposed as the type strain of the species . On the basis of polyphasic characterization, especially the inability to grow at temperatures higher than 32 degrees C on Vero cells that allow growth of other Rickettsia to at least 35 degrees C, it is confirmed that this agent, although different from other recognized rickettsial species, is genotypically indistinguishable from bacteria previously detected within cat fleas and provisionally named ELB . Comparison of the phenotypic characteristics previously described for R . felis and those observed for the isolate in this study indicated some differences, although concurrent analysis of the two was not possible as no extant isolates of the first isolate of R . felis exist.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 1997 - 2002
Classification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Mycobacterium sp . strain PYR-1, as Mycobacterium vanbaalenii sp . nov; Khan AA et al.; A polycyclic aromatic hydrocarbon (PAH)-utilizing Mycobacterium strain, PYR-1(T), was isolated from petroleum-contaminated estuarine sediments and has been shown by 16S rRNA gene sequencing to be closely related to Mycobacterium aurum ATCC 23366(T) and Mycobacterium vaccae ATCC 15438(T) . In this investigation, the 16S rDNA, fatty acid methyl esters, DNA-DNA hybridization, PFGE analysis of restriction-digested total genomic DNA and biochemical tests were used to determine the taxonomic relationship of strain PYR-1(T) to other closely related Mycobacterium species . The sequence of the 16S rRNA gene of strain PYR-1(T) was similar to that of Mycobacterium austroafricanum ATCC 33464(T), except for one gap at position 43 . Fatty acid methyl ester analysis also showed similarity to M . austroafricanum ATCC 33464(T); however, the Euclidean distance was greater than 4.0, indicating that these strains were not identical . Dot-blot DNA-DNA hybridization of strain PYR-1(T) with M . austroafricanum indicated less than 40% relatedness . When the total chromosomal DNA of M . aurum ATCC 23366(T), M . austroafricanum ATCC 33464(T) and strain PYR-1(T) was digested with restriction enzyme Xbal and analysed by PFGE, all three organisms gave different restriction patterns . Previous studies from our laboratory have shown that the reverse-phase HPLC elution profiles of mycolic acids of strain PYR-1(T) and M . austroafricanum ATCC 33464(T) have different patterns . Based on phylogenetic analysis using 165 rRNA gene sequences, fatty acid analysis, DNA-DNA hybridization and PFGE analysis and physiological and chemotaxonomic characteristics, it is concluded that strain PYR-1(T) (= DSM 7251(T) = NRRL B-24157(T)) represents a novel species of the genus Mycobacterium, for which the name Mycobacterium vanbaalenii sp . nov . is proposed.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 1937 - 44
Stenotrophomonas rhizophila sp . nov., a novel plant-associated bacterium with antifungal properties; Wolf A et al.; A polyphasic taxonomic study was performed on 16 Stenotrophomonas strains from environmental and clinical sources . A group of three plant-associated isolates were shown to be phenotypically different from the other strains . This group formed a separate physiological cluster (B1) with 42% heterogeneity to the other isolates . The defining characteristics of the new species were as follows: growth at 4 degrees C and the absence of growth at 40 degrees C; the utilization of xylose as a carbon source; lower osmolytic tolerance (< 4.5% NaCl, w/v), although the isolates can produce trehalose and glucosylglycerol as osmoprotective substances; the absence of lipase and beta-glucosidase production; and antifungal activity against plant-pathogenic fungi . The whole-cell fatty acid profile of this group was different and characterized by the main fatty acids iso-C15:0 and anteiso-C15:0 . Numerical analysis of the fatty acid profiles of the strains examined supports the differentiation of the physiological B1 group . By 16S rDNA analysis, three clusters were distinguished . The three strains of the B1 group formed a separate environmental cluster (E1) . They showed a mean similarity of 99.5% within the cluster, and differed from strains of a second environmental cluster (E2) by 2.2% and from the clinical cluster (C) by about 3.0% . DNA-DNA hybridization data supported the taxonomic differentiation . All results led to the proposal of a new species, Stenotrophomonas rhizophila sp . nov., with strain e-p10(T) (= DSM 14405(T) = ATCC BAA-473(T)) as the type strain.

Bioresour Technol, 2003 May, 87(3), 295 - 8
Thermo-acido-tolerant phytase production from a soil bacterium in a medium containing rice bran and soybean meal extract; Popanich S et al.; A bacterial strain capable of producing a thermo-acido-tolerant phytase was isolated from soil around haystacks and designated as strain PH01 . The phytase produced was purified to homogeneity as determined by native PAGE . From SDS-PAGE, it was 30 kDa in size . The purified phytase was a thermo-acido-tolerant enzyme . A complex medium for the PH01 phytase production was developed . The medium, "PheB", was composed of 2% glucose, 0.2% CaCl(2), 0.5% NH(4)NO(3), 0.05% KCl, 0.05% MgSO(4).7H(2)O, 0.001% FeSO(4).7H(2)O, 0.001% MnSO(4).H(2)O in rice bran plus soybean meal extract containing 3% (v/v) phosphate solution (7.3% NaHPO(4)+3.2%KH(2)PO(4), pH 7.2) . Cultivation was done at 37 degrees C with aeration for 48 h which produced phytase at 10 U/ml . Exposure of the phytase to 1% bile salt; i.e., taurocholate or deoxycholate, caused less than 15% reduction of activity . Potential application of PH01 phytase as a feed supplement was suggested.

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2314 - 22
Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase, that needs product of upstream gene, sldB, for activity; Shinjoh M et al.; The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions . The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium . The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol . The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase . The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did . SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.

Toxicol Lett, 2003 Jan 13, 136(3), 217 - 27
Effect of Bisphenol A on non-specific immunodefenses against non-pathogenic Escherichia coli; Sugita-Konishi Y et al.; We examined the effect of Bisphenol A (BPA) on non-specific defense in experiments with a non-pathogenic bacterium, Escherichia coli K-12 . Mice were pretreated by a subcutaneous route with BPA (5 mg/kg body weight) for 5 consecutive days in the back and 3 days after the last treatment, injected by the intra-peritoneal route with E . coli K-12 . BPA pretreatment caused a decrease of T and B cell populations in the spleen of treated mice . After the challenge with E . coli, the activity to eliminate bacteria from the peritoneal cavity in the early stage of infection (within 24 h) was diminished compared with non-treated mice . BPA induced the migration of excess neutrophils into the peritoneal cavity, but reduced their phagocytic activity against E . coli K-12 . For macrophages and lymphocytes, BPA reduced the population in the spleen and the accumulation at infection foci . The production of MCP-1 was enhanced by BPA treatment, but that of IL-6 was suppressed after infection . These results suggest that BPA possessed immunotoxicity and reduced the non-specific host defense as an acute toxicity.

J Infect, 2003 Jan, 46(1), 49 - 55
Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability; Saito N et al.; OBJECTIVES: Helicobacter pylori (H . pylori), present in a half of the world's population, is a very successful pathogen . The infection by this bacterium causes several gastric diseases in human . H . pylori is morphologically divided into two types; a spiral and a coccoid form . Both types are observed in human stomach . Although the former is converted into the latter in vitro, the process of coccoid formation remains obscure . Furthermore, whether coccoid forms possess viability arouses much controversy among scientists . We investigated both the process of coccoid formation by electron microscopy and the viability of coccoid H . pylori . METHODS: A laboratory strain, ATCC43504, was cultured in liquid medium (Brucella Broth medium with 10% heat inactivated horse serum) for seven days . In each culture day, the organisms were observed by scanning-, conventional transmission- and immuno-electron microscopy and were simultaneously examined the culturability of H . pylori to identify the viability of coccoid form . RESULTS: As the days went by, spiral forms were replaced by coccoid forms in each medium which possessed culturability to some degree until the 4th day . By the detailed observation of ultrastructural features, coccoid forms were classified into two types on the ground that represents different transformation-processes and distinctive ultrastructures . One was the coccoid form that we named Type A with an irregularly surface- and intracytoplasmic-structure which adhered to one another . The other was Type B having a smooth surface with flagella coiled around its own body and the strictly membranous structure . This type was not adhered to another organism . In later days, each type came to be similar external structure and lost the culturability . CONCLUSIONS: The coccoid forms of H . pylori can be classified into two types by electron microscopy which represent different transformation-processes and consist of the dying bacteria, the living ones with culturability and the viable but non-culturable ones.

Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 351 - 6
A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis; Shibata Y et al.; It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth . In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined . We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P . gingivalis and seemed to confer colonizing activities . Recently, we have constructed the P . gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis . The beige mutant exhibited little co-aggregation and the virulence was also decreased . Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site . The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin . These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P . gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P . gingivalis.

J Nat Prod, 2002 Dec, 65(12), 1956 - 62
Plant-like biosynthetic pathways in bacteria: from benzoic acid to chalcone; Moore BS et al.; Although phenylpropanoids and flavonoids are common plant natural products, these major classes of biologically active secondary metabolites are largely absent from bacteria . The ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are key biosynthetic catalysts in phenylpropanoid and flavonoid assembly, respectively . Until recently, few bacterial counterparts were known, thus reflecting the dearth of these plant natural products in bacteria . This review highlights our progress on the biochemical and genetic characterization of recently identified streptomycete biosynthetic pathways to benzoic acid and type III polyketide synthase (PKS)-derived products . The sediment-derived bacterium "Streptomyces maritimus" produces benzoyl-CoA in a plant-like manner from phenylalanine involving a PAL-mediated reaction through cinnamic acid during the biosynthesis of the polyketide antibiotic enterocin . All but one of the genes encoding benzoyl-CoA biosynthesis in "S . maritimus" have been cloned, sequenced, and inactivated, providing a model for benzoate biosynthesis not only in this bacterium, but in plants where benzoic acid is an important constituent of many products . The recent discovery that bacteria harbor homodimeric PKSs belonging to the plant CHS superfamily of condensing enzymes has further linked the biosynthetic capabilities of plants and bacteria . A bioinformatics approach led to the prediction that the model actinomycete Streptomyces coelicolor A3(2) contains up to three type III PKSs . Biochemical analysis of one of the recombinant type III PKSs from S . coelicolor demonstrated activity as a 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) . A homology model of THNS based upon the known three-dimensional structure of CHS was constructed to explore the structural and mechanistic details of this new subclass of bacterial PKSs.

J Gen Appl Microbiol, 1998 Aug, 44(4), 289 - 295
Moritella japonica sp . nov., a novel barophilic bacterium isolated from a Japan Trench sediment; Nogi Y et al.; Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356 m . Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina . Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M . marina and Shewanella barophilic species . Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M . marina . Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species . The name Moritella japonica is proposed . The type strain is JCM 10249.

Biochemistry, 2002 Dec 31, 41(52), 15524 - 35
Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability; Lebbink JH et al.; Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability . While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles . In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193 . The X-ray structure of the domain was solved at 1.43 A and reveals the salt-bridge network with surrounding solvent molecules in detail . All three participating charges in the network were mutated to alanine in all combinations . The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein . Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue . Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea . Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol . The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.

Biofizika, 2002 Nov-Dec, 47(6), 1015 - 20
{Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids}; Krikunova MA et al.; Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids . LH2 preparations isolated from wild-type and carotenoid-depleted cells were used . 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation . It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone . It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 139 - 41 Epub 2002 Dec 19.
Crystallization and preliminary X-ray analysis of Alicyclobacillus acidocaldarius endoglucanase CelA; Eckert K et al.; Crystallization of a family 9 beta-1,4-glucanase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius CelA is reported . Thin plates can be obtained by hanging-drop vapour-diffusion crystallization in high concentrations (60%) of MPD . These crystals are unusual in that they do not bind the dye IZIT in the mother liquor and do not appear to dissolve in water after three weeks or in the storage buffer after 2 d . The crystals diffract weakly and the diffraction pattern is compatible with crystal disorder in one direction . After testing several crystals at the ESRF beamlines ID14-1 and ID14-2, a crystal was found which gave ordered diffraction in all directions . A full data set was collected to 3.0 A resolution, which allowed unambiguous determination of the space group as P2(1)2(1)2 and the unit-cell parameters as a = 85, b = 129.7, c = 48.6 A . Initial promising results from molecular-replacement searches are reported.

Infect Immun, 2003 Jan, 71(1), 354 - 64
Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination; Jeevan A et al.; To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library . The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively . Spleen or lymph node cells from naive and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M . tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone . Compared to the IFN-gamma mRNA expression in cells of naive animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination . However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M . tuberculosis bacterium per 10 cells . The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis . Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD . This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination . The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.

Infect Immun, 2003 Jan, 71(1), 335 - 42
Role for recombinant gamma-glutamyltransferase from Treponema denticola in glutathione metabolism; Chu L et al.; Volatile sulfur compounds, including hydrogen sulfide (H(2)S), have been implicated in the development of periodontal disease . Glutathione is an important thiol source for H(2)S production in periodontal pockets . Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H(2)S . In this pathway, gamma-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation . We have cloned the gene of GGT from T . denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids . Transformation of this gene into Escherichia coli led to the expression of a recombinant protein . After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-gamma-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine . L-Cysteine is not a substrate of GGT . Importantly, GNA, when added to T . denticola, was able to compete with glutathione and inhibit the production of H(2)S, ammonia, and pyruvate . This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria . Purified GGT was inactivated by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and proteinase K treatment . However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol . Our further experiments showed that the addition of recombinant GGT to Porphyromonas gingivalis, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H(2)S, ammonia, and pyruvate . This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities . Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.

Nucleic Acids Res, 2002 Dec 15, 30(24), 5579 - 92
Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis; Journet EP et al.; We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices . We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression . Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process . These clusters were manually annotated, using a specifically developed annotation interface . Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction . These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula . A searchable database has been built and can be accessed through a public interface.

Evolution Int J Org Evolution, 2002 Nov, 56(11), 2290 - 5
How can sex ratio distorters reach extreme prevalences? Male-killing Wolbachia are not suppressed and have near-perfect vertical transmission efficiency in Acraea encedon; Jiggins FM et al.; Maternally transmitted bacteria that kill male hosts early in their development are found in many insects . These parasites typically infect 1-30% of wild females, but in a few species of insects, prevalences exceed 95% . We investigated one such case in the butterfly Acraea encedon, which is infected with a male-killing Wolbachia bacterium . We measured three key parameters that affect the prevalence of the parasite: transmission efficiency, rate of survival of infected males, and the direct cost of infection . We observed that all wild females transmit the bacterium to all their offspring and that all infected males die in wild populations . We were unable to detect any physiological cost to infection in lab culture . These observations explain the high prevalence of the A . encedon male killer, as theory predicts that under these conditions the parasite will spread to fixation . This will occur provided the death of males provides some benefit to the surviving infected females . The problem therefore becomes why the bacterium has not reached fixation and driven the butterfly extinct due to the shortage of males . We therefore investigated whether males choose to mate with uninfected rather than infected females, as this would prevent the bacterium from reaching fixation . We tested this hypothesis in the "lekking swarms" of virgin females found in the most female-biased populations, and were unable to detect any evidence of mate choice . In conclusion, this male killer has spread to high prevalence because it has a high transmission efficiency and low cost, but the factors maintaining uninfected females in the population remain unknown.

J Bacteriol, 2003 Jan, 185(1), 325 - 31
Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti; Marketon MM et al.; Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants . This invasion process requires the synthesis, by S . meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II . We have previously shown that the sinRI locus of S . meliloti encodes a quorum-sensing system that plays a role in the symbiotic process . Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis . Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis . This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression . This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C(16:1)-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression . Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

J Bacteriol, 2003 Jan, 185(1), 71 - 9
Function of oxygen resistance proteins in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough; Fournier M et al.; Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed . Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase . The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain . No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D . vulgaris genome . Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod . The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE) . A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D . vulgaris . Thus, Sor plays a key role in oxygen defense of D . vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm . Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.

Oral Microbiol Immunol, 2002 Dec, 17(6), 354 - 9
Sequence diversity in the major fimbrial subunit gene (flp-1) of Actinobacillus actinomycetemcomitans; Kaplan JB et al.; Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces . Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell . The flp-1 gene encodes the major pilin protein component of A . actinomycetemcomitans fimbriae . In this study we compared flp-1 DNA sequences from 43 strains of A . actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes . DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes . Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili . flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains . At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes . Four of the seven flp-1 classes were present in geographically diverse isolates . Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay . Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential . Our findings provide a framework for future biochemical, immunological and genetic studies of A . actinomycetemcomitans fimbriae.

Biochemistry, 2002 Dec 24, 41(51), 15234 - 44
Characterization of the redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum; McDevitt CA et al.; Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide . Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI) . Variable temperature X-band EPR spectra (120-122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), {3Fe-4S}(+), {4Fe-4S}(+), and a b-type heme . A pH-dependent EPR study of the Mo(V) center in (1)H(2)O and (2)H(2)O revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH(2), Mo(V)-anion, and Mo(V)-OH . Above pH 8.2 the dominant species was Mo(V)-OH . The maximum specific activity occurred at pH 9.27 . Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG . A sequence comparison of DdhB and NarH has predicted the presence of four {Fe-S} clusters in DdhB . A {3Fe-4S}(+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH . A {4Fe-4S}(+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand . The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH {Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P . (1992) Eur . J . Biochem . 307, 63-68} . Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase . Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis . Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E degrees = +315 mV, pH 8).

J Gen Appl Microbiol, 2000 Apr, 46(2), 59 - 67
Metabolism of gamma-hexachlorocyclohexane by Arthrobacter citreus strain BI-100: Identification of metabolites; Datta J et al.; Growth characteristics of the aerobic bacterial strain Arthrobacter citreus BI-100 in mineral salts medium with gamma-hexachlorocyclohexane (gamma-HCH) as the sole source of carbon and degradation of gamma-HCH by the strain are reported . The highest yield of the bacteria is observed at a gamma-HCH concentration of 100 mg/L . At this concentration, the bacteria entered the exponential phase of growth without any lag . At 8 h of growth, no residual HCH, but its metabolites, was detectable in the medium . The bacterium attained its stationary phase at 48 h and at 72 h; no metabolite of gamma-HCH could be detected by gas chromatography . Six metabolic intermediates of gamma-HCH produced by A . citreus BI-100 at different periods of growth were characterized by using gas chromatography-mass spectrometry and high-performance liquid chromatography, which furnished evidence for the presence of gamma-1,3,4,5,6-pentachlorocyclohexene, tetrachlorocyclohexene, trichlorocyclohexa-diene, 2-chlorophenol, phenol, and catechol, among others.

Microbiology, 2002 Dec, 148(Pt 12), 3813 - 25
Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated 'KatA-associated protein', KapA (HP0874); Harris AG et al.; Helicobacter pylori infection elicits an aggressive inflammatory response that the bacterium is able to resist by virtue of its well-adapted antioxidant defence mechanisms . Catalase (KatA) appears to be a key enzyme in this resistance . Upstream of katA, a low-affinity ferric uptake regulator (Fur)-box has been identified . Downstream of katA, an ORF (HP0874) with no known function has also been identified . Non-polar isogenic mutants of katA, fur and HP0874 were constructed by allelic exchange . The impact of these mutations on the catalase activities and bacterial viability following exposure to hydrogen peroxide was studied . Concurrently, the effect of variation in the iron content of the media used to grow the cells was determined . The data showed that catalase-deficient isolates of H . pylori were hypersensitive to hydrogen peroxide, whereas wild-type cells could resist approximately approximately 100 mM hydrogen peroxide . Fur-deficient mutants and cells grown on low-iron-containing medium showed a distinct reduction in catalase activity and increased sensitivity to hydrogen peroxide . The data suggest a direct or indirect effect of Fur and iron on the activity of catalase . HP0874-deficient mutants showed no reduction in catalase activity but showed an increased sensitivity to hydrogen peroxide . That is, the protein encoded by HP0874 appears to have a role in resistance to hydrogen peroxide not directly related to catalase activity . This is the first report of a functional relationship of the product of this ORF . There is evidence of protein-protein interaction between KatA and the product encoded by HP0874, and the name 'KatA-associated protein' (KapA) is proposed.

Dev Cell, 2002 Dec, 3(6), 763 - 4
Hijacking the host cell proteasome; Waksman G; Uropathogenic Escherichia coli subvert host cell signaling mechanisms to induce cellular responses that facilitate bacterial invasion and colonization . A recent publication in the November 15 issue of Cell shows that the bacterium may accomplish such a feat by hijacking the proteasome machinery.

Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 82 - 9
Exceptional stability of a {2Fe-2S} ferredoxin from hyperthermophilic bacterium Aquifex aeolicus; Higgins CL et al.; Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type {2Fe-2S} ferredoxin (Aae Fd1) . This unique protein contains two cysteines, in addition to the four that act as ligands of the {2Fe-2S} cluster, which form a disulfide bridge . We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic {2Fe-2S} ferredoxin from spinach . Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state . Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times . Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7) . The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity . Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.

J Biol Chem, 2003 Feb 28, 278(9), 7531 - 9 Epub 2002 Dec 09.
The structure of a cold-adapted family 8 xylanase at 1.3 A resolution . Structural adaptations to cold and investgation of the active site; Van Petegem F et al.; Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures . The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs . Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 A resolution . Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8 . The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel . Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues . The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively . Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite . A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.

Biochemistry, 2002 Dec 17, 41(50), 15085 - 92
A light-dependent mechanism for massive accumulation of manganese in the photosynthetic bacterium Synechocystis sp . PCC 6803; Keren N et al.; Manganese is an essential micronutrient for many organisms . Because of its unique role in the water oxidizing activity of photosystem II, manganese is required for photosynthetic growth in plants and cyanobacteria . Here we report on the mechanism of manganese uptake in the cyanobacterium Synechocystis sp . PCC 6803 . Cells grown in 9 microM manganese-containing medium accumulate up to 1 x 10(8) manganese atoms/cell, bound to the outer membrane (pool A) . This pool could be released by EDTA treatment . Accumulation of manganese in pool A was energized by photosynthetic electron flow . Moreover, collapsing the membrane potential resulted in the immediate release of this manganese pool . The manganese in this pool is mainly Mn(II) in a six-coordinate distorted environment . A distinctly different pool of manganese, pool B ( approximately 1.5 x 10(6) atoms/cell), could not be extracted by EDTA . Transport into pool B was light-independent and could be detected only under limiting manganese concentrations (1 nM) . Evidently, manganese uptake in Synechocystis 6803 cells occurs in two steps . First, manganese accumulates in the outer membrane (pool A) in a membrane potential-dependent process . Next, manganese is transported through the inner membrane into pool B . We propose that pool A serves as a store that allows the cells to overcome transient limitations in manganese in the environment.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 263 - 8
Overexpression of a heterologous protein, haloalkane dehalogenase, in a poly-beta-hydroxybutyrate-deficient strain of the facultative methylotroph Methylobacterium extorquens AM1; FitzGerald KA et al.; Using an expression vector containing p(mxaF'), a strong native promoter, expression of a model heterologous protein, haloalkane dehalogenase, from Xanthobacter autotrophicus GJ10 was achieved in the methylotrophic bacterium, Methylobacterium extorquens AM1 . Although expression using the wild-type strain was <5% of total cell protein, expression at a level of 10% of the total cell protein was achieved in a mutant unable to synthesize poly-beta-hydroxybutyrate granules . Two other tested heterologous proteins, catechol dioxygenase and green fluorescent protein, were expressed at moderate levels in both wild-type and the PHB-negative strain . These results suggest that the M . extorquens PHB-negative strain is a possible platform for overexpression of heterologous proteins with labeled or unlabeled methanol as a starting material .

Microb Pathog, 2002 Nov, 33(5), 225 - 37
Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions; Kim S et al.; Brucella infects macrophages by swimming internalization, after which it is enclosed in macropinosomes . We investigated the role of the uptake pathway in phagosome trafficking, which remains unclear . This study found membrane sorting during swimming internalization and is essential in intracellular replication of Brucella . The B . abortus virB mutant replicated intracellularly when it was in the macropinosome established by wild-type B . abortus that retained its ability to alter phagosome trafficking . Lipid rafts-associated molecules, such as GM1 ganglioside, were selectively included into macropinosomes, but Rab5 effector early endosome autoantigen (EEA1) and lysosomal glycoprotein LAMP-1 were excluded from macropinosomes containing B . abortus induced by swimming internalization . In contrast, when the swimming internalization was bypassed by phorbol myristate acetate (PMA)-induced macropinocytosis, lipid raft-associated molecules were excluded, and EEA1 and LAMP-1 were included into macropinosomes containing bacteria . The phosphatidylinositol 3-kinase inhibitor wortmannin that inhibits PMA-induced macropinocytosis blocked internalization of virB mutant, but not of wild-type of B . abortus and wortmannin treatment did not affect intracellular replication . Our results suggest that membrane sorting requires swimming internalization of B . abortus and decides the intracellular fate of the bacterium, and that Brucella -induced macropinosome formation is a different mechanism from PMA-induced macropinocytosis.

Arch Microbiol, 2002 Dec, 179(1), 15 - 25 Epub 2002 Nov 07.
New genes involved in chromate resistance in Ralstonia metallidurans strain CH34; Juhnke S et al.; Chromate resistance in Ralstonia metallidurans CH34 is based on chromate efflux catalyzed by ChrA efflux pumps . The bacterium harbors two chromate resistance determinants, the previously known chr(1) on plasmid pMOL28 (genes chrI, chrB(1), chrA(1), chrC, chrE, chrF(1)) and chr(2) on the chromosome (genes chrB(2), chrA(2), chrF(2)) . Deletion of the genes chrI, chrC, chrA(2), chrB(2) and chrF(2) influenced chromate resistance and transcription from a chrBp(1) ::lacZ fusion . Deletion of the plasmid-encoded gene chrB(1) did not change chromate resistance or chrBp(1) regulation . Northern hybridization and primer-extension experiments were used to study transcription of the plasmid-encoded chr(1) determinant . Transcription of chrB(1), chrA(1) and chrC was induced by chromate . The presence of sulfate influenced transcription positively . The chrBp(1), chrAp(1) and chrCppromoters showed some similarity to heat-shock promoters . Transcription of the gene rpoH encoding a putative heat-shock sigma factor was also induced by chromate, but rpoH was not essential for chromate resistance . The ChrC protein was purified as a homotetramer and exerted superoxide dismutase activity . Thus, possible regulators for chromate resistance (ChrI, ChrB(1), ChrB(2), ChrF(1), and ChrF(2)) and an additional detoxification system (ChrC) were newly identified as parts of chromate resistance in R . metallidurans.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1227 - 32
Optimization of Allium sativum solvent extraction for the inhibition of in vitro growth of Helicobacter pylori; Canizares P et al.; Helicobacter pylori (Hp) is the bacterium responsible for serious gastric diseases such as ulcers and cancer . The work described here involved the study of the inhibitory power of Allium sativum extracts against the in vitro growth of Hp (Hp ivg) . We used purple garlic of the "Las Pedroneras" variety for this study . The effects of two different extraction methods (Soxhlet, stirred tank extractor) and four solvents with different characteristics (water, acetone, ethanol, and hexane) were investigated in terms of the efficiency of the extraction process . Satisfactory results were obtained in most cases in the activity tests, indicating that different extracts gave rise to good inhibitory activity against Hp ivg . The extracts that showed the highest bacteriostatic activities were selected to evaluate the influence of the most important operation variables on the extraction yield: stirring speed, operation time, garlic conditioning, and garlic storage time . The best results were obtained using ethanol and acetone as solvents in a stirred tank . The inhibitory powers of these extracts were compared to those shown by some commercial antibiotics used in the medical treatment of Hp infections . The results of this study show that garlic extracts produce levels of inhibition similar to those of the commercial materials . These extracts were also tested against other common bacteria, and equally satisfactory results were obtained . The research described here represents an important starting point in the fight against and/or prevention of peptic ulcers, as well as other pathologies associated with Hp infections such us gastric cancer . The extracted material can be used by direct application and involves a simple and economical extraction procedure that avoids isolation or purification techniques.

Acta Neurol Scand, 2002 Dec, 106(6), 371 - 3
Mononeuritis multiplex caused by Coxiella burnetii infection (Q fever); Sommer JB et al.; After 1 week of flu-like illness, a 64-year-old man developed rapidly progressive mononeuritis multiplex involving the right arm and both legs . Serologic studies identified Coxiella burnetii as the cause of the febrile disease (Q fever) . Fourteen days doxycycline treatment (200 mg daily) induced rapid and complete recovery . After 6 months, flu-like symptoms, weakness and hypalgesia of the right leg reappeared . Antibody titers again identified Q fever . Doxycycline was re-established and induced prompt recovery . Q fever has been associated with various neurologic complications such as meningoencephalitis, cerebellitis, optic neuritis or polyneuroradiculitis . This is the first report on Q fever related mononeuritis multiplex . Prolonged antibiotic treatment may be required to prevent relapsing infection from the resistant bacterium . Copyright Blackwell Munksgaard 2002

Biochimie, 2002 Aug, 84(8), 713 - 22
tRNA maturation in Aquifex aeolicus; Willkomm DK et al.; Several tRNAs in the hyperthermophilic bacterium Aquifex aeolicus are encoded in clusters and as part of ribosomal RNA operons, implying the requirement for tRNA processing by ribonuclease P (RNase P) . Intriguingly, neither a gene for the RNA nor the protein component of this ubiquitous ribonucleoprotein enzyme has been hitherto identified in the sequenced genome of A . aeolicus, despite extensive data mining . As a result of the present study, primer extension analysis revealed that tRNAs in A . aeolicus possess canonical mature 5' ends; yet we were unable to demonstrate RNase P holoenzyme or RNase P RNA alone activity in A . aeolicus extracts under a variety of reaction conditions utilizing mono- and dimeric ptRNA substrates . Processing of dimeric ptRNA transcripts in extracts of A . aeolicus disclosed at least one endoribonuclease which cleaves in the A/U-rich spacer of the tandem ptRNA, reminiscent of bacterial RNase E-like enzymes.

Front Biosci, 2003 Jan 01, 8, a40 - 7
Moraxella catarrhalis induces mast cell activation and nuclear factor kappa B-dependent cytokine synthesis; Krishnaswamy G et al.; Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response . Recent studies have suggested a prominent role for mast cells in host defense . In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells . Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria . Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1) . Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-kappaB) nuclear binding activity . In some experiments, NF-kappaB inhibitor, Bay-11 was added to determine functional significance . Both M . catarrhalis and N . cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1 . This was accompanied by NF-kappaB activation . Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells . Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces . The NF-kappaB inhibitor, Bay-11, inhibited expression of MCP-1 . These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

J Clin Microbiol, 2002 Dec, 40(12), 4789 - 91
Roseomonas gilardii infection: case report and review; Shokar NK et al.; Roseomonas gilardii is a bacterium that has been indicated as a rare cause of human infections . The case of a patient presenting with cellulitis and bacteremia secondary to R . gilardii is described together with the clinical characteristics of infection with this organism obtained from a review of cases previously reported.

Biochemistry, 2002 Dec 3, 41(48), 14403 - 11
Selective protein extraction from Chlorobium tepidum chlorosomes using detergents . Evidence that CsmA forms multimers and binds bacteriochlorophyll a; Bryant DA et al.; Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins . Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood . Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters . When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted . However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes . Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers . Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a . Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed . These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes . Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2030 - 5
Ferrous-iron-dependent uptake of L-glutamate by a mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9; Inoue T et al.; Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth . The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes . L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture . Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM . The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0 . Uptake activity was dependent on the concentration of the L-glutamate . The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively . L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake . L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM . 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake . The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity . The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome . The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells . The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 596 - 603
{Physiological, biochemical, and cytological characteristics of a halotolerant and alkalitolerant methanotroph grown on methanol}; Eshinimaev BTs et al.; The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M) . At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h-1) was twice as high as on methane (0.1 h-1) . The maximum growth rate increased with increasing medium salinity and was lower at neutral than at alkaline pH . The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively . The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars . The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine . The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold . The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells . The data obtained suggest that the high methanol tolerance of M . buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 587 - 97
Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments; Sobolev D et al.; The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY) . The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved 02 and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment-water interface, and which support comparable numbers (10(5)-10(6) mL(-1)) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing . The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O2 input from the atmosphere and measurable dissolved O2 to a depth of 2 mm below the sand-water interface . In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(IllI)-reducing bacteria alone . Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand . Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 283 - 91
Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans; de Bok FA et al.; The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases . All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm . These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations . These findings suggest that both H2 and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 43 - 9
Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila; Forst S et al.; Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae . Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect . The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph . OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria . To study the role of ompR in the lifecyle of XenorhabdIs, an ompR -minus strain was constructed . The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype . Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC . The ompR mutant strain was virulent towards insect hosts . However, when nematodes were grown on a mixture of the wild-type and the omnpR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode . The role of ompR in the symbiosis between the bacterium and the nematode is under investigation.

J Bacteriol, 2002 Dec, 184(24), 7025 - 41
Ultrastructural analysis of differentiation in Legionella pneumophila; Faulkner G et al.; Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts . Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Garduno et al., p . 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation . In following the fine-structural pathway of L . pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy . Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane . These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections . Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions . Our results confirm that L . pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L . pneumophila . Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L . pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.

Biochim Biophys Acta, 2002 Dec 16, 1601(2), 192 - 9
Expression and localization of alpha- and beta-carbonic anhydrase in Helicobacter pylori; Chirica LC et al.; Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (alpha and beta), catalyzing the reversible hydration of CO(2) . Presumably, the high CO(2) requirement of H . pylori implies an important role for this enzyme in the bacterial physiology . In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO(2) concentration in the investigated range (0.1-10%) . Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration . Moreover, the localization and distribution of the alpha-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the beta-form of the enzyme . The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures . The resulting electron microscopy images indicate a localization of the beta-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space . The alpha-enzyme was found attached to the surface of the bacterium.

J Chem Inf Comput Sci, 2002 Nov-Dec, 42(6), 1311 - 9
Conformation analysis of carotenoids in the purple bacterium Rhodobium marinum based on NMR spectroscopy and AM1 calculation; Qian P et al.; Five carotenoids existing in the purple bacterium of Rhodobium marinum, lycopene, anhydrorhodovibrin, spirilloxanthin, rhodopin, and rhodovibrin, were isolated and purified . Their configurations in the chromophore region and conformations of the terminal part were determined by 1D, 2D 1H and 13C NMR spectroscopy . The semiempirical quantum chemical calculation AM1 was subsequently performed using the rough 3-D structures established by NOE correlations as an initial input . The final optimized structures are coincident with 1H-1H NOE correlations and match with the X-ray crystallographic data of carotenoids . The calculation results show that chemically symmetrical carotenoids have a Ci point group . The Ci point group of molecules was destroyed by asymmetrical terminal part although the polyene chain still keeps it roughly . The polyene region of investigated carotenoids are in all-trans with slightly twisted in-plane and slight out-plane forming s-shape carbon backbone due to the spatial interaction of the methyl groups . Terminal parts, on the other hand, have several stable conformers due to the freely rotatable single bonds, but they prefer to take extended conformations.

Mol Cells, 2002 Oct 31, 14(2), 245 - 54
Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp . strain SS1 DSM 11726; Eom CY et al.; Three structural genes that consist of a dnaK operon in a restricted facultative methylotrophic bacterium Methylovorus sp . strain SS1 DSM 11726 were cloned and characterized . The genes were clustered in the transcription order grpE-dnaK-dnaJ . The cloned grpE, dnaK, and dnaJ genes had open-reading frames of 474, 1,926, and 1,116 nucleotides, coding for proteins with calculated molecular masses of 17,390, 69,761, and 41,050, respectively . The overall identities in the deduced amino acid sequences of GrpE, DnaK, and DnaJ with those of the Escherichia coli homologs were 45.2, 74.5, and 61.2%, respectively . Northern blot analyses with grpE-, dnaK-, and dnaJ-specific probes revealed that the three genes are co-transcribed as a 4.0-kb mRNA . A primer extension analysis revealed that the transcription of the dnaK operon started at the nucleotide A that is located 28 bp upstream of the grpE start codon . The transcription start site was preceded by a putative promoter region 15'-CCCCGCTTGAA(13-bp)CCCCAATTT-3'}, which is highly homologous to the consensus sequences of the E . coli sigma32-type heat shock promoter . The putative promoter worked under both normal and heat shock conditions in E . coli . The nature of the nucleotide sequence in the second half of the -35 region played a critical role during transcription . The heat shock mRNA was maximally produced at about 10 min after transfer of the Methylovorus sp . strain SS1 from 30 to 42 degrees C . The dnaK operon was also induced by ethanol, hydrogen peroxide, and NaCl shocks . The cloned dnaK operon complemented the E . coli dnaK mutant.

Mol Cells, 2002 Oct 31, 14(2), 192 - 7
Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes; Lee Y et al.; A robust and fast DNA chip method was developed in order to detect the various beta-lactam antibiotic-resistance genes in one slide . These genes included PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY, and AmpC . beta-lactam antibiotic-resistance genes were labeled with a fluorescent nucleotide by a multiplex polymerase chain reaction using a mixture of specific primer sets for each gene . This labeled target was hybridized with a DNA chip that contained the spots of the specific probe DNAs for each beta-lactam antibiotic-resistance gene . This technique made it possible to detect the specific resistance gene, even in a single bacterium.

Bull Soc Belge Ophtalmol, 2002, (285), 37 - 40
{Cause of unusual unilateral vitreous hemorrhage: neuroretinitis of Cat-Scratch disease . Presentation of a case}; Degueldre F et al.; A 49 years old woman presents an unilateral decrease of vision . An important vitreous hemorrhage obscures the fundus . After partial resorption a typical aspect of neuroretinitis is seen: macular and disc edema with macular star formation . Cat-Scratch disease is suspected after general examination of the patient . This diagnosis will be confirmed by the laboratory investigation . This disease is not so rare and represents one of the principal causes of neuroretinitis . The vitreous hemorrhage can be due to vascular insult by the bacterium responsible for Cat-Scratch disease which one has an important tropism for endothelial cells and can be responsible for vascular occlusive phenomena . Clinical signs, treatment and evolution of this disease are going to be discussed in this article.

Infect Immun, 2002 Dec, 70(12), 6846 - 52
Invasion activity of a Mycobacterium tuberculosis peptide presented by the Escherichia coli AIDA autotransporter; Casali N et al.; The mce1A gene of Mycobacterium tuberculosis was initially identified by its ability to promote uptake of Escherichia coli into HeLa cells . It was subsequently shown that this activity was confined to a 58-amino-acid region of the protein . A 72-amino-acid fragment (InvX) incorporating this active peptide was expressed in E . coli as a fusion to the AIDA (adhesin involved in diffuse adherence) autotransporter translocator, and its stable expression on the surface of the bacterium was demonstrated . Recombinant E . coli expressing InvX-AIDA showed extensive association with HeLa cells, and InvX was shown to be sufficient for internalization . Uptake was found to be both microtubule and microfilament dependent and required the Rho family of GTPases . Thus, the E . coli AIDA system facilitated both the qualitative and quantitative analysis of the functional domain of a heterologous protein.

Infect Immun, 2002 Dec, 70(12), 6751 - 60
Decreased infectivity despite unaltered C3 binding by a DeltahbhA mutant of Mycobacterium tuberculosis; Mueller-Ortiz SL et al.; HbhA of Mycobacterium tuberculosis is a multifunctional binding protein, binding to both sulfated sugars such as heparin and to human complement component C3 . HbhA may therefore interact with host molecules and/or host cells during M . tuberculosis infection and play a role in the pathogenesis of this bacterium . The purpose of this study was to use allelic exchange to create an M . tuberculosis strain deficient in expression of HbhA to determine whether this protein's C3-binding activity plays a role in the pathogenesis of M . tuberculosis . An in-frame, 576-bp unmarked deletion in the hbhA gene was created using sacB as a counterselectable marker . Southern blotting and PCR analyses confirmed deletion of hbhA in the DeltahbhA mutant . The DeltahbhA mutant strain grew at a rate similar to that of the parent in broth culture and in J774.A1 murine macrophage-like cells but was deficient in growth compared to the parent strain in the lungs, liver, and spleen of infected mice . In addition, the DeltahbhA mutation did not reduce binding of M . tuberculosis to human C3 or to J774.A1 cells in the presence or absence of serum, suggesting that in the absence of HbhA, other molecules serve as C3-binding molecules on the M . tuberculosis surface . Taken together, these data indicate that HbhA is important in the infectivity of M . tuberculosis, but its ability to bind C3 is not required for mycobacterial adherence to macrophage-like cells . Using the DeltahbhA mutant strain, a second M . tuberculosis C3-binding protein similar in size to HbhA was identified as HupB, but the role of HupB as a C3-binding protein in intact organisms remains to be determined.

Biochemistry, 2002 Nov 26, 41(47), 14028 - 32
High-potential iron-sulfur protein (HiPIP) is the major electron donor to the reaction center complex in photosynthetically growing cells of the purple bacterium Rubrivivax gelatinosus; Nagashima KV et al.; A gene encoding the high-potential iron-sulfur protein (HiPIP) was cloned from the purple photosynthetic bacterium Rubrivivax gelatinosus . An insertional disruption of this gene by a kanamycin resistance cartridge resulted in a significant decrease in the growth rate under photosynthetic growth conditions . Flash-induced kinetic measurements showed that the rate of reduction of the photooxidized reaction center is greatly diminished in the mutant depleted in the HiPIP . On the other hand, mutants depleted in the low- and high-potential cytochromes c(8), the two other soluble electron carriers, which have been shown to donate an electron to the reaction center in Rvi . gelatinosus, showed growth rates similar to those of the wild type under both photosynthetic and respiratory growth conditions . It was concluded that HiPIP is the major physiological electron donor to the reaction center in Rvi . gelatinosus cells grown under photosynthetic conditions.

Biol Chem, 2002 Jul-Aug, 383(7-8), 1223 - 30
Roles for Arg- and Lys-gingipains in the disruption of cytokine responses and loss of viability of human endothelial cells by Porphyromonas gingivalis infection; Baba A et al.; Accumulating evidence indicates that periodontal disease is associated with human cardiovascular diseases . The periodontal pathogen Porphyromonas gingivalis was shown to be present in atherosclerotic plaques in addition to periodontal pockets . This bacterium is known to produce two individual cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp) . Here we show that these two enzymes are responsible for either the disruption of cytokine responses in human umbilical vein endothelial cells (HUVEC) to the bacterium infection or the loss of cell viability . The expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA in HUVEC was greatly induced when infected with the wild-type strain, nevertheless, their protein levels in the culture medium were markedly decreased . This decrease was completely abolished in the cells infected with the Rgp/Kgp-null mutant, but not in either the Rgp- or Kgp-null mutants . Loss of the adhesion activity and viability of HUVEC were greatly induced by the culture supernatant of the wild-type strain and strongly inhibited by either a combination of the Rgp- and the Kgp-specific inhibitors or the deficiency of the Rgp- and Kgp-encoding genes . These findings indicate that P . gingivalis modulates the cytokine response in the cells and disrupts the adhesion activity and the viability through the cooperative action of Rgp and Kgp and thereby may contribute to pathogenesis of cardiovascular diseases as well as periodontal disease.

Biotechnol Annu Rev, 2002, 8, 167 - 81
Separation of biosynthetic polyunsaturated fatty acid (PUFA) with supercritical fluid; Wang L et al.; The separation of biosynthetic polyunsaturated fatty acid (PUFA) was undertaken with supercritical fluid . The polyunsaturated fatty acid was synthesised by WL-1021, which is a strain of marine bacterium isolated from marine fish . The polyunsaturated fatty acid can be very efficiently extracted from WL-1021 that has the characteristics of rapid growth in the artificial medium . The PUFA in WL-1021 has been successfully separated by supercritical fluid extraction (SFE) . The SFE process parameters including pressure, temperature and culture temperature variations have been measured by High Performance Liquid Chromatography (HPLC), GC/FT-IR and GC/MS.

Rev Hosp Clin Fac Med Sao Paulo, 2002 Sep-Oct, 57(5), 205 - 8
Omeprazole, furazolidone, and tetracycline: an eradication treatment for resistant H . pylori in Brazilian patients with peptic ulcer disease; Silva FM et al.; OBJECTIVES: To determine the efficacy of a simple, short-term and low-cost eradication treatment for Helicobacter pylori (H . pylori) using omeprazole, tetracycline, and furazolidone in a Brazilian peptic ulcer population, divided into 2 subgroups: untreated and previously treated for the infection . PATIENTS AND METHODS: Patients with peptic ulcer disease diagnosed by endoscopic examination and infected by H . pylori diagnosed by the rapid urease test (RUT) and histological examination, untreated and previously unsuccessfully treated by macrolides and nitroimidazole, were medicated with omeprazole 20 mg daily dose and tetracycline 500 mg and furazolidone 200 mg given 3 times a day for 7 days . Another endoscopy or a breath test was performed 12 weeks after the end of treatment . Patients were considered cured of the infection if a RUT and histologic examination proved negative or a breath test was negative for the bacterium . RESULTS: Sixty-four patients were included in the study . The women were the predominant sex (58%); the mean age was 46 years . Thirty-three percent of the patients were tobacco users, and duodenal ulcer was identified in 80% of patients . For the 59 patients that underwent follow-up examinations, eradication was verified in 44 (75%) . The eradication rate for the intention-to-treat group was 69% . The incidence of severe adverse effects was 15% . CONCLUSION: The treatment provides good efficacy for H . pylori eradication in patients who were previously treated without success, but it causes severe adverse effects that prevented adequate use of the medications in 15% of the patients.

Int J Epidemiol, 2002 Oct, 31(5), 1001 - 7
Seroprevalence of IgG antibodies against Chlamydia pneumoniae in Chinese, Malays and Asian Indians in Singapore; Koh WP et al.; BACKGROUND: Chlamydia pneumoniae, a bacterium that causes respiratory infections, is probably under-diagnosed . There is also interest in its possible role in the aetiology of coronary heart disease . This is the first population-based seroprevalence survey of C . pneumoniae infection in Singapore . METHODS: A random sample of 1,068 people aged 18-69 years was selected from the participants of the Singapore National Health Survey conducted in 1998 . Sera and data on certain clinical measurements and conditions had been collected . IgG antibodies for C . pneumoniae were detected using an indirect microimmunofluorescence test and positivity graded . Seropositivity was defined as IgG titre >/=1:16 . RESULTS: There were no statistically significant differences in the prevalence rates of seropositivity to C . pneumoniae for age group 18-69 years among the three ethnic groups, i.e . Chinese (males 76.7%, females 68.3%), Malays (males 75.4%, females 59.1%), and Asian Indians (males 74.6%, females 59.4%) . The seropositivity rate for people aged 18-69 years in Singapore was 75.0% for males and 65.5% for females (difference of 9.5%, P < 0.001) . In both genders combined, seropositivity increased from 46.5% in the age group 18-29 to reach a plateau of 78.9% in the age group 40-49, which remained stable to 60-69 years . There was no association of seropositivity with smoking, diabetes mellitus, hypertension or body mass index after adjustment for age and gender . CONCLUSION: The high prevalence rates in our study population and the higher rate in males compared to females are consistent with studies from other parts of the world . No significant difference in prevalence rates was observed among Chinese, Malays and Indians . The pattern of rising and levelling off of seropositivity with age suggests that C . pneumoniae infection occurs early in life, and in older ages the high level of seropositivity is probably maintained by re-infections or chronic infections . Chlamydia pneumoniae infection was not found to be associated with the cardiovascular risk factors examined.

FEMS Microbiol Lett, 2002 Nov 5, 216(2), 151 - 8
The H-NS-like protein HvrA modulates expression of nitrogen fixation genes in the phototrophic purple bacterium Rhodobacter capsulatus by binding to selected nif promoters; Raabe K et al.; Genetic analyses based on chromosomal lac fusions to nitrogen fixation (nif) genes demonstrated that NifA-dependent transcriptional activation of expression of Rhodobacter capsulatus nifH and nifB1 was negatively modulated by HvrA, whereas regulation of rpoN, nifA1, and nifA2 was independent of HvrA . Expression of hvrA itself was not influenced by a mutation in ntrC, which is absolutely essential for N(2) fixation . Furthermore, HvrA accumulated to comparable levels in the presence and absence of ammonium, suggesting that the amount of HvrA in the cells does not differ under nitrogenase-repressing or -derepressing conditions . In addition, competitive gel retardation studies with HvrA-His(6) purified from R . capsulatus were carried out, demonstrating preferential binding of HvrA to the nifH promoter region.

Naturwissenschaften, 2002 Sep, 89(9), 428 - 32 Epub 2002 Aug 15.
Increased acidification in the rhizosphere of cactus seedlings induced by Azospirillum brasilense; Carrillo AE et al.; Acidification of the rhizosphere of cactus seedlings (giant cardon, Pachycereus pringlei) after inoculation with the plant growth-promoting bacterium Azospirillum brasilense Cd, in the presence or absence of ammonium and nitrate, was studied to understand how to increase growth of cardon seedlings in poor desert soils . While ammonium enhanced rhizosphere and liquid culture acidification, inoculation with the bacteria enhanced it further . On the other hand, nitrate increased pH of the rhizosphere, but combined with the bacterial inoculation, increase in pH was significantly smaller . Bacterial inoculation with ammonium enhanced plant growth.

Altern Lab Anim, 2002 Sep-Oct, 30(5), 539 - 50
A comparison of ecotoxicological tests; Botsford JL; A simple, inexpensive and rapid method of determining toxicity by using a bacterium as the indicator organism was developed and compared with 23 other tests . The average correlation coefficient when comparing these 23 tests with the present test was 0.800, ranging from 0.580 to 0.950 . Eleven of the tests were compared in detail by using 35 of the chemicals on the Multicentre Evaluation of In Vitro Cytotoxicity list of test chemicals . Comparing results from the present test with test results for these 35 chemicals with Microtox, Biotox, Daphnia magna, rat hepatocytes and ascites tumour cell resulted in correlation coefficients ranging from 0.871 to 0.933 . Comparisons of the test data with rodent LD50 values, human lethal dose estimates from autopsies and human lethal doses obtained from the literature provided correlation coefficients ranging from 0.580 to 0.770, indicating that the test compares less favourably with these methods . This test provides data comparable to data from other ecotoxicological tests.

Biochim Biophys Acta, 2002 Nov 19, 1601(1), 1 - 8
A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus; Pereira MM et al.; A {3Fe-4S}(1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus . It is a small protein, with an apparent molecular mass of 9 kDa . Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters . However, upon purification, it contains a single {3Fe-4S}(1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6 . {1H}NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre . When purified under anaerobic conditions, the EPR {3Fe-4S}(1+/0) centre signal is also observed . However, it can now be reduced by dithionite and a new signal attributed to a {4Fe-4S}(2+/1+) cluster develops . This can also be observed upon reconstitution of the prosthetic groups . The function of this ferredoxin in R . marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism.The thermodynamic stability of the R . marinus ferredoxin was also investigated and was shown to be high . Thermal and chemical unfolding reactions appear as single, cooperative transitions . The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7) . Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C) . The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.

Microbiology, 2002 Nov, 148(Pt 11), 3531 - 7
Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima; Nanavati D et al.; In this study, high-affinity maltose- and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected; these activities were distinct and specific . At the gross level, the expression of binding-protein activities was repressed by growth of T . maritima in the presence of the cognate sugar . Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity . In competition assays, these binding activities showed distinct patterns of substrate specificity: whereas the maltose-binding activity showed specificity for alpha-linked glucosides, the glucose-binding activity showed a broader specificity . All maltose- and glucose-binding activity was found in the supernatant retrieved following centrifugation (100,000 g) of the cell-free extracts prepared by French-pressure-cell treatment; no activity was found in an octyl-glucoside-treated extract of the membrane fraction . The maltose-binding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T . maritima cells using a newly developed freeze-thaw procedure . Annotation of the complete genome sequence of T . maritima suggests that there may be at least two maltose-binding proteins, MalE1 and MalE2, encoded in the genome . The maltose-binding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins . These data demonstrate that the hyperthermophilic bacterium T . maritima possesses separate maltose- and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

FEMS Microbiol Lett, 2002 Oct 29, 216(1), 49 - 54
Inhibition of acetate and propionate assimilation by itaconate via propionyl-CoA carboxylase in isocitrate lyase-negative purple bacterium Rhodospirillum rubrum; Berg IA et al.; Itaconate is known as a potent inhibitor of isocitrate lyase . Unexpectedly, itaconate was a strong inhibitor of acetate and propionate assimilation in isocitrate lyase-negative purple non-sulfur bacterium Rhodospirillum rubrum . It was shown that in cell extracts of R . rubrum itaconate inhibited propionyl-CoA carboxylase (PCC) activity . The participation of PCC in propionate assimilation in R . rubrum is well-documented, but the inhibition of acetate assimilation suggests that PCC is also involved in acetate metabolism . PCC is one of the enzymes of the citramalate cycle, the anaplerotic pathway proposed for R . rubrum as a substitute for the glyoxylate cycle . These results provide further support for the hypothesis of the occurrence of the citramalate cycle in R . rubrum . PCC from other isocitrate lyase-negative phototrophs, Rhodobacter sphaeroides and Phaeospirillum fulvum, was not inhibited by itaconate.

Eur J Biochem, 2002 Nov, 269(22), 5722 - 30
Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidans; Correia IJ et al.; Trihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium . Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D . acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum . The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre . The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems . This analysis indicates that the electrons enter the cytochrome mainly via haem I . The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied . Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp . and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.

Folia Microbiol (Praha), 2002, 47(4), 455 - 7
Isolation of Carnobacterium piscicola from human pus--case report; Chmelar D et al.; Carnobacterium piscicola was first described in 1984 . These bacteria are often isolated from fish afflicted with bacterial infections . To date, there has been no reported isolation of this bacterium from human specimens . We report here the isolation of C . piscicola from the pus following traumatic amputation of the right hand in the wrist of a 35-year-old man . The traumatic amputation occurred with an industrial water sawmill . The identity of the human strain was determined biochemically, by 16S rDNA sequence similarity and by fatty-acid methyl-ester profile from bacterial cell.

Mol Microbiol, 2002 Nov, 46(4), 1081 - 94
The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis; Porter SL et al.; The purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR . Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis . The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp) . Here, we show that every protein encoded by this locus is essential for normal chemotaxis . Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp . The two putative CheA proteins encoded in this locus are unusual . CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding . These data indicate that the minimal set of proteins required for normal chemotaxis in R . sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R . sphaeroides are not redundant.

J Clin Invest, 2002 Nov, 110(9), 1329 - 37
A novel dispersin protein in enteroaggregative Escherichia coli; Sheikh J et al.; Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture . We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model . We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control . The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium . EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria . Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent . Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae . We have therefore named the aap gene product "dispersin," and we propose that it may be representative of a functional class of colonization factors . Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen.

FEBS Lett, 2002 Nov 6, 531(2), 375 - 80
Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM); Chhabra SR et al.; The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously . The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus . The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C . The cel74 gene was previously found to be induced during T . maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch . However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch . Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes . As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate . Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.

Vet Microbiol, 2002 Dec 20, 90(1-4), 281 - 97
Brucella intracellular life: from invasion to intracellular replication; Gorvel JP et al.; Brucella organisms are pathogens that ultimate goal is to propagate in their preferred niche, the cell . Upon cell contact the bacteria is internalized via receptor molecules by activating small GTPases of the Rho subfamily and by a moderate recruitment of actin filaments . Once inside cells, Brucella localizes in early phagosomes, where it avoids fusion with late endosomes and lysosomes . These early events require the control of Rab small GTPases, and cytokines such as the G-CSF . Then, the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum, where it extensively replicates . Some of the bacterial molecular determinants involved in the internalization and early events after ingestion are controlled by the BvrS/BvrR two component regulatory system, whereas the intracellular trafficking beyond this early compartments are controlled by the VirB type IV secretion system . Once inside the endoplasmic reticulum, Brucella extensively replicates without restricting basic cellular functions or inducing obvious damage to cells . The integrity of Brucella LPS on the bacterial surface is one of the required factors for Brucella intracellular survival, and therefore for virulence .

Biochem J, 2003 Mar 1, 370(Pt 2), 489 - 95
Thermodynamic characterization of a tetrahaem cytochrome isolated from a facultative aerobic bacterium, Shewanella frigidimarina: a putative redox model for flavocytochrome c3; Pessanha M et al.; The facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions . This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c -type haem groups . The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques . This is the first detailed thermodynamic study performed on a tetrahaem cytochrome isolated from a facultative aerobic bacterium and reveals that this protein presents unique features . The redox centres have negative and different redox potentials, which are modulated by redox interactions between the four haems (covering a range of 8-56 mV) and by redox-Bohr interactions between the haems and an ionizable centre (-4 to -36 mV) located in close proximity to haem III . All of the interactions between the five centres are clearly dominated by electrostatic effects and the microscopic reduction potential of haem III is the one most affected by the oxidation of the other haems and by the protonation state of the molecule . Altogether, this study indicates that the tetrahaem cytochrome isolated from S . frigidimarina (Sfc) has the thermodynamic properties to work as an electron wire between its redox partners . Considering the high degree of sequence identity between Sfc and the cytochrome domain of flavocytochrome c(3), the structural similarities of the haem core, and that the macroscopic potentials are also identical, the results obtained in this work are rationalized in order to put forward a putative redox model for flavocytochrome c(3).

EMBO J, 2002 Nov 1, 21(21), 5599 - 610
Structural basis for the oxidation of thiosulfate by a sulfur cycle enzyme; Bamford VA et al.; Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains . This metabolism is a key component of the biogeochemical sulfur cycle . The SoxAX protein is a heterodimeric c-type cytochrome involved in thiosulfate oxidation . The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 A resolution in the oxidized state and at 1.5 A resolution in the dithionite-reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate . The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination . This unusual post-translational modification is also seen in sulfurtransferases such as rhodanese . Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential . These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation . The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33.

Nucleic Acids Res . 2002 Nov 1;30(21):e117.
Simultaneous determination of different DNA sequences by mass spectrometric evaluation of Sanger sequencing reactions; Kaetzke A et al.; All currently available DNA sequencing protocols rest fundamentally upon the homogeneity of the template . In this paper we describe the parallel DNA sequencing of various templates in one sample by a combination of the Sanger method and MALDI-TOF mass spectrometric analysis of the products . PCR-amplified hypervariable 16S rDNA fragments of the bacterium Escherichia coli DF1020 and cDNA of the 6-phosphofructo-1-kinase isoenzymes (PFK-1, EC 2.7.1.11) in rat brain were chosen as model systems for essentially heterogeneous templates . Avoiding cloning of the inhomogeneous PCR products we were able to read three sequences for both the 16S rDNA fragment of E.coli DF1020 and the cDNA of 6-phosphofructo-1-kinase from the peak lists of the Sanger sequencing reactions . Short sequences with a length between 21 and 25 nt were sufficient to reflect the heterogeneity of the 16S rDNA genes in E.coli and the existence of three isoenzymes of PFK-1 in rat brain.

Scand J Gastroenterol Suppl, 2002, (236), 15 - 8
Helicobacter pylori and gastro-oesophageal reflux disease: association and clinical implications . To treat or not to treat with anti-H . pylori therapy?
Loffeld RJ, van der Hulst RW.
BACKGROUND: It has been reported that patients are at risk of developing reflux oesophagitis after successful anti-Helicobacter pylori therapy, and the presence of the bacterium might be protective against the development of reflux oesophagitis . METHODS: Review of the literature . RESULTS: H . pylori is relevant to the management of oesophagitis because it increases the pH-elevating effect of proton-pump inhibitors . which increase the tendency of H . pylori gastritis to progress to atrophic gastritis, and because eradication of H . pylori increases the likelihood of oesophagitis . H . pylori increases basal gastrin levels, basal acid output, meal-stimulated maximal acid output and 24-h intragastric acidity . The effects on gastric acid production depend on the distribution of gastritis in the stomach . CONCLUSION: H . pylori eradication may induce or exacerbate gastro-oesophageal reflux by its influence on gastric acidity and the antisecretory action of proton-pump inhibitors.

Dig Liver Dis, 2002 Sep, 34 Suppl 2, S72 - 7
Consequences of Helicobacter pylori infection on the absorption of micronutrients; Annibale B et al.; Recent studies have suggested a relationship between Helicobacter pylori infection and various important micronutrients, including iron and vitamin B12, suggesting likely biological factors in the association between Helicobacter pylori and microcytic or macrocytic anaemia . There is some evidence that direct or indirect consequences of Helicobacter pylori gastritis on acid secretion account for the role of the bacterium in the absorption process of iron and Vitamin B12 . The plasma, intragastric and mucosal concentration of different antioxidant compounds such as ascorbic acid, a-tocopherol and beta-carotene is also affected by Helicobacter pylori gastritis supporting the possible role of Helicobacter pylori in the multistep cascade leading to gastric carcinogenesis . The relationship between Helicobacter pylori infection and micronutrients is, therefore, a promising and, until now, poorly investigated field of research.

Appl Environ Microbiol, 2002 Nov, 68(11), 5746 - 9
Uptake rates of oxygen and sulfide measured with individual Thiomargarita namibiensis cells by using microelectrodes; Schulz HN et al.; Gradients of oxygen and sulfide measured towards individual cells of the large nitrate-storing sulfur bacterium Thiomargarita namibiensis showed that in addition to nitrate oxygen is used for oxidation of sulfide . Stable gradients around the cells were found only if acetate was added to the medium at low concentrations.

Appl Environ Microbiol, 2002 Nov, 68(11), 5563 - 70
A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium, Alteromonas sp . strain O-7; Miyamoto K et al.; Alteromonas sp . strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction . In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain . The gene encoding MprIII was cloned in Escherichia coli . The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da . Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension . The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function . Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose . Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp . strain O-7 and characterized . The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa . The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate . Pretreatment of native chitin with MprIII significantly promoted chitinase activity . Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.

Mol Ecol, 2002 Nov, 11(11), 2425 - 34
Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae); Riegler M et al.; Wolbachia is an obligately intracellular, maternally inherited bacterium which has been detected in many arthropods . Wolbachia infections disperse in host populations by mechanisms such as cytoplasmic incompatibility (CI) . CI leads to embryonic mortality which occurs when infected males mate with uninfected females or females with a different Wolbachia strain . Populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae) were found to be infected by two different Wolbachia strains, wCer1 and wCer2 . Superinfections with both strains occurred throughout southern and central Europe and infections with wCer1 were found in northern, western and eastern Europe . Strong unidirectional CI between European populations of R . cerasi were first reported in the 1970s . From the conformity in the recent geographical distribution of the Wolbachia infections and the CI expression patterns found 25 years ago it was deduced that wCer2 potentially causes CI in R . cerasi . The comparison of the geographical distributions indicated that wCer1 + 2 must have spread into wCer1-infected populations in some areas . In other regions, a spread of wCer1 + 2 was probably prevented by dispersal barriers . There, a sharp transition from infected to superinfected populations suggested regional isolation between wCer1 and wCer1 + 2-infected populations.

MMWR Morb Mortal Wkly Rep, 2002 Oct 18, 51(41), 924 - 7
Q fever--California, Georgia, Pennsylvania, and Tennessee, 2000-2001; Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker; Department of Biological Sciences, Bowling Green State University, OH 43403, USAStudies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40-80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells . To further verify if A . vinelandii indeed contains 40-80 copies of its chromosome per cell, we have developed an 'in vivo chromosome counting' technique . The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium . The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA . To do this we used an A . vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s) . The plasmids pRK293 and pKT230, which can replicate in A . vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A . vinelandii BG102), served as the extrachromosomal elements with known copy number . Southern blotting and hybridization analysis of the total DNA, isolated from A . vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A . vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230 . We believe that this 'in vivo chromosome counting' technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.

J Bacteriol, 2002 Nov, 184(22), 6182 - 9
Involvement of two putative alternative sigma factors in stress response of the radioresistant bacterium Deinococcus radiodurans; Schmid AK et al.; Two genes bearing similarity to alternative sigma factors were identified in the Deinococcus radiodurans genome sequence and designated sig1 and sig2 . These genes were cloned and inactivated, and both were found to be important for survival during heat and ethanol stress, although the sig1 mutants displayed a more severe phenotype than the sig2 mutants . Reporter gene fusions to the groESL and dnaKJ operons transformed into these mutant backgrounds indicated that sig1 is required for the heat shock induction of groESL and dnaKJ, whereas sig2 mutants show a more moderate defect in dnaKJ induction and are not impaired for groESL induction . Essentiality tests suggested that neither sig1 nor sig2 is essential under all conditions . Sequence comparisons demonstrated that the sig1 gene product is classed distinctly with extracytoplasmic function (ECF) sigma factors, whereas Sig2 appears to be a more divergent sigma factor ortholog . These results suggest that sig1 encodes the major ECF-derived heat shock sigma factor in D . radiodurans and that it plays a central role in the positive regulation of heat shock genes . sig2, in contrast, appears to play a more minor role in heat shock protection and may serve to modulate the expression of some heat protective genes.

J Bacteriol, 2002 Nov, 184(22), 6174 - 81
Poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens AM1: identification and mutation of gap11, gap20, and phaR; Korotkova N et al.; Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates . Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C(1) and C(2) metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N . Korotkova and M . E . Lidstrom, J . Bacteriol . 183:1038-1046, 2001; N . Korotkova, L . Chistoserdova, V . Kuksa, and M . E . Lidstrom, J . Bacteriol . 184:1750-1758, 2002) . In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR . We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C(2) compounds, while they show wild-type growth characteristics on C(1) or multicarbon compounds . The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C(1) compounds and has defects in PHB accumulation but grows normally on C(3) and C(4) compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C(2) compounds . Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C(1), C(2), and heterotrophic metabolism in M . extorquens AM1, as well as the entry metabolite for the PHB cycle . Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

FEMS Microbiol Lett, 2002 Oct 8, 215(2), 285 - 9
Movement on the cell surface of the gliding bacterium, Mycoplasma mobile, is limited to its head-like structure; Miyata M et al.; Mycoplasma mobile cells glide on solid surfaces such as glass with a fast and continuous motion in the direction of the membrane protrusion (head-like structure) at one cell pole . To examine its cell-surface movement, a latex bead was attached to a cell and behavior in gliding was monitored . The bead was carried without movement relative to the cell body, suggesting that the cell does not roll around the cell axis and the surface movement is limited to a small area . A small percentage of cells showed an elongated head-like structure in an old batch culture . The head-like structure moved forward, sometimes leaving the cell body in one position, resulting in a stretching of this head-like structure . These results indicate that the head-like structure drags the cell body, leading us to conclude that the force for gliding is generated at the head-like structure.

FEMS Microbiol Lett, 2002 Oct 8, 215(2), 221 - 7
The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression; Drepper T et al.; The Rhodobacter capsulatus nrfA gene product exhibits extensive similarity to the nif (nitrogen fixation) regulatory factor NrfA of Azorhizobium caulinodans and the nucleoid-associated protein Hfq of Escherichia coli . Mutational analysis revealed that, in contrast to the situation in A . caulinodans, NrfA is not essential for diazotrophic growth of R . capsulatus, but it is required for maximal growth rates with N(2) as sole nitrogen source via either molybdenum nitrogenase or the alternative nitrogenase . NrfA was shown to control N(2) fixation in R . capsulatus at the level of expression of the regulatory genes nifA1, nifA2 and anfA, encoding the transcriptional activators of all the other nitrogen fixation genes.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 149 - 55
KN-62 enhances Chlamydia pneumoniae-induced p44/p42 mitogen-activated protein kinase activation in murine fibroblasts and attenuates in vitro infection; Haralambieva I et al.; Chlamydia pneumoniae elementary bodies were demonstrated to increase the proliferation of murine fibroblast cell line L-929 and rapidly activate p44/p42 mitogen-activated protein kinase (MAPK) in a protein kinase C (PKC) and protein kinase A (PKA)-independent way . Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) inhibitor KN-62 significantly enhanced C . pneumoniae-induced MAPK phosphorylation, suggesting negative control of CaM kinase pathway on the MAPK cascade . In in vitro infection assay, the upstream MAPK kinase 1/2 inhibitor U0126 increased 2.5-fold C . pneumoniae infectivity in L-929 cells, while KN-62 reduced the infection by 36% . Our findings provide insight into the molecular mechanisms of bacterium-host cell interactions and demonstrate the protective role of MAPK in murine fibroblasts, suggesting novel therapeutic approaches to the treatment and prevention of chlamydial infections.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 75 - 80
Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum; Fukamachi H et al.; Hydrogen sulfide and methyl mercaptan are the two major compounds associated with oral malodor . These compounds are highly toxic, and are thought to play an important role in periodontal disease . Fusobacterium nucleatum, an oral bacterium, produces large amounts of hydrogen sulfide from L-cysteine by the enzymatic action of L-cysteine desulfhydrase . We cloned and sequenced the cdl gene encoding L-cysteine desulfhydrase from F . nucleatum ATCC 10953, and revealed that the structural cdl gene consists of 921 bp and encodes a 33.4-kDa protein . The cloned gene was inserted into an expression vector, pDEST17, and expressed in Escherichia coli as a fused protein . The purified enzyme was tested for substrate specificity using various SH-containing compounds . Only L-cysteine served as a substrate for L-cysteine desulfhydrase to produce hydrogen sulfide.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 69 - 74
Chlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell line; Carratelli CR et al.; Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis . Here we show that infection with C . pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal . Analysis by flow cytometry at different post-infection times revealed that 50+/-7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C . pneumoniae-infected cultures (multiplicity of infection, MOI=30) displayed only 18+/-4% of cells in apoptosis . At MOI=20 and MOI=10 the cells susceptible to apoptosis at 48 h were 28+/-5% and 35+/-6% respectively . Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI=30), while UV-treated Chlamydia did provide a degree of protection against apoptosis . These data suggest that the anti-apoptotic effect of C . pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14428 - 33 Epub 2002 Oct 21.
Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites; Higashi H et al.; Helicobacter pylori is a causative agent of gastritis and peptic ulcer . cagA(+) H . pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma . The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation . The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells . CagA proteins of most Western H . pylori isolates have a 34-amino acid sequence that variably repeats among different strains . Here, we show that the repeat sequence contains a tyrosine phosphorylation site . CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes . In contrast, predominant CagA proteins specified by H . pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA . This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA . Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified . Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites . The presence of distinctly structured CagA proteins in Western and East Asian H . pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jan, 22(1), 20 - 1
Bacterium culture and flora analysis of gunshot wound on limbs of dogs in hot and humid environment; Wei KH et al.; OBJECTIVE: To investigate the time of bacterial invasion into the blood and conduct flora analysis of the blood and secretions in the gunshot wound in the limbs of dogs in hot and humid environment . METHODS: Gun-shot wound was induced in 8 dogs who were subsequently assigned at random into hot and humid environment (HHE) group and normal environment (NE) group for observation . At the time points of 0, 4, 6, 8, 12, 24 h after injury, body temperature was measured and bacterial culture and flora analysis performed . RESULTS: Body temperature elevation occurred earlier and lasted for longer time in HEE group than in NE group . Bacterial invasion into the blood occurred at 12 h after the injury in NE group, but at 8 h in HHE group . In the wound tracts of the dogs, surface bacteria were found in both groups . Surface bacteria were also found in the blood of dogs in both groups, but in HHE group, even intestinal flora were identified . CONCLUSION: Body temperature elevation and bacterial invasion into the blood occurred at an earlier time in hot and humid environment where migration of intestinal bacteria into the blood easily takes place, indicating the early application of broad-spectrum antibiotics under such environmental conditions.

Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 413 - 6
Expression and purification of recombinant bone morphogenetic protein-2 in E.coli; Xiong SH et al.; OBJECTIVE: To explore the method for producing human bone morphogenetic protein-2 (hBMP-2) by gene engineering techniques . METHODS: E.coli BL21 was transformed with recombinant plasmid pYR (pBV220-hBMP-2) under different conditions, and SDS-PAGE analysis was conducted to observe the effects of the activation status and induction time of the bacterium on the target protein expression . The inclusion bodies obtained from E.coli were purified by anion exchange chromatography DEAE and molecular sieve S-300, and the recombinant protein was renatured by dialyse . RESULTS: SDS-PAGE analysis showed a conspicuous band after induction signifying a new foreign protein with relative molecular mass of approximately 13 000 . After activation of the bacteria when D600 was about 0.45, most efficient expression of rhBMP-2 was achieved which reached the peak 4 h after induction with heat . Implantation of the purified recombinant hBMP-2 resulted in proliferation of mesenchymal cells and new cartilage and bone formation, as shown by histological analysis 4 weeks after implantation . CONCLUSION: hBMP-2 produced by gene engineering techniques possesses the biological capacity of ectopic bone formation.

Biochemistry, 2002 Oct 29, 41(43), 12921 - 7
Vibrational spectroscopy favors a unique QB binding site at the proximal position in wild-type reaction centers and in the Pro-L209 --> Tyr mutant from Rhodobacter sphaeroides; Breton J et al.; In the various X-ray structures of native reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides, two distinct main binding sites (distal and proximal) for the secondary quinone Q(B) have been described in the literature . The movement of Q(B) from its distal to proximal position has been proposed to account for the conformational gate limiting the rate of the first electron transfer from the primary quinone Q(A-) to Q(B) . Recently, Q(B) was found to bind in the proximal binding site in the dark-adapted crystals of a mutant RC where Pro-L209 was changed to Tyr {Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G . (2001) Biochemistry 40, 4253-4260} . To test the structural and functional implications of the distal and proximal sites, a comparison of the FTIR vibrational properties of Q(B) in native RCs and in the Pro-L209 --> Tyr mutant was performed . Light-induced FTIR absorption changes associated with the reduction of Q(B) in Pro-L209 --> Tyr RCs reconstituted with 13C-labeled ubiquinone (Q3) at the 1 or 4 position show a highly specific IR fingerprint for the C=O and C=C modes of Q(B) upon selective labeling at C1 or C4 . This IR fingerprint is very similar to that of native RCs, demonstrating that equivalent interactions occur between neutral Q(B) and the protein in native and mutant RCs . Consequently, Q(B) occupies the same binding site in all RCs . Since the FTIR data fit the description of Q(B) bonding interactions in the proximal site, it is therefore concluded that neutral Q(B) also binds to the proximal site in native functional RCs . The implication of these new results for the conformational gate of the first electron transfer to Q(B) is outlined.

Evolution Int J Org Evolution, 2002 Sep, 56(9), 1735 - 42
Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D . sechellia; Charlat S et al.; The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI) . CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains . The present study focuses on this phenomenon in two Drosophila species: D . simulans and D . sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including wHa and wNo . Drosophila sechellia is infected by only two Wolbachia: wSh and wSn . In both Drosophila species, double infections with Wolbachia are found . As indicated by several molecular markers, wHa is closely related to wSh, and wNo to wSn . Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely siI (associated with wHa and wNo in D . simulans) and se (associated with wSh and wSn in D . sechellia) . To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected wSh and wSn into D . simulans, to compare their CI properties to those of their sister strains wHa and wNo, respectively, in the same host genetic background . We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible . We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.

J Infect, 2002 Oct, 45(3), 135 - 8
Chlamydia pneumoniae in community-acquired pneumonia: seven years of experience; Monno R et al.; OBJECTIVES: To determine the prevalence of Chlamydia pneumoniae in community-acquired pneumonia during a period of seven years . METHODS: Serum samples from 311 patients with pneumonia were evaluated using microimmunofluorescence assay to detect C . pneumoniae -specific IgG and IgM antibodies . RESULTS: Thirty nine patients (12.5%) complied with the diagnostic criteria of acute C . pneumoniae infection (a four-fold rise in the titer of IgG antibody, or a single IgG titer > or = 1:512, or a single IgM titer > or = 1:16) . All patients were diagnosed as having pneumonia . Co-infection with other respiratory tract pathogens was found in four patients . CONCLUSIONS: C . pneumoniae is an important cause of pneumonia also in our area . Pneumonia due to this bacterium occurs in the cold months and in early spring; in addition we have observed periods of increased incidence of one years duration and periods of low incidence lasting one-two years . Therapy with macrolides and levofloxacin was effective in all patients with C . pneumoniae infection.

Acta Virol, 2002, 46(2), 121 - 4
Analysis of phospholipids from Coxiella burnetii by fast atom bombardment mass spectrometry . A rapid method for differentiation of virulent phase I and low virulent phase II cells; Domingues P et al.; Phospholipids extracted from the Coxiella burnetii strain Nine Mile virulent phase I and low-virulent phase II cells were directly analyzed by fast atom bombardment mass spectrometry (FAB-MS) . Constant neutral loss (CNL) scanning mass spectra (MS) were acquired to identify various phospholipids within phospholipid classes . Phospholipids from the phase I C . burnetii cells were much more complex than those from the phase II cells . Moreover, in the latter, the absence of phospholipids of the phosphatidylinositol class could be noticed . The results indicate that CNL scanning of phospholipid samples provides a rapid and simple method for identification of the phase state of the bacterium.

Clin Infect Dis, 2002 Nov 1, 35(9), e99 - 102 Epub 2002 Oct 03.
Human monocytic ehrlichiosis presenting as acute appendicitis during pregnancy; Smith Sehdev AE et al.; Human monocytic ehrlichiosis (HME) is caused by Ehrlichia chaffeensis, an obligate intracellular bacterium that infects mononuclear phagocytic cells . We report a unique case of HME diagnosed in a woman who presented with abdominal pain and acute appendicitis during early pregnancy and whose condition progressively deteriorated to adult respiratory distress syndrome.

Extremophiles, 2002 Oct, 6(5), 397 - 405 Epub 2002 May 23.
Purification and characterization of a cold-adapted isocitrate lyase and expression analysis of the cold-inducible isocitrate lyase gene from the psychrophilic bacterium Colwellia psychrerythraea; Watanabe S et al.; Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized . The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs . The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C . Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C . These features indicate that ICL from this bacterium is a typical cold-adapted enzyme . A partial amino acid sequence of the C . psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris . Expression of the gene encoding the C . psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium . The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.

Extremophiles, 2002 Oct, 6(5), 369 - 75 Epub 2002 Apr 18.
F- and V-type ATPases in the hyperthermophilic bacterium Thermotoga neapolitana; Iida T et al.; Two gene clusters encoding F- or V-type ATPases were found in genomic DNA of the hyperthermophilic bacterium Thermotoga neapolitana . The subunit genes of each ATPase formed an operon . While the gene arrangement in the operon of the F-type ATPase resembled those in eukaryotic organelles and bacteria, that of the V-type ATPase was different from those reported for archaea, bacteria, or eukaryotes . Both ATPases were found to be expressed in the cells of T . neapolitana by Western blot analysis . Although V-type ATPase could not be rendered soluble, F-type ATPase was solubilized with 1% Triton X-100 and characterized . This is the first report of the coexistence of both F- and V-type ATPases in hyperthermophilic bacteria . It has recently been shown by a genome analysis that Thermotoga maritima has no V-type ATPase gene cluster but does have an F-type ATPase gene cluster; however, part of a gene for the D-subunit of the V-type ATPase gene has been reported in the T . maritima genome . Evolution of the two types of ATPases in Thermotoga is discussed.

J Mol Biol, 2002 Oct 18, 323(2), 225 - 36
Identification and structure of the anti-sigma factor-binding domain of the disulphide-stress regulated sigma factor sigma(R) from Streptomyces coelicolor; Li W et al.; The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis . Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis . Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity . Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site . We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association . We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A . sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

Infect Immun, 2002 Nov, 70(11), 6330 - 8
In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions; Fenhalls G et al.; We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients . The M . tuberculosis genes chosen fall into two classes . Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure . Both necrotic and nonnecrotic granulomas were identified in all of the patients . Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region . Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas . Mycobacterial gene expression was associated with CD68-positive myeloid cells . Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area . There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present . The differential expression of genes within granulomas demonstrates that M . tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments . These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Aug, 22(8), 678 - 83
{Expression and identification of phage display library for Fab fragments of colorectal cancer-related antibodies}; Sun X et al.; OBJECTIVE: To express the original human Fab antibody phage display library with positive recombined bacterium XL1-blue-Pcomb3 and identify its specific binding activity with colorectal cancer cells in vitro after screening with human colorectal cancer-related antigens . METHOD: The recombination rate of Fd fragment of the heavy chain and insertion of kappa chain of the antibodies was determined with PCR, and the original Fab library was expressed . The antigens were extracted from 3 sensitized colorectal cancer tissues previously used for construction of the original Fab library and from 13 non-sensitized colorectal cancer tissues, along with the antigens from LoVo, HT-29 and LS-174T cells cultured in vitro . The original Fab antibody library was screened with the 3 groups of mixed antigens derived in preceding procedure and 3 tertiary Fab antibody libraries were obtained, which were then mixed in equal volume for subsequent tests of binding activity with human colorectal cancer tissues and cells in vitro using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining . Specimens of gastric and esophageal carcinomas and normal intestinal mucosa, together with liver cancer cells and gastric cancer cells were utilized as control . RESULT: The recombination rate of Fd and kappa chain were 40 % and 70 % respectively, and the rate of their simultaneous insertion into Pcomb3 vector was 28% . The capacity of library for Fab fragment genes was 2.1x10(6), and the original antibody libraries screened with the 3 groups of mixed antigens were enriched to varied degrees, which all displayed relatively specific binding activity with human colorectal cancer tissue and cells in vitro . CONCLUSION: Colorectal cancer-related antibody Fab fragments are obtained through screening phage display library, which show relatively specific binding activity with human colorectal cancer tissues and cells.

Analyst, 2002 Sep, 127(9), 1247 - 51
An indirect conductimetric screening method for the detection of antibiotic residues in bovine kidneys; Myllyniemi AL et al.; An indirect conductimetric screening method using three test bacterium-medium combinations was developed for rapid detection of antibiotic residues in bovine carcasses . The detection time (DT), i.e . the point when the growth of the test bacterium was detected, was determined by observing the rate of change in the conductance plotted against time . This detection time averaged half of the reference time recorded by the instrument software . Total change in conductance (TC) was used as a further measure of growth . Threshold values for DT and TC were determined with inhibitor-free kidney samples . The presence of a residue was indicated if the DT exceeded the respective threshold value and was confirmed if the TC remained below the TC threshold value . The limits of detection (LODs) determined with fortified samples were at about or below the MRLs for cephalexin, chlortetracycline, ciprofloxacin, dihydrostreptomycin, enrofloxacin, oxytetracycline and penicillin G . The LODs for penicillin G, oxytetracycline and the sum of enrofloxacin and ciprofloxacin were also estimated with incurred samples; these samples were also analysed using liquid chromatography . The LODs determined with fortified and incurred samples were in close agreement . Given its rapid detection, good sensitivity to a wide range of antibiotics and ease of performance, the indirect conductimetric method developed here would seem to offer an appealing alternative to agar diffusion tests.

J Bacteriol, 2002 Nov, 184(21), 6069 - 72
groEL expression in gyrB mutants of Borrelia burgdorferi; Alverson J et al.; GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease . Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B . burgdorferi . Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B . burgdorferi . To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium.

FEBS Lett, 2002 Oct 9, 529(2-3), 208 - 14
A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite:cytochrome c oxidoreductase from Starkeya novella; Kappler U et al.; The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions . Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism . A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c . The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme . The pDorEX system therefore seems to be potentially useful for heterologous expression of multi-subunit proteins containing complex redox cofactors.

Microbiology, 2002 Oct, 148(Pt 10), 2997 - 3006
Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis; Santangelo MP et al.; mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium . Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature . To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc(2)155 and M . tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used . The presence of the Rv1963 gene in the strains greatly reduced beta-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon . Expression of mce3 by recombinant M . tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions . However, no lifting of repression was induced . The mce3 promoter was defined by deletion and cloning of the Rv1963-Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon . Gel-shift experiments determined that the Rv1963-binding site was located in this region . These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.

J Periodontal Res, 2002 Oct, 37(5), 353 - 9
Release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of Actinobacillus actinomycetemcomitans; Claesson R et al.; Matrix metalloproteinase 8 (MMP 8) degrades type I collagen and may be involved in the pathogenesis of periodontitis . Latent MMP 8 is stored in neutrophil granules and can be activated when released extracellularly . The periodontitis-associated bacterium Actinobacillus actinomycetemcomitans produces an RTX-toxin, leukotoxin, that degranulates and lyses human neutrophils . This study deals with the ability of leukotoxic A . actinomycetemcomitans to trigger the release and activation of MMP 8 . Whole bacteria of three A . actinomycetemcomitans strains or leukotoxin purified from the highly toxic strain HK 1519 were incubated with human neutrophils . The extracellularly released latent and active forms of MMP 8 were detected by an immunoblot technique using specific antibodies against the protease . The activity of MMP 8 was determined by a collagen degradation assay . All strains induced release and activation of MMP 8 . The effect was more pronounced under aerobic than anaerobic conditions and correlated with the leukotoxicity of the strains . Pure leukotoxin also induced MMP 8 release and activation in a concentration-dependent manner . Under aerobic conditions, oxidising substances formed by the neutrophils contributed to the rapid activation of the latent enzyme . Upon anaerobic incubation, the activation was slow and mainly caused by other proteases released during neutrophil degranulation . The activation was totally abolished in the presence of serum, probably due to the serum-protease inhibitors . Compared to the calcium ionophore A 23187, a well-known stimulus of neutrophil degranulation, leukotoxin was a more powerful inducer of MMP 8 release, since it triggered the process at a 1000-fold lower concentration . The present findings reveal a specific mechanism that can be induced by A . actinomycetemcomitans leukotoxin and which may contribute to the degradation of periodontal tissues under certain conditions.

Mol Microbiol, 2002 Oct, 46(1), 75 - 85
The four different sigma(54) factors of Rhodobacter sphaeroides are not functionally interchangeable; Poggio S et al.; The sigma(54) factor is highly conserved in a large number of bacterial species . From the complete genome sequence of Rhodobacter sphaeroides, it was possible to identify four different sequences encoding potentially functional sigma(54) factors . In this work, we provide evidence that one of these copies (rpoN2) is specifically required to express the flagellar genes in this bacterium . A mutant strain carrying a lesion in the rpoN2 gene was unable to swim even though the RpoN1 and RpoN3 proteins were present in the cytoplasm . The possibility that the different copies of the sigma(54) factor might be specific for the transcription of a particular subset of sigma(54) promoters was reinforced by the fact that a mutant strain carrying a lesion in rpoN1 showed a severe growth defect in nitrogen-free culture medium, even though the rpoN2 and rpoN4 genes were actively transcribed from a plasmid or from the chromosome . Different mechanisms that might be responsible for this specificity are discussed.

Cell Microbiol, 2002 Oct, 4(10), 663 - 76
Activation of Rho and Rab GTPases dissociates Brucella abortus internalization from intracellular trafficking; Chaves-Olarte E et al.; Brucella abortus is an intracellular pathogen that relies on unconventional virulence factors to infect hosts . In non-professional phagocytes, Rho GTPases-activation by the Escherichia coli cytotoxic necrotizing factor (CNF) promoted massive Brucella entrance by membrane ruffling, a mechanism that differs from the common mode of entrance used by this bacterium in non-treated cells . Cytotoxic necrotizing factor treatment, however, did not alter the intracellular route followed by the wild type or non-virulent defined mutants . In contrast, expression of a constitutively active Rab5Q79L GTPase did not alter cell-invasion by Brucella but hampered its ability to reach the endoplasmic reticulum . The CNF-induced Brucella super-infection did not reduce the ability of host cells to synthesize DNA and progress through the cell cycle . Furthermore, CNF-treatment increased the isolation of Brucella-containing compartments by a factor of 15 . These results demonstrate that in non-professional phagocytic cells, Brucella manipulates two different sets of GTPases during its biogenesis, being internalization and intracellular trafficking two consecutive but independent processes . Besides, CNF-induced super-infection demonstrates that Brucella does not interfere with crucial cellular processes and has shown its potential as tool to characterize the intracellular compartments occupied by this bacterium.

Clin Microbiol Rev, 2002 Oct, 15(4), 631 - 46
Tularemia; Ellis J et al.; Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals . In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal . The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent . The diagnosis of disease is not straightforward . F . tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel . Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia . Little is known about the virulence mechanisms of F . tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages . An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia . Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F . tularensis . In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia.

Dis Aquat Organ, 2002 Aug 29, 51(2), 85 - 92
Relationship between antigen concentration and bacterial load in Pacific salmon with bacterial kidney disease; Hamel OS et al.; Using data collected to test spawning female Pacific salmon (Oncorhynchus kisutch and O . tshawytscha for the presence and severity of bacterial kidney disease (BKD), a mathematical model of the relationship between bacterial load and antigen concentration in tissues and ovarian fluid is developed . Renibacterium salmoninarum, the causative agent of BKD, secretes large amounts of a 57 kDa protein ('p57'), its major soluble antigen, which eventually breaks down or is otherwise removed from free circulation . Bacterial load and soluble antigen concentration in tissues are strong indicators of fish health, while in ovarian fluid they are predictors of the success of offspring . Model results indicate either an exponentially increasing antigen removal rate or an exponentially decreasing per-bacterium antigen secretion rate with increasing antigen concentration . Possible mechanisms underlying the observed relationship include a nonlinear increasing autolytic rate of the 'p57' antigen and a bacterium-antigen interaction threshold which prevents bacterial antigen secretion.

Acta Microbiol Pol, 2002, 51(2), 121 - 9
Comparison of multiplex PCR, and an immunochromatographic method sensitivity for the detection of Escherichia coli O157:H7 in minced beef; Gryko R et al.; In this study, the sensitivities of multiplex PCR and an immuno-chromatographic methods to detect Escherichia coli O157:H7 in minced beef were compared . The detection of Escherichia coli O157:H7 in minced beef inoculated with 1-100 cells of this bacterium was possible after enrichment of culture and subsequent analysis by either of the two methods . Enrichment conditions were eight hours of incubation at 37 degrees C or 42 degrees C in a non-selective medium (Buffered Peptone Water) . Multiplex PCR analysis was performed using three primer sets with analysis by gel electrophoresis . The Quix immuno-chromatographic assay which is a new kit being marketed by New Horizons Diagnostics, Columbia, MD, was used for immunological analysis of the enriched broths.The sensitivity of both tests was similar . The results depended on the concentration of the specific bacterium in the culture since the influence of the proportion of other bacteria to the E . coli O157:H7 was not observed . The data suggests that either method or used together, when coupled with an enrichment technique, could provide a rapid mean to detect the presence of this pathogen in minced meat samples.

Proteomics, 2002 Sep, 2(9), 1288 - 303
Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting; Mathesius U et al.; We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species . First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability . Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities . In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities . However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species . Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing . While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases . In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1687 - 93
Sinorhizobium morelense sp . nov., a Leucaena leucocephala-associated bacterium that is highly resistant to multiple antibiotics; Wang ET et al.; Sinorhizobium morelense sp . nov . is described to designate a group of bacteria isolated from root nodules of Leucaena leucocephala . S . morelense shows 98% 16S rRNA gene sequence similarity to some Sinorhizobium species and to Ensifer adhaerens . This novel species is distinguished from other Sinorhizobium species and from E . adhaerens by DNA-DNA hybridization, 165 rRNA gene restriction fragments and sequence and some distinctive phenotypic features . Strains of this species are highly resistant to some antibiotics, such as carbenicillin (1 mg ml(-1)), kanamycin (500 microg ml(-1)) and erythromycin (300 microg ml(-1)) . They do not form nodules, but a nodulating strain, Lc57, is closely related to the novel species . Strain Lc04T (= LMG 21331T = CFN E1007T) is designated as the type strain of this novel species.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1527 - 32
Psychromonas kaikoae sp . nov., a novel from the deepest piezophilic bacterium cold-seep sediments in the Japan Trench; Nogi Y et al.; Two strains of obligately piezophilic bacteria were isolated from sediment collected from the deepest cold-seep environment with chemosynthesis-based animal communities within the Japan Trench, at a depth of 7434 m . The isolated strains, JT7301 and JT7304T, were closely affiliated with members of the genus Psychromonas on the basis of 16S rDNA sequence analysis . Hybridization values for DNA-DNA relatedness between these strains and the Psychromonas antarctica reference strain were significantly lower than that accepted as the phylogenetic definition of a species . The optimal temperature and pressure for growth of the isolates were 10 degrees C and 50 MPa and they produced both eicosapentaenoic acid (C20:5omega3) and docosahexaenoic acid (C22:6) in the membrane layer . Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Psychromonas species . The name Psychromonas kaikoae sp . nov . (type strain JT7304T = JCM 11054T = ATCC BAA-363T) is proposed . This is the first proposed obligately piezophilic species of the genus Psychromonas.

Lett Appl Microbiol, 2002, 35(4), 331 - 7
PCR-based assay for the detection of Curtobacterium flaccumfaciens pv . flaccumfaciens in bean seeds; Tegli S et al.; AIMS: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv . flaccumfaciens (Cff) in bean seeds . METHODS AND RESULTS: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using Cff DNA as template . Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h . CONCLUSIONS: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved . SIGNIFICANCE AND IMPACT OF THE STUDY:Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques.

Cytometry, 2002 Oct 1, 49(2), 49 - 55
Evaluation of several flow cytometric assays for the analysis of T-cell responses in goats; Totte P et al.; BACKGROUND: Flow cytometry (FCM) provides an alternative to radioactive methods for the analysis of T-cell responses . However, a comparative study of common FCM assays in an outbred ruminant model is lacking, which motivated this work . METHODS: Goats immunized with the obligate intracellular bacterium Cowdria ruminantium, inactivated and emulsified in oil-based adjuvants, were used as a model to study T-cell recall responses in vitro . FCM-based methods to measure Cowdria-induced lymphoblastogenesis, DNA synthesis, and interleukin-2 receptor (IL-2R) expression by T-cell subsets were compared . RESULTS: IL-2R expression was the most sensitive and reliable method provided that the number of molecules per cell was analyzed and not simply the percentage of positive cells of a given phenotype . Despite high background due to adjuvant and low proliferation, this method could detect antigen-specific activation of immune CD4(+) and CD8(+) T cells . CONCLUSIONS: FCM-based measurement of lymphoblastogenesis and DNA synthesis are not the most appropriate methods to analyze T-lymphocyte activation during vaccination of outbred animals . On several occasions, analysis of IL-2R expression was the only assay capable of discriminating between vaccinated and naive animals in this model .

Biochem J, 2003 Jan 1, 369(Pt 1), 153 - 61
Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens; Lloyd JR et al.; A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized . The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans . The gene encoding PpcA was identified from the G . sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49% identity to the c (7) cytochrome of D . acetoxidans . In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method . Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant . When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored . The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered . The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor . When taken together with previous studies on other electron transport proteins in G . sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.

J Clin Microbiol, 2002 Oct, 40(10), 3871 - 3
Dialister pneumosintes associated with human brain abscesses; Rousee JM et al.; In this report, we review two cases of brain infection due to Dialister pneumosintes in previously healthy patients . The bacterium was isolated from the first patient by blood culture and directly from a brain abscess in the second patient . In both cases, the infection was suspected to be of nasopharyngeal or dental origin . The patients had favorable outcomes following surgical debridement and antibiotic treatment . After in vitro amplification and partial sequencing of the 16S rRNA gene, two strains were classified as D . pneumosintes . However, traditional biochemical tests were not sufficient to identify the bacteria . In addition to causing periodontal and opportunistic infections, D . pneumosintes, contained in mixed flora, may behave as a clinically important pathogen, especially in the brain . In addition to phenotypic characterization, 16S rRNA partial sequencing was used to identify D . pneumosintes definitively.

J Clin Microbiol, 2002 Oct, 40(10), 3720 - 8
Real-time quantitative PCR for detection of Helicobacter pylori; He Q et al.; Helicobacter pylori is one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma . Humans are the bacterium's only host . Here we report the development of a real-time quantitative (Q) PCR-based assay to measure ureC gene copy number to detect H . pylori, based on the fact that there is only one copy of the ureC gene per bacterium . Upon optimization of LightCycler Q-PCR conditions, we obtained a standard curve with a linear range (correlation coefficient = 1) across six logs of DNA concentration . We were able to accurately quantify as few as 1,000 bacteria in our assay . Analysis of variance on 15 randomly selected clinical samples showed good reproducibility of this assay . Comparison of Q-PCR results with bacterial culture and histopathological results from an additional 85 clinical biopsy samples showed a significant difference for the presence of H . pylori . Many samples that were negative for H . pylori by culture and histopathology were positive by Q-PCR . Contamination of PCR by H . pylori or H . pylori genetic material could not be ruled out . In summary, we developed a rapid, sensitive, and real-time Q-PCR method for detecting H . pylori . This technique offers a significant improvement over other available methods for detecting H . pylori in clinical and research samples.

Mol Microbiol, 2002 Sep, 45(6), 1589 - 98
Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin-filamentous haemagglutinin interaction; Zaretzky FR et al.; Adenylate cyclase (AC) toxin from Bordetella pertussis is unusual in that, unlike most other members of the repeats-in-toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface . In this study, we investigated the nature of the association of this toxin with the surface of B . pertussis . AC toxin was extracted from crude outer membrane preparations of B . pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane . B . pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin . AC toxin distribution was unaffected by the absence of pertactin, or full-length lipopolysaccharide, or a defect in secretion of pertussis toxin . Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA . Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B . pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell.

Oral Microbiol Immunol, 2002 Oct, 17(5), 321 - 3
Isolation of the provisionally named Desulfovibrio fairfieldensis from human periodontal pockets; Loubinoux J et al.; Sulfate-reducing bacteria have recently been associated with periodontitis and proposed to play a role in the pathogenesis of this chronic inflammatory process . Eight isolates of sulfate-reducing bacteria belonging to the genus Desulfovibrio were obtained from the periodontal pockets of five out of seven patients presenting with active periodontitis . A multiplex PCR was devised for their identification at the species level . All isolates were identified as Desulfovibrio fairfieldensis, a recently proposed new species . This finding reinforces the suggestion that Desulfovibrio fairfieldensis is a human bacterium that may present a pathogenic potential.

Oral Microbiol Immunol, 2002 Oct, 17(5), 290 - 5
Cloning and expression of a Porphyromonas gingivalis gene for protoporphyrinogen oxidase by complementation of a hemG mutant of Escherichia coli; Kusaba A et al.; Porphyromonas gingivalis, a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX . By complementation with a P . gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli . The DNA sequence analysis of this clone revealed three open reading frames (ORFs) . ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da . The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues) . When the ORF3 gene was overexpressed in E . coli, the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO . Thus, ORF3 was named PgHemG . Furthermore, several porphyrin-related genes, including hemD, hemN and hemH, were identified in the data bases on the websites available on-line . We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P . gingivalis.

Biosci Biotechnol Biochem, 2002 Aug, 66(8), 1677 - 81
Effects of acetan on production of bacterial cellulose by Acetobacter xylinum; Ishida T et al.; Acetan is a water-soluble polysaccharide produced by a bacterial cellulose (BC) producer, Acetobacter xylinum . An acetan-nonproducing mutant, EP1, was generated from wild-type A . xylinum BPR2001 by the disruption of aceA, which may act to catalyze the first step of the acetan biosynthetic pathway in this bacterium . EP1 produced less BC than the wild-type strain . However, when EP1 was cultured in a medium containing acetan, BC production was stimulated and the final yield of BC was equivalent to that of BPR2001 . The culture broth containing acetan was more viscous and the free cell number was higher than that of the broth without the polysaccharide, so acetan may hinder the coagulation of BC in the broth . The addition of 1.5 g/l agar also increased BC production; we concluded that acetan and BC syntheses were not directly related on the genetic level.

Biosci Biotechnol Biochem, 2002 Aug, 66(8), 1646 - 51
Isolation from Nocardioides sp . strain CT16, purification, and characterization of a deoxycytidine deaminase extremely thermostable in the presence of D,L-dithiothreitol; Sakai T et al.; A deoxycytidine deaminase that was extremely thermostable in the presence of dithiothreitol was found in a mesophilic bacterium isolated from soil . The bacterium was classified as a Nocardioides sp . The enzyme was purified to a homogeneous protein by treatment at 100 degrees C, ammonium sulfate precipitation, and chromatography on DEAE-Toyopearl, hydroxyapatite, and then Sephacryl S-100 . Twenty micrograms of the pure enzyme was obtained from 811 mg of the starting crude protein . After treatment at 50 degrees C for 15 min in the absence of dithiothreitol, enzyme activity was 44% of the starting activity; after treatment at 100 degrees C for 2 h in the presence of 50 mm dithiothreitol, activity was 56% of the starting activity . Dithiothreitol greatly stabilized the enzyme . Activity was maximum at 99 degrees C . The Km values for deoxycytidine, cytidine, and methyl-deoxycytidine were 55.2, 140, and 130 microM, respectively . The molecular mass was estimated to be 52 kDa by gel permeation chromatography . The enzyme molecule was dissociated into two subunits each of 18 kDa subunit when reduced with mercaptoethanol.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 289 - 294
Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii; Kawahara K et al.; The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy . One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before . In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.

J Nutr Sci Vitaminol (Tokyo), 2002 Jun, 48(3), 242 - 6
Purification and characterization of methylmalonyl-CoA mutase from a methanol-utilizing bacterium, Methylobacterium extorquens NR-1; Miyamoto E et al.; High activity (about 50 mU x mg protein(-1)) of methylmalonyl-CoA mutase (82-95% apo-enzyme) was constantly found during the cell growth of a methanol-utilizing bacterium, Methylobacterium extorquens NR-1 . The apo-enzyme was purified to homogeneity and characterized . The purified enzyme was colorless . An apparent Mr of M . extorquens NR-1 enzyme was calculated to be 150,000 +/- 5,000 by Superdex 200 HR gel filtration . SDS-polyacrylamide gel electrophoresis of the purified enzyme gave two protein bands with an apparent Mr of 85.000 +/- 2,000 and 70,000 +/- 2,000, indicating that the M . extorquens NR-1 enzyme is composed of two nonidentical subunits . NH2-terminal amino acid sequences of the small and large subunits of M . extorquens NR-1 enzyme showed no significant homology to those of the enzyme from other species . Some enzymological properties of the M . extorquens NR-1 enzyme were studied.

J Biol Chem, 2002 Nov 29, 277(48), 46110 - 5 Epub 2002 Sep 24.
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase; D'Amico S et al.; Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation . In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas . This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft . We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme . Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T(m)) was not increased . Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated alpha-amylase . However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C as well as the appearance of a less stable calorimetric domain . It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution.

Appl Environ Microbiol, 2002 Oct, 68(10), 5104 - 12
Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation; Hiraoka Y et al.; Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs . In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome . In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50% . In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs . In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content . These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content . To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement . The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP) . When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP . When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP . Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes . Electron microscopy revealed that cell-cell separation was inhibited in this culture . This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.

Appl Environ Microbiol, 2002 Oct, 68(10), 4809 - 11
Isolation of elemental sulfur as a self-growth-inhibiting substance produced by Legionella pneumophila; Inoue H et al.; The addition of HP-20 resin to a medium could enhance the growth of Legionella species . Elemental sulfur was isolated as a self-growth-inhibiting substance produced endogenously by Legionella pneumophila from methanol extracts of the resins used to culture the bacterium . Elemental sulfur shows strong growth-inhibiting activity toward all Legionella species tested.

Mol Ecol, 2002 Oct, 11(10), 2123 - 35
Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid, Acyrthosiphon pisum; Tsuchida T et al.; In addition to the essential intracellular symbiotic bacterium Buchnera, several facultative endosymbiotic bacteria called collectively secondary symbionts (S-symbionts) have been identified from the pea aphid Acyrthosiphon pisum . We conducted an extensive and systematic survey of S-symbionts in Japanese local populations of A . pisum using a specific PCR detection technique . Five S-symbionts of A . pisum, PASS, PAUS, PABS, Rickettsia and Spiroplasma, and two facultative endosymbionts universally found in various insects, Wolbachia and Arsenophonus, were targeted . Of 119 isofemale strains originating from 81 localities, 66.4% of the strains possessed either of four S-symbionts: PASS (38.7%); PAUS (16.0%); Rickettsia (8.4%); and Spiroplasma (3.4%), while 33.6% of the strains contained only Buchnera . PABS, Wolbachia and Arsenophonus were not detected from the Japanese strains of A . pisum . In order to understand intra- and interpopulational diversity of S-symbiont microbiota in detail, 858 insects collected from 43 localities were examined for infection with the four S-symbionts . It was demonstrated that different S-symbionts coexist commonly in the same local populations, but double infections with two S-symbionts were rarely detected . Notably, the S-symbionts exhibited characteristic geographical distribution patterns: PASS at high frequencies all over Japan; PAUS at high frequencies mainly in the northeastern part of Japan; and Rickettsia and Spiroplasma at low frequencies sporadically in the southwestern part of Japan . These results indicate that the geographical distribution and infection frequency of the S-symbionts, in particular PAUS, might be affected by environmental and/or historical factors . Statistical analyses suggested that the distribution of PAUS infection might be related to host plant species, temperature and precipitation.

CVI Forum, 1994 Aug, (7), 7 - 10
A Forum brief on nucleic acid vaccines; Tuberculosis and children: the missing diagnosis . A growing problem; PIP: About 33% of the world's population (2 billion people) are infected with Mycobacterium tuberculosis . Annually, 3 million people die from tuberculosis (TB) and 8 million acquire TB . Most TB cases are in developing countries . TB can attack the lungs or other organs . Pulmonary TB is most common in adults . Extrapulmonary TB, which is not infectious, is most common in children . Adults are the main source of TB infection in the community . When a TB-infected adult coughs or sneezes, he/she sprays many M bacterium into the air in tiny droplets . TB is curable, yet it is responsible for more deaths in adults than any other infectious disease . About 170,000 children die each year from tuberculous meningitis and disseminated TB disease . The increase in TB in adults will put more children at risk . Children most vulnerable to TB's effects are those younger than 2 and those whose parents suffer or die from TB . Increasing poverty, neglect of TB programs, and the spread of HIV account for the increase in TB cases . TB spreads best in overcrowded, badly ventilated places and among the malnourished . Health systems worldwide have undergone deep cuts and wide-ranging reforms, resulting in reduced access to vital TB services for the poorest members of society . Effective TB control requires properly operating, well-managed health services with adequate diagnostic facilities, trained staff, and available drugs . Limited community education results in people with active TB not seeking treatment and continuing to infect 20-28 others, including children . The HIV epidemic is causing an increase in TB in adults and in young children in some countries . Children infected with both HIV and TB have a poor prognosis . Health workers must be able to identify and treat TB with antibiotics . Proper treatment makes TB patients no longer infectious after 2-3 weeks . The BCG vaccination can protect children against the most severe forms of TB .

J Bacteriol, 2002 Oct, 184(20), 5686 - 95
Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones; Marketon MM et al.; Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa . Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S . meliloti strains, Rm1021 and Rm41 . In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length . Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures . This is the first report of AHLs having acyl chains longer than 14 carbons . We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs . Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis . We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL . We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S . meliloti."

J Bacteriol, 2002 Oct, 184(20), 5654 - 60
A CheW homologue is required for Myxococcus xanthus fruiting body development, social gliding motility, and fibril biogenesis; Bellenger K et al.; In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes . Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues . It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M . xanthus social gliding (S) motility and development . Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils . In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE . We showed that difC mutations resulted in defects in M . xanthus developmental aggregation, sporulation, and S motility . We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production . We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC . The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M . xanthus fibril biogenesis and S motility.

Vet Microbiol, 2002 Oct 22, 89(2-3), 161 - 6
Coxiella burnetii plasmid types QpDG and QpH1 are closely related and likely identical; Jager C et al.; Strains and isolates of Coxiella burnetii, an obligate intracellular bacterium, carry a single plasmid or a plasmid-homologous sequence integrated into the chromosome . The plasmids QpH1, QpRS, QpDV and the chromosome-integrated plasmid-homologous region have been completely sequenced, whereas no sequence data are available for the QpDG plasmid . In this study, we used total genomic DNA from reference strain C . burnetii Dugway 5J108-111 to demonstrate and characterize the QpDG plasmid by pulsed-field gel electrophoresis (PFGE) and Southern hybridization . Primers derived from regions shared among C . burnetii plasmids were used to construct a physical map of the QpDG plasmid by extra long (XL) PCR . Both approaches, Southern hybridization and XL PCR indicated that QpDG and QpH1 represent a closely related and likely identical plasmid.

J Vet Med B Infect Dis Vet Public Health, 2002 Aug, 49(6), 308 - 9
Investigation of Helicobacter pylori in raw sheep milk samples; Turutoglu H et al.; It is known that Helicobacter pylori is a cause of chronic gastritis and peptic ulcer disease in humans . However the origin and transmission of this bacterium has not been clearly explained . One of the suggested theories is transmission via raw milk from animals to human beings . In this study, the presence of H . pylori was investigated in sheep milk that is commonly consumed as human food . For this purpose, a total of 440 raw sheep milk samples collected from the Burdur region of Turkey were examined by specific cultural procedures, however, H . pylori was not isolated in any sample.

Plant Cell, 1996 Nov, 8(11), 1991 - 2001
Inhibition of Programmed Cell Death in Tobacco Plants during a Pathogen-Induced Hypersensitive Response at Low Oxygen Pressure; Mittler R et al.; The hypersensitive response (HR) of plants to invading pathogens is thought to involve a coordinated activation of plant defense mechanisms and programmed cell death (pcd) . To date, little is known about the mechanism underlying death of plant cells during this response . In addition, it is not known whether suppression of pcd affects the induction of other defense mechanisms during the HR . Here, we report that death of tobacco cells (genotype NN) infected with tobacco mosaic virus (TMV) is inhibited at low oxygen pressure . In contrast, virus replication and activation of defense mechanisms, as measured by synthesis of the pathogenesis-related protein PR-1a, were not inhibited at low oxygen pressure . Bacterium-induced pcd was also inhibited at low oxygen pressure . However, pcd induced by TMV or bacteria was not inhibited in transgenic tobacco plants expressing the mammalian anti-pcd protein Bcl-XL . Our results suggest that ambient oxygen levels are required for efficient pcd induction during the HR of plants and that activation of defense responses can be uncoupled from cell death . Furthermore, pcd that occurs during the interaction of tobacco with TMV or bacteria may be distinct from some cases of pcd or apoptosis in animals that are insensitive to low oxygen or inhibited by the Bcl-XL protein.

Biochem J, 2003 Jan 1, 369(Pt 1), 185 - 9
New enzyme belonging to the family of molybdenum-free nitrate reductases; Antipov AN et al.; A novel molybdenum-free nitrate reductase was isolated from the obligate chemolithoautotrophic and facultative anaerobic, (halo)alkaliphilic sulphur-oxidizing bacterium Thioalkalivibrio nitratireducens strain ALEN 2 . The enzyme was found to contain vanadium and haem c as cofactors . Its native molecular mass was determined as 195 kDa, and the enzyme consists of four identical subunits with apparent molecular masses of 57 kDa . Apart from nitrate, the enzyme can utilize nitrite, chlorate, bromate, selenate and sulphite as electron acceptors . Moreover, it also has a haloperoxidase activity.

J Wildl Dis, 2002 Jul, 38(3), 644 - 8
Evidence of Helicobacter sp . in dental plaque of captive dolphins (Tursiops gephyreus); Goldman CG et al.; Gastrointestinal lesions have been extensively reported in wild and captive marine mammals . However, their etiology remains unclear . In humans and other animals, chronic gastritis and peptic ulcers have been associated with Helicobacter sp . Therefore, the aim of our study was to investigate the presence of Helicobacter sp . in the gastric juice, dental plaque, and saliva of marine mammals living in a controlled environment . Five dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), three sea lions (Otaria flavescens), two elephant seals (Mirounga leonina), and two fur seals (Arctocephalus australis) were studied . Saliva, dental plaque, and gastric juice samples were examined for Helicobacter sp . using polymerase chain reaction . None of the gastric juice or saliva samples were positive for Helicobacter sp . However, Helicobacter sp . DNA was detected in dental plaque from two dolphins, suggesting the oral cavity might be a reservoir of this bacterium.

Acta Vet Hung, 2002, 50(3), 297 - 305
Clinical signs and mortality caused by Ornithobacterium rhinotracheale in turkey flocks; Szalay D et al.; Upper respiratory tract disease (manifesting itself in rhinitis, tracheitis and conjunctivitis) and mortality associated with Ornithobacterium rhinotracheale infection were observed in four flocks of 2- to 3-week-old turkeys . In a 15- to 16-week-old turkey flock bilateral catarrhal-croupous pneumonia was found in the dead birds . In a further 5-week-old flock and in three 16- to 20-week-old turkey flocks mortality was preceded by nervous signs (motor disturbances, recumbency, abnormal carriage of the head) and was found to be associated with fibrinopurulent inflammation of the cranial bones and meningitis . The bacterium O . rhinotracheale was isolated from the affected organs in the different disease conditions . The isolated strains did not differ markedly in cultural, morphological and biochemical properties . This is the first report of a turkey disease manifesting itself in nervous signs associated with O . rhinotracheale infection.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 894 - 906
Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67; James EK et al.; A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs . IR42 and IR72 . Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI . GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots . Bacteria entered the roots via cracks at the points of lateral root emergence, with cv . IR72 appearing to be more aggressively infected than cv . IR42 . H . seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue . Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed . Dense colonies of H . seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI . Epiphytic bacteria were also seen . Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased . Only cv . IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.

Plant Physiol, 1994 Aug, 105(4), 1139 - 1147
A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast); Dong Z et al.; The intercellular spaces of sugarcane (Saccharum officinarum L.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation . It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 104 bacteria/mL extracted fluid) on N-poor medium containing 10% Suc (pH 5.5) . This bacterium was identical with the type culture of Acetobacter diazotrophicus, a recently discovered N2-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air . Similar bacteria were observed in situ in the intercellular spaces . This demonstrates the presence of an N2-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium . The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop . This endogenous culture broth consisting of substrate and N2-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers . It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.

Plant Physiol, 1995 Jul, 108(3), 961 - 968
Preincubation of Bradyrhizobium japonicum with Genistein Accelerates Nodule Development of Soybean at Suboptimal Root Zone Temperatures; Zhang F et al.; In the soybean (Glycine max {L.} Merr.) N2-fixing symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development, especially at temperatures below 17{deg}C . A step in the infection process that occurs within the first 24 h is particularly sensitive to suboptimal RZT . The first phase in the establishment of the soybean-Bradyrhizobium japonicum symbiosis is the exchange of recognition molecules . The most effective plant-to-bacterium signal is genistein . Binding of genistein to B . japonicum activates many of the B . japonicum nod genes . To our knowledge, the potential of sub-optimal RZT to disrupt this interorganismal signaling has not previously been investigated . Controlled environment experiments were conducted to determine whether the preincubation of B . japonicum with genistein increases soybean nodulation and N2 fixation at suboptimal RZT and whether the time between inoculation and root-hair curling is shortened by genistein application . The results of these experiments indicated that (a) genistein application increased soybean nodulation at suboptimal RZTs (17.5 and 15{deg}C) but not at the optimal RZT (25{deg}C); (b) the period between inoculation and root-hair curling was shortened by inoculation with bradyrhizobia preincubated with genistein; (c) at 17.5 and 15{deg}C RZT, the onset of N2 fixation occurred earlier in plants that received genistein-treated bradyrhizobia than in plants inoculated with untreated bradyrhizobia; (d) over the tested concentration range, genistein application at 15 to 20 {mu}M was the most effective in stimulating nodulation; and (e) between 25 and 15{deg}C, as RZT decreased, there was an increase in the nodulation-stimulating potential of genistein.

Infect Immun, 2002 Oct, 70(10), 5816 - 21
Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics; Beron W et al.; The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles . To further characterize these vacuoles, HeLa cells were infected with C . burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles . Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation . These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway . The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C . burnetii phase II . Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy . Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N . The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt . These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

Infect Immun, 2002 Oct, 70(10), 5562 - 7
Cytokine profiles of patients infected with Mycobacterium ulcerans and unaffected household contacts; Gooding TM et al.; Mycobacterium ulcerans, the cause of Buruli ulcer, is an environmental mycobacterium with a distinct geographic distribution . The reasons why only some individuals who are exposed to M . ulcerans develop ulcers are not known but are likely to reflect individual differences in the immune response to infections with this bacterium . In this study, we investigated cytokine profiles of peripheral blood mononuclear cells (PBMC) from 23 Buruli ulcer patients and 25 household contacts in a region of Australia where Buruli ulcer is endemic . The results showed that following stimulation with M . ulcerans or Mycobacterium bovis BCG, PBMC from Buruli ulcer patients mounted a Th2-type response, which was manifested by the production of mRNA for interleukin 4 (IL-4), IL-5, IL-6, and IL-10, whereas unaffected contacts responded mainly with the Th1 cytokines gamma interferon (IFN-gamma) and IL-12 . For example, mRNA for IL-4 was detected in 18 of 23 patients but in only 3 of 25 control subjects (P < 0.0001) . By contrast, PBMC from 21 of 25 unaffected individuals produced IFN-gamma compared with 3 of 23 patients (P < 0.0001) . IFN-gamma release following stimulation with mycobacteria was markedly reduced in affected subjects . Frequencies of antibodies to M . ulcerans in serum samples from affected and unaffected subjects were similar, indicating that many of the control subjects had been exposed to this bacterium . Together, these findings suggest that a Th1-type immune response to M . ulcerans may prevent the development of Buruli ulcer in people exposed to M . ulcerans, but a Th-2 response does not.

Orig Life Evol Biosph, 2002 Jun, 32(3), 237 - 45
The asymmetry of organic aerosol fission and prebiotic chemistry; Donaldson DJ et al.; We examine the prebiotic applicability of our recent analysis of the fission of an atmospheric aerosol particle coated with an organic film . The fission is made possible by the free energy change upon compression of the exterior monolayer film on the parent particle, which overcomes the increase in surface area associated with the production of two spherical daughter particles . Asymmetric division into a larger and a smaller particle becomes possible following surfactant film collapse . The size of the airborne parent particle is determined by the balance between aerodynamics and gravity, while the ratio of the radii of the daughters is determined by the compression characteristics of the amphiphilic molecules comprising the parent film . For an Earth atmosphere of one bar surface pressure, the larger and smaller daughters have the sizes of a single-celled bacterium and of a virus respectively . Chemical differentiation between the daughters is possible.

AAOHN J, 2002 Aug, 50(8), 373 - 7; quiz 378-9
Tularemia . A pathogen in nature and a biological weapon; Altman GB; Tularemia as a potential biological weapon is of great concern because F . tularensis is a hardy organism that can be spread with a small inoculum . In addition, tularemia can be contracted through nature, predominately in rural areas . This disease can be spread by a wide variety of animals and can range from skin lesions to multi-organ involvement . The severity varies with amount of inocula, the virulence of the bacterium, and the port of entry . Exposure to aerosolized forms of F . tularensis, the major concern with bioterroism, can rapidly lead to respiratory failure and death . Untreated, other forms of tularemia can spread through the blood stream to other organs, leading to sepsis and death . Early recognition and treatment is tantamount to treatment and prevention of morbidity and mortality . Occupational health nurses are on the front line and must be assertive in identifying risk factors associated with exposure . Furthermore, education of the general population about exposure through nature can potentially decrease the incidence of tularemia . Occupational health nurses, as one of the largest health specialties in the workplace, may be the first contact for the exposed individual . Tularemia is treatable with knowledge of prevention, astute assessment, prompt identification, and treatment . Combined, they are powerful nursing tools in achieving optimal outcomes.

Can J Gastroenterol, 2002 Aug, 16(8), 559 - 63
Two decades of Helicobacter pylori: a review of the Fourth Western Pacific Helicobacter Congress; Fallone CA et al.; From March 3 to 6, 2002, Helicobacter enthusiasts gathered in Perth, Australia for the Fourth Western Pacific Helicobacter Congress to celebrate the 20th anniversary of the modern discovery of this organism by Barry Marshall and Robin Warren . The meeting included state-of-the-art lectures highlighting the breakthroughs that have occurred since the discovery of this bacterium . As well, advances from the forefront of current Helicobacter pylori research were presented, particularly in the realm of genomics and molecular biology . A symposium about vaccines and trends for future H pylori research completed this congress . The purpose of the present review is to summarize the highlights from this conference, emphasizing new advances.

J Biol Chem, 2002 Nov 15, 277(46), 44440 - 7 Epub 2002 Sep 10.
Proteinase-mediated release of epithelial cell-associated CD44 . Extracellular CD44 complexes with components of cellular matrices; Cichy J et al.; CD44 is a receptor for the matrix glycosaminoglycan hyaluronan . Proteoglycan forms of CD44 also exhibit affinity for fibronectin and collagen as well as chemokines and growth factors . CD44 plays a role in autoimmunity, inflammation, and tumor progression . Soluble CD44 (sCD44) is found in plasma, and the levels of sCD44 correlate with immune function and some malignancies . The mechanisms by which sCD44 is generated and its function are unknown . We demonstrate here that normal bronchial epithelial cells spontaneously release sCD44 . Exposure to phagocyte- and bacterium-derived proteinases markedly increased the release of sCD44 from epithelial cells . The spontaneously released sCD44 was incorporated into high molecular mass complexes derived from the matrix that also contained chondroitin sulfate, fibronectin, hyaluronan, and collagens I and IV . Enzymatic digestion with proteinases liberated sCD44 from the high molecular mass complex . Consistent with the homology of CD44 to proteoglycan core and link proteins, these data suggest that CD44 spontaneously released from normal bronchial epithelial cells can accumulate as an integral component of the matrix, where it may play a role in the organization of matrices and in anchoring growth factors and chemokines to the matrix . Increases in plasma CD44 during immune activation and tumor progression therefore may be a manifestation of the matrix remodeling that occurs in the face of the enhanced proteolytic activity associated with infection, inflammation, and tumor metastasis, leading to alterations in cell-matrix interactions.

Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1546 - 51
Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans; Takeda M et al.; An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans . Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC) . The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide . The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance . Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS . The resulting data showed that EPS produced by S . natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below . -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.

J Microbiol Methods, 2002 Nov, 51(3), 349 - 59
Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates; Poussier S et al.; Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil) . In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked . Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample . Consequently, preparation of samples prior to PCR depended upon sample origin . Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful . DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R . solanacearum in environmental samples . However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R . solanacearum were inefficient . This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R . solanacearum populations in their natural habitats .

Comp Biochem Physiol B Biochem Mol Biol, 2002 Sep, 133(1), 29 - 34
A bacterial TMAO transporter; Raymond JA et al.; Trimethylamine oxide (TMAO) acts as an osmolyte in a wide variety of marine organisms, but little is known about the mechanisms by which it accumulates in certain tissues . To determine whether a TMAO-specific transporter occurs in Nature, we examined a bacterium Aminobacter aminovorans that is known to be able to subsist on methylamine as the sole carbon source . We found that A . aminovorans is also able to grow on TMAO as the sole carbon source, and that it takes up {14C}labeled TMAO at a rate of approximately 50 pmol min(-1) x mg protein(-1) . TMAO uptake was strongly inhibited by unlabeled TMAO (5 mM) but not by related compounds such as methylamine, betaine or gamma-amino-n-butyric acid (GABA), indicating that a TMAO-specific transporter is present . The TMAO transporter appears to have an ATP requirement but no ion exchange requirement . This appears to be the first evidence of a TMAO-specific transporter in any organism . The TMAO-grown cells also expressed transporters that were specific to betaine and trimethylamine . Madin-Darby canine kidney (MDCK) cells, which have a betaine transporter that is also capable of transporting GABA, were unable to take up TMAO.

Biochemistry, 2002 Sep 17, 41(37), 11211 - 7
Mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit and cytochrome c2 in the purple bacterium Rhodovulum sulfidophilum; Masuda S et al.; The purple photosynthetic bacterium Rhodovulum sulfidophilum has an unusual reaction center- (RC-) bound cytochrome subunit with only three hemes, although the subunits of other purple bacteria have four hemes . To understand the electron-transfer pathway through this subunit, three mutants of R . sulfidophilum were constructed and characterized: one lacking the RC-bound cytochrome subunit, another one lacking cytochrome c(2), and another one lacking both of these . The mutant lacking the RC-bound cytochrome subunit was grown photosynthetically with about half the growth rate of the wild type, indicating that the presence of the cytochrome subunit, while not indispensable, is still advantageous for the photosynthetic electron transfer to support its growth . The mutant lacking both the cytochrome subunit and cytochrome c(2) showed a slower rate of growth by photosynthesis (about a fourth of that of the wild type), indicating that cytochrome c(2) is the dominant electron donor to the RC mutationally devoid of the cytochrome subunit . On the other hand, the mutant lacking only the cytochrome c(2) gene grew photosynthetically as fast as the wild type, indicating that cytochrome c(2) is not the predominant donor to the RC-bound triheme cytochrome subunit . We further show that newly isolated soluble cytochrome c-549 with a redox midpoint potential of +238 mV reduced the photooxidized cytochrome subunit in vitro, suggesting that c-549 mediates the cytochrome c(2)-independent electron transfer from the bc(1) complex to the RC-bound cytochrome subunit . These results indicate that the soluble components donating electrons to the RC-bound triheme cytochrome subunit are somewhat different from those of other purple bacteria.

Biochemistry, 2002 Sep 17, 41(37), 11134 - 42
Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases; Parsiegla G et al.; Cellulases cleave the beta-1.4 glycosidic bond of cellulose . They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate . Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism . The bacterium C . celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9 . As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains . The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate . The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively . Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C . thermocellum and the processive endo-cellulase Cel9A from T . fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain . These enzymes differ in their activity on substrates with specific macroscopic appearances . The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 37 - 43
The bc(1) complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction . Is there a second bc(1) complex?
Brasseur G, Bruscella P, Bonnefoy V, Lemesle-Meunier D.
Acidithiobacillus ferrooxidans is an acidophilic chemolithotrophic bacterium that can grow in the presence of either a weak reductant, Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+) . Here we first review the latest findings about the uphill electron transfer pathway established in iron-grown A . ferrooxidans, which has been found to involve a bc(1) complex . We then provide evidence that this bc(1) complex cannot function in the forward direction (exergonic reaction), even with an appropriate substrate . A search for the sequence of the three redox subunits of the A . ferrooxidans bc(1) complex (strain ATCC 19859) in the complete genome sequence of the A . ferrooxidans ATCC 23270 strain showed the existence of two different bc(1) complexes in A . ferrooxidans . Cytochrome b and Rieske protein sequence comparisons allowed us to point out some sequence particularities of these proteins in A . ferrooxidans . Lastly, we discuss the possible reasons for the existence of two different "classical" bc(1) complexes and put forward some suggestions as to what role these putative complexes may play in this acidophilic chemolithotrophic bacterium.

Evolution Int J Org Evolution, 2002 Jul, 56(7), 1331 - 9
Within- and between-population variation for Wolbachia-induced reproductive incompatibility in a haplodiploid mite; Vala F et al.; Wolbachia pipientis is a bacterium that induces cytoplasmic incompatibility (CI), the phenomenon in which infected males are reproductively incompatible with uninfected females . CI spreads in a population of hosts because it reduces the fitness of uninfected females relative to infected females . CI encompasses two steps: modification (mod) of sperm of infected males and rescuing (resc) of these chromosomes by Wolbachia in the egg . Infections associated with CI have mod+ resa+ phenotypes . However, mod- resc+ phenotypes also exist; these do not result in CI . Assuming mod/resc phenotypes are properties of the symbiont, theory predicts that mod- resc+ infections can only spread in a host population where a mod+ resc+ infection already occurs . A mod- resc+ infection spreads if the cost it imposes on the infected females is lower than the cost inflicted by the resident (mod+ resc+) infection . Furthermore, introduction of a mod- Wolbachia eventually drives infection to extinction . The uninfected population that results can be recolonized by a CI-causing Wolbachia . Here, we investigated whether variability for induction of CI was present in two Tetranychus urticae populations . In one population all isofemale lines tested were mod- . In the other, mod+ resc+ and mod- resc+ isofemale lines coexisted . We found no evidence for a cost difference to females expressing either type (mod-/-) . Infections in the two populations could not be distinguished based on sequences of two Wolbachia genes . We consider the possibility that mod- is a host effect through a population dynamics model . A mod- host allele leads to infection extinction in the absence of fecundity differences . Furthermore, the uninfected population that results is immune to reestablishment of the (same) CI-causing Wolbachia.

Rev Inst Med Trop Sao Paulo, 2002 Jul-Aug, 44(4), 217 - 24
Infectious agents in coronary atheromas: a possible role in the pathogenesis of plaque rupture and acute myocardial infarction; Higuchi Mde L et al.; In this review we report our recent findings of histopathological features of plaque instability and the association with Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP) infection, studying thrombosed coronary artery segments (CAS) of patients who died due to acute myocardial infarction . Vulnerable plaques are known to be associated with fat atheromas and inflammation of the plaque . Here we demonstrated that vulnerability is also related with focal positive vessel remodeling that maintains relatively well preserved lumen even in the presence of large atheromatous plaques . This phenomena may explain why the cinecoronariography may not detect large and dangerous vulnerable plaques . Greater amount of these bacteria in vulnerable plaques is associated with adventitial inflammation and positive vessel remodeling: the mean numbers of lymphocytes were significantly higher in adventitia than in the plaque, good direct correlation was obtained between numbers of CD20 B cells and numbers of CP infected cells in adventitia, and between % area of MP-DNA in the plaque and cross sectional area of the vessel, suggesting a cause-effect relationship . Mycoplasma is a bacterium that needs cholesterol for proliferation and may increase virulence of other infectious agents . In conclusion, co-infection by Mycoplasma pneumoniae and Chlamydia pneumoniae may represent an important co-factor for plaque instability, leading to coronary plaque thrombosis and acute myocardial infarction, since larger amount of these bacteria strongly correlated with histological signs of more vulnerability of the plaque . The search of CMV and Helicobacter pilori in these tissues resulted negative.

J Bacteriol, 2002 Oct, 184(19), 5426 - 35
Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids; Cordova CM et al.; Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis . The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools . In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene . The predicted M . pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA . However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region . Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M . pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them . An internal fragment of the M . pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M . pulmonis . Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented . These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.

J Bacteriol, 2002 Oct, 184(19), 5385 - 92
Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti; Bringhurst RM et al.; The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources . As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose . We isolated lacR mutants in a genetic screen designed to find S . meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides . The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose . For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants . However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present . To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression . Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose . This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose . Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.

J Econ Entomol, 2002 Aug, 95(4), 739 - 47
Temporal distribution of Chaetocnema pulicaria (Coleoptera: Chrysomelidae) populations in Iowa; Esker PD et al.; In 1999 and 2000, yellow sticky cards and sweep net samples were used to document the occurrence of an overwintering adult generation of Chaetocnema pulicaria Melsheimer, corn flea beetle, followed by two distinct populations peaks during the growing season in Iowa Emergence of the overwintering adult generation started in mid-April and continued until early June in both years, with populations as high as 45 +/- 7.9 per 10 sweeps . Periods that ranged from 14 to 32 d were observed in 1999 and 2000 when C . pulicaria was not found following the overwintering generation . The first summer peak of C pulicaria was observed between the end of June into the middle of July, with the highest observed peak at 16.70 +/- 1.42 C . pulicaria per 10 sweeps in cornfields . The second summer peak of C pulicaria was observed between the middle into early September, with populations as high as 27.80 +/- 2.76 C . pulicaria per 10 sweeps . During the growing season, more C . pulicaria were caught on yellow sticky cards originating from soybean borders than from grass borders . There were significantly greater numbers of C . pulicaria on yellow sticky cards located in grass borders adjacent to cornfields at the end of the growing season, compared with yellow sticky cards located within cornfields, indicating the movement of C . pulicaria from the cornfield back into the grass borders at the end of the growing season . In 2000, from August to the end of the corn growing season, significantly more C . pulicaria were found in grass borders than in the cornfields . Based on this new quantitative information, planting time could be altered to avoid the emergence of the overwintering generation of C . pulicaria . In addition, knowledge concerning the seasonalities of the first and second population peaks of C pulicaria during the corn growing season could be used to recommend optimal timing for foliar-applied insecticide applications . This new knowledge concerning the seasonal dynamics of C pulicaria will help to improve management recommendations for Stewart's disease of corn, caused by the bacterium Pantoea stewartii, and that is vectored by C pulicaria.

Microbiology, 2002 Sep, 148(Pt 9), 2911 - 4
Disruption analysis of DR1420 and/or DR1758 in the extremely radioresistant bacterium Deinococcus radiodurans; Nishida H et al.; The extremely radioresistant bacterium Deinococcus radiodurans encodes two genes that are homologous to those involved in bacterial lysine biosynthesis . In lysine biosynthesis, these genes are involved in the aminoadipate pathway and the diaminopimelate (DAP) pathway . DR1420 is homologous to lysZ, which is essential for bacterial lysine biosynthesis via the aminoadipate pathway, and DR1758 is homologous to lysA, which is essential for lysine biosynthesis via the DAP pathway . In this study, DR1420 and/or DR1758 were disrupted . Each disruptant of DR1420 and DR1758, and of DR1420 or DR1758 grew in a minimal medium, as did the wild-type . These results show that D . radiodurans performs lysine biosynthesis in a unique way.

Microbiology, 2002 Sep, 148(Pt 9), 2869 - 82
Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae; Mikael LG et al.; The bacterium Actinobacillus pleuropneumoniae, a swine pathogen, utilizes ferrichrome as an iron source . This study details the molecular cloning and sequencing of the genes involved in the uptake of this hydroxamate siderophore . Four ferric hydroxamate uptake (fhu) genes, fhuC, fhuD, fhuB and fhuA, were identified in a single operon, and these were found to encode proteins homologous to proteins of the fhu systems of several bacteria, including Escherichia coli . The fhuA gene encodes the 77 kDa outer-membrane protein (OMP) FhuA, the receptor for ferrichrome . FhuD is the 35.6 kDa periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane . FhuC (28.5 kDa) and FhuB (69.4 kDa) are cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes the ferric hydroxamate . Reference strains of A . pleuropneumoniae that represented serotypes 1 to 12 of this organism all tested positive for the four fhu genes . When A . pleuropneumoniae FhuA was affinity-tagged with hexahistidine at its amino terminus and expressed in an E . coli host, the recombinant protein reacted with an mAb against E . coli FhuA, as well as with a polyclonal pig serum raised against an A . pleuropneumoniae infection . Hence, the authors conclude that fhuA is expressed in vivo by A . pleuropneumoniae . Three-dimensional modelling of the OMP FhuA was achieved by threading it to the X-ray crystallographic structure of the homologous protein in E . coli . FhuA from A . pleuropneumoniae was found to have the same overall fold as its E . coli homologue, i.e . it possesses an N-terminal cork domain followed by a C-terminal beta-barrel domain and displays 11 extracellular loops and 10 periplasmic turns.

Ultramicroscopy, 2002 May, 91(1-4), 157 - 64
Direct observation of polyhydroxyalkanoate chains by atomic force microscopy; Sudesh K et al.; Atomic force microscopy in the tapping mode was used to investigate aqueous acetone-treated polyhydroxyalkanoate (PHA) inclusions freshly isolated from a recombinant bacterium . The PHA is a copolymer containing about 95 mol% 3-hydroxybutyrate units while the rests are units of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate . Polymer chains extending to several micrometers in length were observed on glass cover slips upon the evaporation of the aqueous acetone . The polymer chains seem to exist in the form of fibrillar aggregates . The height of the microfibrils was about 1 nm . Upon prolonged standing at ambient conditions, the microfibrils dissociated into finer strands of about 0.5 nm in height . The results suggest that biosynthesized PHA are stored in the inclusions in an amorphous state but with minimal chain entanglement . This is possible because the PHA chains exist in the form of fibrillar aggregates that may be the product of a special biosynthesis mechanism.

Comp Med, 2002 Aug, 52(4), 313 - 5
Evaluation of sensitivity and specificity of a Mycoplasma haemomuris-specific polymerase chain reaction test; Zhang C et al.; BACKGROUND AND PURPOSE: Mycoplasma haemomuris, a small pleomorphic bacterium parasitic of red blood cells, often causes chronic and subclinical infection of rodents . Mycoplasma haemomuris is uncultivable, and a serologic testing method is not readily available . The purpose of the study reported here was to develop a sensitive and specific polymerase chain reaction (PCR) test for detection of M . haemomuris in blood samples . METHODS: On the basis of the regions of the M . haemomuris 16S rRNA gene most divergent from corresponding regions of related bacteria, M . haemomuris-specific primers were designed so that these primers could selectively amplify M . haemomuris DNA . A PCR test was performed, using blood samples from BALB/c mice infected with M . haemomuris strains TR 8564, TR 8563, and TR 8556 . RESULTS: Use of the PCR test enabled detection of M . haemomuris DNA in a minimum of 0.0001 microl of infected mouse blood . The test also was specific for M . haemomuris and did not amplify closely related species, such as M . haemofelis, M . haemosuis, M . orale, or Anaplasma marginale . CONCLUSION: This method is sensitive and specific for detection of M . haemomuris.

Biotechnol Bioeng, 2002 Oct 20, 80(2), 195 - 200
Sensitivity function-based model reduction: A bacterial gene expression case study; Smets I et al.; Mathematical models used to predict the behavior of genetically modified organisms require 1) . a (rather) large number of state variables, and 2) . complicated kinetic expressions containing a large number of parameters . Since these models are hardly identifiable and of limited use in model-based optimization and control strategies, a generic methodology based on sensitivity function analysis is presented to reduce the model complexity at the level of the kinetics, while maintaining high prediction power . As a case study to illustrate the method and results obtained, the influence of the dissolved oxygen concentration on the cytN gene expression in the bacterium Azospirillum brasilense Sp7 is modeled . As a first modeling approach, available mechanistic knowledge is incorporated into a mass balance equation model with 3 states and 14 parameters . The large differences in order of magnitude of the model parameters identified on the available experimental data indicate 1) . possible structural problems in the kinetic model and, associated with this, 2) . a possibly too high number of model parameters . A careful sensitivity function analysis reveals that a reduced model with only seven parameters is almost as accurate as the original model .

J Virol, 2002 Oct, 76(19), 9695 - 701
Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein; Bendtsen JD et al.; Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli . A phylogenetic analysis indicated that the P1 ssb gene coexists with its E . coli counterpart as an independent unit and does not represent a recent acquisition of the phage . The P1 and E . coli SSB proteins are fully functionally interchangeable . SSB-P1 is nonessential for phage growth in an exponentially growing E . coli host, and it is sufficient to promote bacterial growth in the absence of the E . coli SSB protein . Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E . coli . Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary phase . These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids.

Clin Diagn Lab Immunol, 2002 Sep, 9(5), 1079 - 84
Cytokine gene expression by peripheral blood leukocytes in horses experimentally infected with Anaplasma phagocytophila; Kim HY et al.; Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila . The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE . In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A . phagocytophila or by allowing feeding by infected ticks . The p44 genes encoding the major outer membrane protein P44s of A . phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure . During the 20-day infection period, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses . Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each . IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression, however, could not be detected in the PBLs of any of the four horses . These results suggest that IL-1beta, TNF-alpha, and IL-8 generation during A . phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation.

Carbohydr Res, 2002 Sep 9, 337(16), 1427 - 33
Exopolygalacturonate lyase from Thermotoga maritima: cloning, characterization and organic synthesis application; Parisot J et al.; A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells . A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues . The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield . The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C . The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.

J Biol Inorg Chem, 2002 Sep, 7(7-8), 815 - 22 Epub 2002 Apr 27.
Structure-function relationship in type II cytochrome c(3) from Desulfovibrio africanus: a novel function in a familiar heme core; Pereira PM et al.; NMR and visible spectroscopy were used to characterize the type II tetraheme cytochrome c(3) isolated from the periplasmic space of Desulfovibrio africanus, a sulfate-reducing bacterium . Although structurally similar to other cytochromes c(3), this protein displays distinct functional properties . Proton NMR signals from the four hemes were assigned to the structure in the ferri- and ferrocytochromes using two-dimensional NMR experiments . The thermodynamic parameters of the hemes and of an acid-base center in the type II cytochrome c(3) were determined using NMR and visible spectroscopies . The thermodynamic features indicate that electrostatic effects dominate all of the interactions between the centers and no positive cooperativity between hemes is observed . The redox-Bohr effect in this protein is associated with the acid-base equilibrium of a propionate of heme II instead of propionate 13 of heme I as is the case for all of the type I cytochromes c(3) . These novel functional properties are analyzed together with the redox-linked structural differences reported in the literature and reveal a mechanistic basis for type II cytochromes c(3) having a physiological function that is different from that of type I cytochromes c(3).

J Clin Microbiol, 2002 Sep, 40(9), 3427 - 31
Actinomyces cardiffensis sp . nov . from human clinical sources; Hall V et al.; Eight strains of a previously undescribed catalase-negative Actinomyces-like bacterium were recovered from human clinical specimens . The morphological and biochemical characteristics of the isolates were consistent with their assignment to the genus Actinomyces, but they did not appear to correspond to any recognized species . 16S rRNA gene sequence analysis showed the organisms represent a hitherto unknown species within the genus Actinomyces related to, albeit distinct from, a group of species which includes Actinomyces turicensis and close relatives . Based on biochemical and molecular genetic evidence, it is proposed that the unknown isolates from human clinical sources be classified as a new species, Actinomyces cardiffensis sp . nov . The type strain of Actinomyces cardiffensis is CCUG 44997(T).

Appl Environ Microbiol, 2002 Sep, 68(9), 4187 - 93
Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus; Sauer C et al.; The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization . A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males . Large amounts of bacteria were also found in the oocytes of workers and queens . In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males . Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals . During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages . However, during larval development, bacteria could also be detected in other tissues although to a lesser extent . Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes . Tetracycline and rifampin efficiently cleansed C . floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased . Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.

BMC Evol Biol . 2002 Aug 22;2(1):12.
Long-term experimental evolution in Escherichia coli . X . Quantifying the fundamental and realized niche; Cooper VS; BACKGROUND: Twelve populations of the bacterium, Escherichia coli, adapted to a simple, glucose-limited, laboratory environment over 10,000 generations . As a consequence, these populations tended to lose functionality on alternative resources . I examined whether these populations in turn became inferior competitors in four alternative environments . These experiments are among the first to quantify and compare dimensions of the fundamental and realized niches . RESULTS: Three clones were isolated from each of the twelve populations after 10,000 generations of evolution . Direct competition between these clones and the ancestor in the selective environment revealed average fitness improvements of approximately 50% . When grown in the wells of Biolog plates, however, evolved clones grew 25% worse on average than the ancestor on a variety of different carbon sources . Next, I competed each evolved population versus the ancestor in four foreign environments (10-fold higher and lower glucose concentration, added bile salts, and dilute LB media) . Surprisingly, nearly all populations were more fit than the ancestor in each foreign environment, though the margin of improvement was least in the most different environment . Most populations also evolved increased sensitivity to novobiocin . CONCLUSIONS: Reduced functionality on numerous carbon sources suggested that the fundamental niche of twelve E . coli populations had narrowed after adapting to a specific laboratory environment . However, in spite of these results, the same populations were competitively superior in four novel environments . These findings suggest that adaptation to certain dimensions of the environment may compensate for other functional losses and apparently enhance the realized niche.

J Biol Chem, 2002 Nov 1, 277(44), 41857 - 64 Epub 2002 Aug 24.
Crystal structure of the 47-kDa lipoprotein of Treponema pallidum reveals a novel penicillin-binding protein; Deka RK et al.; Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum . T . pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for beta-lactams are still unknown . We previously identified the T . pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP) . Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs . Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47 . The crystal structure of Tp47 at a resolution of 1.95 A revealed a fold different from any other known PBP; Tp47 is predominantly beta-sheet, in contrast to the alpha/beta-fold common to other PBPs . It comprises four distinct domains: two complex beta-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds . The three hypothetical PBP signature motifs do not come together to form a typical PBP active site . Furthermore, Tp47 is unusual in that it displays beta-lactamase activity (k(cat) for penicillin = 271 +/- 6 s(-1)), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques . Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.

J Biol Chem, 2002 Nov 1, 277(44), 41590 - 6 Epub 2002 Aug 25.
Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus; Xu MG et al.; In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain . By PCR, using genome DNA of A . aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli . The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure . The beta subunit was inactive in catalysis but was able to bind tRNA(Leu) . Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E . coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer . The kinetics of A . aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A . aeolicus and E . coli were studied . Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively . Under optimal conditions, A . aeolicus alphabeta-LeuRS is stable up to 65 degrees C.

Biochem Soc Trans, 2002 Aug, 30(4), 669 - 72
Molecular and atomic analysis of uranium complexes formed by three eco-types of Acidithiobacillus ferrooxidans; Merroun M et al.; A combination of EXAFS, transmission electron microscopy and energy-dispersive X-ray was used to conduct a molecular and atomic analysis of the uranium complexes formed by Acidithiobacillus ferrooxidans . The results demonstrate that this bacterium accumulates uranium as phosphate compounds . We suggest that at toxic levels when the uranium enters the bacterial cells, A . ferrooxidans can detoxify and efflux this metal by a process in which its polyphosphate bodies are involved.

Biochem Soc Trans, 2002 Aug, 30(4), 638 - 42
Novel domain packing in the crystal structure of a thiosulphate-oxidizing enzyme; Bamford VA et al.; A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains . The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide . The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold . The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.

Int J Med Microbiol, 2002 Jul, 292(2), 127 - 37
Molecular analysis of beta-lactamase structure and function; Majiduddin FK et al.; The extensive and sometimes irresponsible use of beta-lactam antibiotics in clinical and agricultural settings has contributed to the emergence and widespread dissemination of antibiotic-resistant bacteria . Bacteria have evolved three strategies to escape the activity of beta-lactam antibiotics: 1) alteration of the target site (e.g . penicillin-binding protein (PBPs), 2) reduction of drug permeation across the bacterial membrane (e.g . efflux pumps) and 3) production of beta-lactamase enzymes . The beta-lactamase enzymes inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring rendering the antibiotic ineffective . The inactivation of the antibiotic provides resistance to the bacterium . Currently, there are over 300 beta-lactamase enzymes described for which numerous kinetic, structural, computational and mutagenesis studies have been performed . In this review, we discuss the recent work performed on the four different classes (A, B, C, and D) of beta-lactamases . These investigative advances further expand our knowledge about these complex enzymes, and hopefully, will provide us with additional tools to develop new inhibitors and antibiotics based on structural and rational designs.

Eur J Clin Invest, 2002 Aug, 32(8), 549 - 55
The relation between Helicobacter pylori and atherosclerosis cannot be explained by a high homocysteine concentration; Bloemenkamp DG et al.; BACKGROUND: Recent studies have suggested that a chronic infection with Helicobacter pylori might be an independent risk factor for atherosclerosis . However, a direct role in atherogenesis is not plausible, since the bacterium has not been isolated from atherosclerotic lesions . An indirect mechanism that could link H . pylori with atherosclerosis might be through an increase in plasma homocysteine concentration caused by deficiencies of vitamin B12 and folate in plasma . MATERIALS AND METHODS: In 150 female patients with peripheral arterial disease (PAD) and in 412 healthy control women from a nation-wide population-based case-control study, blood samples were collected to determine the antibody titre against H . pylori and to measure plasma homocysteine, folate and vitamin B12 levels . First, the odds ratio for PAD in women with a positive antibody titre against H . pylori was calculated and adjusted for homocysteine level . Secondly, mean concentrations of vitamin B12, folate and homocysteine were compared in healthy controls with a positive or negative antibody titre against H . pylori . Thirdly, the relation between H . pylori and PAD in individuals with a normal or high homocysteine level was investigated . RESULTS: A positive immunoglobulin G antibody titre against H . pylori was found in 42% of the PAD patients and in 27% of the controls . The age- and socio-economic-status (SES) adjusted odds ratio for PAD was 1.5 (95%CI; 1.0-2.2) . Additional adjustment for homocysteine plasma concentration did not essentially change the odds ratio . Secondly, among the healthy controls, the homocysteine plasma concentration did not depend on the immunoglobulin G titre, neither did the folate plasma concentration . The concentration of vitamin B12 was slightly higher in women with a positive titre . Thirdly, H . pylori infection was a risk factor for PAD in subjects with a normal homocysteine concentration {OR 2.0 (95%CI 1.3-3.1)} . CONCLUSIONS: This study shows a relationship between a positive immunoglobulin G antibody titre against H . pylori and PAD in young women . Moreover, this study does not support the hypothesis that H . pylori infection is related to atherosclerosis via an increase in plasma homocysteine concentration.

Mikrobiol Z, 2002 Mar-Apr, 64(2), 28 - 35
{Growth of phototrophic bacterium Rhodobacter sphaeroides and formation of carotenoids on mineral water "Dzhermuk"}; Paronian AKh; A possibility to use thermal carbon sulphate-hydrocarbonate mineral waters "Dzhermuk" as the basis of nutrient medium for growing has been established . No distinct correlation was observed between biomass accumulation and carotenoids formation: maximum yields of the biomass is registered under microaetrophilic growth conditions, while that of carotenoids--under anaerobic conditions . Maximum synthesis of carotenoids is observed at illumination of 1000-1500 lx, more intensive illumination (3000 lx) results in the decrease of their synthesis . Spheroiden, which makes up about 40-45% of the total amount of carotenoids, is predominant . The biomass drying and storage period (2 yr.) affect inconsiderably the quantitative composition of pigments.

Rev Biol Trop, 2001 Sep-Dec, 49(3-4), 1207 - 12
Growth and survival of Escherichia coli O157: H7 in meat, poultry and vegetables mixed with different concentrations of mayonnaise; Arias ML et al.; In the last 20 years Escherichia coli O157: H7 has emerged as a new pathogen, causing worldwide disease, death and economic loss . Different studies have revealed important survival characteristics of this pathogen, although there are divergent criteria about its ability to survive in various mayonnaise formulations . We studied the effect of different mayonnaise concentrations (0%, 18%, 37% and 56%) (weight/weight) over the survival of the bacterium in common foods from a neotropical environment (Costa Rica) . High {10(7)-10(8) Colony Forming Units (CFU)/ml} and low E . coli populations (10(4)-10(6) CFU/ml) were inoculated, (three replicates) in meat, chopped cabbage and poultry, and mixed with commercial mayonnaise to obtain the concentrations specified . They were incubated at 12 degrees C for 24, 48 and 72 hr . The E . coli O157: H7 enumeration was done according to a standard methodology . Populations of E . coli O157: H7 showed an increasing trend during the first incubation period (48 hr), in all the preparations, regardless of the fat concentration used . Our data indicate that E . coli O157: H7 is capable of surviving and growing in meat, cabbage and poultry mixed with mayonnaise, independently of its concentration.

Nippon Rinsho, 2002 Aug, 60(8), 1543 - 8
{Diagnosis of Helicobacter pylori infection}; Fukuda Y et al.; Peptic ulcer in elderly patients may be associated with Helicobacter pylori infection . Diagnosis for H . pylori infection should be needed in these patients . The test may be resulted in false negative in case of invasive diagnostic methods based gastric biopsy, because atrophic gastritis and intestinal metaplasia developed in patients may not be able to detect scarce infection of the bacterium . Urea breath test or stool antigen test are recommended for diagnosis of H . pylori infection in patients . It is suitable for patients, because of the non invasive nature of these diagnostic tests.

Ultrastruct Pathol, 2002 May-Jun, 26(3), 161 - 9
Ultrastructural changes in a standard strain of Bartonella henselae after passages through BALB/cAn mice; Velho PE et al.; Human bartonelloses are a group of illnesses of poorly understood pathogenesis . Bartonella henselae is one of the most studied bacterium of its genus . The objective of this study was to observe whether passages of these bacteria, in vivo, would determine ultrastructural changes in them . For this purpose, isogenic mice were inoculated with a standard strain of B . henselae (I) . These were initially retrieved from genetically immunodeficient animals (II) and then inoculated in immunocompetent ones . The bacterial colonies obtained (III) were compared, by transmission electron microscopy, with colonies I and II . Loss of fimbriae and an abundant bleb formation were the most common morphological changes found in colony III . Also, on day 6 postinfection, the main histological abnormalities were the endothelial proliferation presented in immunodeficient animals and the incipient granulomata reaction found in one of the immunocompetent inoculated mice, which died spontaneously . These features agree with the Bartonella human disease clinical and histological observations . This study demonstrates that B . henselae in vivo passages induce significant morphological changes in the bacteria and that these abnormalities could explain their seemingly greater virulence . Most of these observations have not been previously described . Thus, further studies on the Bartonella species pathogenesis should consider these data.

Infect Immun, 2002 Sep, 70(9), 5307 - 11
Borrelia burgdorferi erp (ospE-related) gene sequences remain stable during mammalian infection; Stevenson B; A number of studies have indicated that Borrelia burgdorferi erp genes need not vary during vertebrate infection . However, it was recently reported that a B . burgdorferi bacterium reisolated from an infected mouse evidenced mutation and recombination events in several erp genes . Reexamination of that reisolate indicates that the previously reported changes were no doubt artifacts of the PCR processes originally used to clone those DNAs . Thus, no evidence has been found of erp gene variation during mammalian infection.

Infect Immun, 2002 Sep, 70(9), 4818 - 25
Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice; Watarai M et al.; Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages . Intracellular replication of B . abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems . In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B . abortus and also contributes to the establishment of bacterial infection in mice . The internalization of B . abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL) . Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B . abortus . Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B . abortus in macrophages . The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B . abortus infection . NPC1-deficient mice were resistant to the bacterial infection . Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B . abortus . Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B . abortus infection.

Pediatr Infect Dis J, 2002 Jun, 21(6), 574 - 5
Cervical lymphadenitis caused by Mycobacterium lentiflavum; Cabria F et al.; We report a case of human infection caused by Mycobacterium lentiflavum and review the literature for infections caused by this bacterium . The patient was a 19-month-old boy with involvement of a cervical lymph node . Surgical removal of the lymphadenopathy was both diagnostic and curative.

Int Health News . 1988 Apr;9(4):2.
Tetanus facts; Resistance to cadmium and zinc in Acidiphilium symbioticum KM2 is plasmid mediated; Indian Institute of Chemical Biology, 4 Raja S . C . Mullick Road, Calcutta-700032, India . nmahapatra@ucsd.edu

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size . The bacterium becomes extremely sensitive to metals when it is cured of its plasmids . A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+ . The Lac(-)Amp(r) transformants of Escherichia coli DH5alpha, isolated after transformation with the library, were counter-selected on Cu(2+), Cd(2+), Ni(2+), and Zn(2+)-containing plates . Only Cd(2+)- and Zn(2+)-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained . The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2 . The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid . These results strongly suggested that resistance to cadmium and zinc in A . symbioticum KM2 is mediated by these plasmids . The smallest insert of 422 bp in pNM206 conferring metal resistance in E . coli has no sequence similarity with the reported metal-resistant genes . All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.

Plant Physiol, 2002 Aug, 129(4), 1788 - 94
Identification of a soybean protein that interacts with GAGA element dinucleotide repeat DNA; Sangwan I et al.; Dinucleotide repeat DNA with the pattern (GA)(n)/(TC)(n), so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein . Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)(n)/(CT)(n) DNA . Soybean GBP was dissimilar from the GAGA factor of Drosophila melanogaster . Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)(n)/(CT)(n) . GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element . Removal of that GAGA element abrogated binding of GBP to the promoter . Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA . Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding . Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum . In addition, Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h . Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well . We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11049 - 54 Epub 2002 Aug 12.
Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags; Lipton MS et al.; Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations . To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry . The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans . This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Microbiology, 2002 Aug, 148(Pt 8), 2579 - 89
The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris; Watanabe S et al.; The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced . The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms . The analyses of amino acid content and primary structure of the C . maris ICL suggested that it possessed many features of a cold-adapted enzyme . Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively . The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature . The induction of icl expression by low temperature took place in the C . maris cells grown on succinate as the carbon source but not acetate . Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract . These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C . maris is important for acetate utilization and cold adaptation.

Microbiology, 2002 Aug, 148(Pt 8), 2457 - 66
Regulation of polyphenol oxidase activities and melanin synthesis in Marinomonas mediterranea: identification of ppoS, a gene encoding a sensor histidine kinase; Lucas-Elio P et al.; Marinomonas mediterranea is a melanogenic marine bacterium that expresses two different polyphenol oxidases . One of them is a multipotent laccase able to oxidize a wide range of substrates . The second enzyme is an SDS-activated tyrosinase . Using transposon mutagenesis, a mutant affected in the regulation of both polyphenol oxidase activities and melanogenesis has been isolated . The sequencing of the gene disrupted by the mini-Tn10 transposon in this mutant indicates that it encodes a hybrid sensor kinase . This sensor kinase shows three phosphorylated conserved domains: the transmitter domain containing a histidine site typical of sensor kinases, a receiver domain with an aspartate residue and an additional phosphotransferase domain with a second conserved histidine . This structural organization is characteristic of kinases participating in a phosphorelay system . Northern blot and lacZ operon fusions indicate that the multipotent laccase activity is regulated not only by PpoS but also by growth phase at the transcriptional level . These results suggest that PPO activities and melanin synthesis play a role in the adaptive response of M . mediterranea to stressful environmental conditions.

J Am Chem Soc, 2002 Aug 21, 124(33), 9845 - 55
X-ray crystal structures of reduced rubrerythrin and its azide adduct: a structure-based mechanism for a non-heme diiron peroxidase; Jin S et al.; Rubrerythrin (Rbr) is a 44-kDa homodimeric protein, found in many air-sensitive bacteria and archaea, which contains a unique combination of a rubredoxin-like {Fe(SCys)(4)} site and a non-sulfur, oxo/dicarboxylato-bridged diiron site . The diiron site structure resembles those found in O2-activating diiron enzymes . However, Rbr instead appears to function as a hydrogen peroxide reductase (peroxidase) . The diferrous site in all-ferrous Rbr (Rbr(red)) shows a much greater reactivity with H2O2 than does the diferric site in all-ferric Rbr (Rbr(ox)), but only the latter structure has been reported . Here we report the X-ray crystal structures of the recombinant Rbr(red) from the sulfate reducing bacterium, Desulfovibrio vulgaris, as well as its azide adduct (Rbr(red)N3) . We have also redetermined the structure of Rbr(ox) to a higher resolution than previously reported . The structural differences between Rbr(ox) and Rbr(red) are localized entirely at the diiron site . The most striking structural change upon reduction of the diferric to the diferrous site of Rbr is a 1.8-A movement of one iron away from a unique glutamate carboxylate ligand and toward a trans-disposed histidine side chain, which replaces the glutamate as a ligand . This movement increases the inter-iron distance from 3.3 to 4 A . Rbr(red)N(3) shows this same iron movement and His-->Glu ligand replacement relative to Rbr(ox), and, in addition, an azide coordinated to the diiron site in a cis mu-1,3 fashion, replacing two solvent ligands in Rbr(red) . Relative to those in O2-activating enzymes, the bridging carboxylate ligation of the Rbr diiron site is less flexible upon diferric/diferrous interconversion . The diferrous site is also much more rigid, symmetrical, and solvent-exposed than those in O2-activating enzymes . On the basis of these unique structural features, a mechanism is proposed for facile reduction of hydrogen peroxide by Rbr involving a cis mu-eta(2) H2O2 diferrous intermediate.

J Appl Microbiol, 2002, 93(3), 497 - 504
Interactions between the unicellular red alga Rhodella reticulata (Rhodophyta) and contaminated bacteria; Toncheva-Panova TG et al.; AIMS: To define the role of the bacterial strains LR1 and LR3 in the Rhodella cell destruction caused by Cytophaga sp.LR2 . METHODS AND RESULTS: The bacteria were obtained from algal culture with destruction . They were isolated in pure culture and tested for biochemical activities using Polymicrotest . The ability of bacteria to degrade and utilize the algal polysaccharide was investigated . The bacteria were grown in a media containing Rhodella polysaccharide as a sole carbon source . The level of the reducing sugars in the culture media was determined . Scanning electron microscopy (SEM) was used to define the location of bacteria in extensively and intensively cultivated Rhodella reticulata previously infected by Cytophaga sp . LR2 . CONCLUSIONS: The lysis of Rhodella reticulata cells is due to the joint action of the three bacterial strains with the former pathogen Cytophaga sp . LR2 playing the main role . The accumulation of the polysaccharide and the excreted metabolites of the strains LR1 and LR3 stimulated the development of Cytophaga sp . LR2 . The adaptation of the strain to particular conditions of alga cultivation and the utilization of polysaccharide as a sole carbon source supported its stable growth in alga suspension and destruction of Rhodella cells . SIGNIFICANCE AND IMPACT OF THE STUDY: The predominance of Cytophaga sp . LR2 over the two other contaminants and the lysis of Rhodella reticulata cells resulted from the ability of the bacterium to attach to the algal polysaccharide sheath . The formation of slime and extrusions facilitated the phenomenon of bacterial adhesion to the algal surface as well as the formation of colonial alga - bacterial spherules . The sedimentation of these aggregates decreased the ability of the algal strain to photosynthesize, led to the lysis of the cells and finally caused the death of Rhodella.

New Microbiol, 2002 Jul, 25(3), 315 - 21
Infection by Helicobacter pylori and acute myocardial infarction . Do cytotoxic strains make a difference?
Pellicano R, Parravicini PP, Bigi R, Gandolfo N, Aruta E, Gai V, Figura N, Angelino P, Rizzetto M, Ponzetto A.
The classical risk factors for acute myocardial infarction (AMI) fail to explain all the epidemiological variations of the disease . Among the risk factors recently reported, several infectious agents appear to increase the risk of AMI . Helicobacter pylori (H . pylori) infection, a bacterium involved in duodenal and gastric ulcer, gastric cancer and MALT-lymphoma, seems to be strongly associated with AMI . More virulent (anti-CagA positive) strains of the bacterium are almost exclusively the causative agents of such diseases . To determine the prevalence of H . pylori infection and of virulent strains, a case-control study was conducted in a group of male patients with AMI . A group of patients consecutively admitted to the Emergency Care Unit served as controls . We studied 223 consecutive male patients, mean age 60.2 (range 40-79) years, admitted for AMI to the Coronary Care Units at Hospitals in two towns of Northern Italy, 223 age matched male patients (mean age 61.8, range 40-79 years) admitted to the Emergency Care Unit, served as control . H . pylori seroprevalence was assessed by presence of antibodies (IgG) against H . pylori and anti-CagA in circulation . Among the patients we investigated the presence of hypertension, levels of cholesterol and glucose in serum, fibrinogen in plasma and smoking habits . H . pylori infection was present in 189/223 (84.7%) of the patients and in 138/223 (61.8%) of the control population (p < 0.0001 OR 3.42 {IC 95% 2.12-5.54}) . The anti-CagA antibodies were detected in 33.8% of infected patients with AMI (64/189) versus 26.8% in the control subjects (37/138) (p:0.17, OR 1.40 {IC 95% 0.84-2.33}) . Classical risk factors for AMI did not differ among patients with and without H . pylori infection . Patients admitted to the Coronary Care Unit for acute myocardial infarction had a notably higher prevalence of anti-H . pylori not restricted to virulent strains, when compared to a population of patients referred to the Emergency Care Unit . The classical risk factors for coronary disease were present in the patients with AMI irrespective of H . pylori status.

Philos Trans R Soc Lond B Biol Sci, 2002 Jul 29, 357(1423), 887 - 93
Low-temperature sensors in bacteria; Eriksson S et al.; Bacteria are ubiquitous colonizers of various environments and host organisms, and they are therefore often subjected to drastic temperature alterations . Temperature alterations set demands on these colonizers, in that the bacteria need to readjust their biochemical constitution and physiology in order to survive and resume growth at the new temperature . Furthermore, temperature alteration is also a main factor determining the expression or repression of bacterial virulence functions . To cope with temperature variation, bacteria have devices for sensing temperature alterations and a means of translating this sensory event into a pragmatic gene response . While such regulatory cascades may ultimately be complicated, it appears that they contain primary sensor machinery at the top of the cascade . The functional core of such machinery is usually that of a temperature-induced conformational or physico-chemical change in the central constituents of the cell . In a sense, a bacterium can use structural alterations in its biomolecules as the primary thermometers or thermostats.

AIDS Treat News . 2000 Jun 2;(344):3.
New oral DNA vaccine funded for trials; James JS; A new kind of vaccine uses a harmless bacterium to deliver selected parts of HIV to immune cells in the intestinal membranes.

J Bacteriol, 2002 Sep, 184(17), 4920 - 4
Membrane association and kinase-like motifs of the RamC protein of Streptomyces coelicolor; Hudson ME et al.; The protein RamC is required for the production of the spore-forming cells called aerial hyphae by the filamentous bacterium Streptomyces coelicolor . We showed that RamC, which contains several weakly predicted membrane-spanning sequences, is located exclusively in the S . coelicolor membrane . By constructing site-directed mutants in the cloned ramC gene and complementing a ramC null mutant, we showed that protein kinase-like sequence motifs in the amino-terminal half of the protein are required for function in vivo . These data suggest that RamC is a membrane-associated receptor kinase.

J Bacteriol, 2002 Sep, 184(17), 4875 - 80
SipY Is the Streptomyces lividans type I signal peptidase exerting a major effect on protein secretion; Palacin A et al.; Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins . The developmental complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (sipW, sipX, sipY, and sipZ) unusually clustered in its chromosome . Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual sip mutants . None of the sip genes seemed to be essential for bacterial growth . Analysis of total extracellular proteins indicated that SipY is likely to be the major S . lividans SPase, since the sipY mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.

Pathol Biol (Paris), 2002 Jul, 50(6), 388 - 93
{Enzymatic resistance of Escherichia coli to beta-lactams and clinical prevalence}; Lavigne JP et al.; Escherichia coli (E . coli) is the most frequent bacterium implicated in community-acquired infection as well as in nosocomial infections . This bacterium is characterised by numerous possibilities to acquire resistance mechanisms, even during antibiotic treatment . The main mechanism of resistance is the production of beta-lactamines, enzymes hydrolysing beta-lactam ring . This paper describes enzymatic mechanisms of resistance of E . coli to beta-lactam and indicates the necessity of a good knowledge of the risk factors for resistance to have an adapted good clinical practice in using antibiotics.

J Gastroenterol Hepatol, 2002 Sep, 17(9), 960 - 5
Influences of Helicobacter pylori on gastric angiogenesis and ulcer healing in mice; Gunawan E et al.; BACKGROUND AND AIMS: Helicobacter pylori infection is associated with peptic ulcers; however, it is unclear whether the bacterium delays ulcer healing . We investigated the influence of H . pylori on ulcer healing in mice . We also examined the influence of H . pylori infection on angiogenesis . METHODS: An acetic acid ulcer was made in male BALB/c mice . Three days later (day 0), the animals were inoculated with H . pylori SS1 strain . The healing process of the ulcer was examined macroscopically and microscopically on days 0, 6 and 9 . The index of angiogenesis was also determined using carmine dye injection . RESULTS: On day 0, angiogenesis began at the ulcer margin while the mucosal epithelia had not yet regenerated . On days 6 and 9, angiogenesis and epithelial regeneration developed and ulcer size reduced . These phenomena were significantly suppressed in mice infected with H . pylori . CONCLUSION: Helicobacter pylori infection significantly suppressed angiogenesis and delayed ulcer healing . These results indicate that H . pylori plays an important role in ulcer healing .

Helicobacter, 2002 Aug, 7(4), 250 - 6
Helicobacter pylori infection in connective tissue disorders is associated with high levels of antibodies to mycobacterial hsp65 but not to human hsp60; Kalabay L et al.; BACKGROUND: To investigate whether the Helicobacter pylori status influences levels of antibodies against mycobacterial heat shock protein (hsp) 65 and human hsp60 in systemic autoimmune diseases and to study the concentration of anti-H . pylori antibodies in autoimmune patients and healthy controls . MATERIALS AND METHODS: Antibodies against human heat-shock protein hsp60, mycobacterial heat-shock protein hsp65 were analyzed by ELISA . Anti-Helicobacter antibodies were determined by enzyme immunoassay . RESULTS: There was a markedly higher prevalence of H . pylori infection in undifferentiated connective tissue disease (82%) (n = 33) and systemic sclerosis (78%) (n = 55) but not in systemic lupus erythematosus (n = 49), polymyositis/dermatomyositis (n = 14), rheumatoid arthritis (n = 21) or primary Raynaud's syndrome (n = 26) compared with controls (59%) (n = 349) . In autoimmune diseases H . pylori infection was associated with elevated levels of antihsp65 (p =.008) but not of antihsp60 . Anti-hsp65 levels were significantly higher in H . pylori-infected (n = 129) than in uninfected patients (n = 69) (p =.0007) . CONCLUSIONS: These findings indicate that in autoimmune diseases the infection with the H . pylori bacterium is associated with increased concentration of antimycobacterial hsp65.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1366 - 9
Cloning and characterization of extracellular metal protease gene of the algicidal marine bacterium Pseudoalteromonas sp . strain A28; Lee SO et al.; The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp . strain A28 DNA library . The recombinant EmpI protein was expressed in E . coli and purified . Paper-disk assays showed that the purified protease had potent algicidal activity . A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.

Cytokine, 2002 Jun 7, 18(5), 242 - 51
Helicobacter pylori virulence genes and host IL-1RN and IL-1beta genes interplay in favouring the development of peptic ulcer and intestinal metaplasia; Zambon CF et al.; Helicobacter pylori infection outcome might depend on genotypic polymorphisms of both the bacterium and the host . We ascertained: (1) the functionality of H . pylori oipA gene; (2) the polymorphism of the hostinterleukin (IL-1beta) gene (-31 C/T) and of the IL-1RN gene (intron 2 VNTR); (3) the association between the above genes and the histological and pathological outcome of H . pylori infection . One hundred and sixty-five H . pylori positive and 137 H . pylori negative subjects (23 gastric adenocarcinoma, 58 peptic ulcer, 221 gastritis) were studied . oipA was sequenced, IL-1beta was RFLP analysed . Antral and body mucosal biopsies were histologically evaluated . Functional oipA genes were correlated with cagA gene; both genes were significantly associated with gastritis activity, peptic ulcer and gastric adenocarcinoma . In these patients heterozygousIL-1RN 1/2 and IL-1beta C/T genotypes were more frequent than in gastritis patients . Intestinal metaplasia was associated with cagA, functional oipA and IL-1RN 2 allele . In conclusion, peptic ulcer and the preneoplastic intestinal metaplasia are associated with H . pylori virulence genes and with IL-1RN 2 host allele . An interplay between bacterial virulence factors and cytokines genotypes, is probably the main route causing H . pylori infection to lead to benign mild disease, benign severe disease or preneoplastic lesions.

Rheumatology (Oxford), 2002 Aug, 41(8), 857 - 68
HLA B27 in health and disease: a double-edged sword?
Bowness P.
The strong association of the HLA class 1 allele HLA B27 with ankylosing spondylitis (AS) has been recognized for over 25 yr, however the pathogenic mechanism linking HLA B27 with AS and other spondyloarthropathies remains a mystery . We now know that the principal natural function of HLA B27 is an immunologic one, namely to bind antigenic peptides and then present them to T lymphocytes . I have shown that HLA B27 functions as an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals . A working molecular model of how T cells recognize HLA B27 has been generated and tested . Evidence that T cells have a role in spondyloarthritis has also been found . First, expanded populations of T lymphocytes were found in both the blood and synovial fluid of patients with reactive arthritis (ReA) . Secondly, a strong cytotoxic T-cell response to an HLA B27-restricted peptide epitope from Chlamydia trachomatis was found in a patient with ReA . This peptide, derived from a bacterium known to trigger ReA, is thus a candidate 'arthritogenic' peptide . We have also found evidence that HLA B27 has an unusual cell biology compared with other HLA molecules . HLA B27 demonstrates an unusual ability to form heavy chain homodimers in vitro . Dimerization is dependent upon disulphide bonding through an unpaired cysteine at position 67 . Remarkably these dimers lack beta2 microglobulin, previously thought to be an essential component of all mature MHC class 1 molecules . HLA B27 homodimer formation has also been demonstrated in certain cell lines in vivo, and preliminary data suggest that significant numbers of T cells from patients with spondyloarthropathy express a ligand for HLA B27 homodimers . These findings have extended our understanding of the beneficial immunologic function of HLA B27, and have also led us to propose the testable new hypothesis that HLA B27 heavy chain dimerization may be involved in the pathogenesis of spondyloarthritis.

J Biochem (Tokyo), 2002 Aug, 132(2), 183 - 8
Isolation and characterization of the dcw cluster from the piezophilic deep-sea bacterium Shewanella violacea; Ishii A et al.; The dcw cluster of genes involved in cell division and cell wall synthesis from the piezophilic deep-sea bacterium Shewanella violacea was isolated and characterized . It comprises 15 open reading frames, of which the organization is mraZ-mraW-ftsL-ftsI-murE-murF-mraY-murD-ftsW-murG-murC-ftsQ-ftsA-ftsZ-envA, in that order . To analyze transcription upstream from the ftsZ gene, Northern blot and primer extension analyses were performed . The results showed that gene expression is not pressure dependent . Western blot analysis showed that the FtsZ protein is equally expressed under several pressure conditions in the range of atmospheric (0.1 MPa) to high (50 MPa) pressures . Using immunofluorescence microscopy, the FtsZ ring was observed in the center of cells at pressure conditions of 0.1 to 50 MPa . These results imply that the FtsZ protein function is not affected by elevated pressure in this piezophilic bacterium.

Environ Microbiol, 2002 Aug, 4(8), 433 - 42
Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata; Egan S et al.; Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds . In addition to these antifouling compounds, P . tunicata produces at least two pigments . These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance . Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P . tunicata . Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants . The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth . The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms . This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment . Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 782 - 95
Performance of a novel Viresolve NFR virus filter; Brough H et al.; Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs) . The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV) . The NFR designation refers to retrovirus removal in a normal flow format . To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV) . Robust retention was demonstrated over a wide range of process parameters . Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV . In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta . Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies . Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters . Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated . The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.

Water Res, 2002 May, 36(10), 2636 - 42
Corrosion by bacteria of concrete in sewerage systems and inhibitory effects of formates on their growth; Yamanaka T et al.; Not only sulfur-oxidizing bacteria but also an acidophilic iron-oxidizing bacterium (or bacteria) were found in the corroded concrete from several sewerage systems in Japan . The surface pH of concrete test piece exposed to an atmosphere containing hydrogen sulfide of the concentrations more than 600 ppm in the systems was usually below 2 after a month . This was attributable to ability of the sulfur-oxidizing bacteria to grow in the thin water layer which contained hydrogen sulfide and covered the piece even when the surface pH of concrete was 12-13 . When the sulfuroxidizing bacteria grew in the surface of concrete and produced sulfuric acid, the pH of the inner parts of concrete was lowered where the bacteria were hardly found . Probably, sulfuric acid formed by the bacteria in the surface parts penetrated into the inner parts . The different species of sulfur-oxidizing bacteria were found in different sewerage systems . The growth of the sulfur-oxidizing and acidophilic iron-oxidizing bacteria was completely inhibited by formates, especially by calcium formate of concentrations more than 50 mM . Calcium formate can protect concrete in sewerage systems from bacterial corrosion.

J Heart Valve Dis, 2002 Jul, 11(4), 492 - 7
Is there an advantage in using homografts in patients with acute infective endocarditis of the aortic valve?
Gulbins H, Kilian E, Roth S, Uhlig A, Kreuzer E, Reichart B.
BACKGROUND AND AIM OF THE STUDY: Acute infective endocarditis is a surgical challenge, particularly when paravalvular abscesses and annular destruction are present . The choice of a homograft or mechanical valve prosthesis is an important issue in these patients . The study aim was to compare the outcome with homografts and mechanical valves in patients with acute infective endocarditis . METHODS: A total of 77 patients (mean age 49+/-9 years) operated on for acute endocarditis of the aortic valve was included in the study and analyzed retrospectively . The causative bacterium was isolated from blood cultures in 71 cases . Preoperatively, 21 patients required artificial ventilation and 24 had inotropic support due to hemodynamic instability . Aortic homografts were implanted in 43 patients, and mechanical valve prostheses in 34 . The two patient groups were similar in terms of gender, age and preoperative inotropic support . In total, 31 patients (44%) had paravalvular abscesses, and a homograft was used significantly more often (77%, p <0.05) in these cases . Follow up examinations (clinical examination, ECG and transthoracic echocardiography) were performed six months postoperatively and continued on an annual basis . Endocarditis relapse was defined as persisting infection, whereas re-endocarditis indicated a new infection after an interval of at least six months . RESULTS: Perioperative mortality was 11.5% (5/43) in homograft patients . In the 38 survivors, follow up was complete and averaged 5.0+/-1.2 years . One patient had an endocarditis relapse three months after surgery . Re-endocarditis occurred in three patients after two or three years . One other patient had pseudoaneurysm formation without a need for intervention, and one had repeat aortic valve replacement due to dysfunction of the graft after four years . The other 33 patients had an uneventful follow up . Echocardiography revealed aortic insufficiency grade 1 in 12 cases (36%), with no progression during follow up . Perioperative mortality in mechanicat valve patients was 20.5% (n = 7) (p <0.05 versus homograft), and in those with paravalvular abscess, perioperative mortality was even higher than in homograft patients (4/7, 57.1% versus 3/24, 12.5%; p <0.05) . When considering only patients without paravalvular abscess, there was no significant difference between groups (10.5% versus 12.5%) . Three relapses occurred in mechanical valve patients (10.3%), but no endocarditis recurred during follow up . One late death (3.7%) occurred due to bleeding complicating long-term anticoagulation . CONCLUSION: The study results do not permit a general recommendation to be made for homograft use in patients with acute endocarditis . In cases with paravalvular abscesses, however, there was a trend towards improved outcome in the homograft group.

J Clin Microbiol, 2002 Aug, 40(8), 2981 - 8
Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients; Lin Q et al.; Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis . Immunodominant 44-kDa outer membrane proteins of A . phagocytophila are encoded by a p44 multigene family . In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized . A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients . The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A . phagocytophila isolate . A . phagocytophila was isolated by cell culture from all of the three 2000 patients . Genomic Southern blot analysis of the three 2000 and two 1995 A . phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A . phagocytophila genome were conserved . Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved . The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains . These results suggest that variations in the sequences of p44 are not random but are restricted . Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

J Clin Microbiol, 2002 Aug, 40(8), 2849 - 53
Role of corpus gastritis and cagA-positive Helicobacter pylori infection in reflux esophagitis; Queiroz DM et al.; Considering that the role of Helicobacter pylori infection in gastroesophageal reflux and reflux esophagitis (GERD) is still controversial and that the role of virulence markers of the bacterium has not been evaluated in most studies of GERD, we investigated the association among H . pylori infection with cagA-positive and -negative strains, corpus gastritis, and GERD in a large group of patients by controlling for confounding factors . We studied prospectively 281 consecutive adult patients: 93 with GERD and 188 controls . H . pylori infection status was diagnosed by culture, by the preformed urease test, with a carbolfuchsin-stained smear, and by histology . The cagA status was determined by PCR of H . pylori isolates and gastric biopsy specimens . H . pylori infection was diagnosed in 191 (68.0%) of 281 patients . Among the 93 patients with GERD, 84 presented with mild or moderate esophagitis and 9 presented with severe esophagitis . In the multivariate analysis, the age of the patients and the degree of oxyntic gastritis were associated with GERD . Among the strains isolated from patients with GERD and from the control group, 24.4 and 66.9%, respectively, were positive for cagA (P < 0.001) . Compared to infection with cagA-negative strains, infection with cagA-positive H . pylori strains was associated with a more intense gastritis in the corpus (P = 0.001) . cagA status (odds ratio {OR} = 0.16, 95% confidence interval {CI} = 0.07 to 0.40), gastritis of the corpus (OR = 0.69, 95% CI = 0.48 to 0.99), and age (OR = 1.04, 95% CI = 1.01 to 1.07) were associated with GERD . In conclusion, the study provides evidence supporting the independent protective roles of cagA-positive H . pylori strains and the degree of corpus gastritis against GERD.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1201 - 3
Actinomyces coleocanis sp . nov., from the vagina of a dog; Hoyles L et al.; A hitherto undescribed Actinomyces-like bacterium was isolated from the vagina of a dog . Biochemical testing and PAGE analysis of whole-cell proteins indicated that the isolate was phenotypically different from previously described Actinomyces species and related taxa . Sequencing of 165 rRNA showed that the unknown bacterium was distinct from all currently known Actinomyces species . Phylogenetically, the unidentified organism displayed a specific association with Actinomyces europaeus, but a sequence divergence of > 5% demonstrated that it represents a distinct species . Based on both phenotypic and 165 rRNA sequence considerations, it is proposed that the unknown strain from a dog be classified as a novel species, Actinomyces coleocanis sp . nov . The type strain is CCUG 41708T (= CIP 106873T).

Appl Environ Microbiol, 2002 Aug, 68(8), 4158 - 61
Inhibitory effects of high pressure and heat on Alicyclobacillus acidoterrestris spores in apple juice; Lee SY et al.; The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated . Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90 degrees C).

Appl Environ Microbiol, 2002 Aug, 68(8), 4136 - 9
Characterization of ISRgn1, a novel insertion sequence of the IS3 family isolated from a bacteriocin-negative mutant of Ruminococcus gnavus E1; Gomez A et al.; ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1 . The copy number of ISRgn1 in R . gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined . Results obtained suggest that ISRgn1 is not indigenous to the R . gnavus phylogenetic group but that it can transpose in this bacterium.

Appl Environ Microbiol, 2002 Aug, 68(8), 3780 - 9
Identification, characterization, and regulation of a cluster of genes involved in carbapenem biosynthesis in Photorhabdus luminescens; Derzelle S et al.; The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics . We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P . luminescens . The cpm cluster differs in several crucial aspects from other car operons . The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P . luminescens genome . Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production . The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon . Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase . The importance of this carbapenem production in the ecology of P . luminescens is discussed.

Huan Jing Ke Xue, 2002 May, 23(3), 84 - 7
{The degradation of phenanthrene and pyrene contaminated soil with immobilized technique}; Wang X et al.; The phenanthrene and pyrene contaminated soil was degradaded by immobilized-imbedding the selected Zoogloea sp . The degradation rate was determined under various amounts of inoculation and concentration, and the result showed that there was the best degradation rate at 5% . After 168 h, the degradation rate of immobilized Zoogloea sp . to phenanthrene and pyrene was 84.89% and 76.94% respectively . While, the degradation rate of the native bacterium was only 27.85% and 19.65% at the same condition . So the Zoogloea sp . had the better degradation ability . And, Zoogloea sp . distributing shape in immobilized carrier was studied with electric scan-photo, it inferred that immobilized bacterium had the better degradation rate.

EMBO J, 2002 Aug 1, 21(15), 3927 - 35
Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution; Jamieson SJ et al.; Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes . Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM) . Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter . Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved . In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal . In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring . This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring . The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.

J Mol Biol, 2002 Aug 9, 321(2), 307 - 16
Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase; Vonck J et al.; The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits . The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy . Here, we present an alpha-carbon model of the c-subunit ring . Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop . The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm) . The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix . Each inner helix is connected by a clearly visible loop to an outer C-terminal helix . The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus . Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface . Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

J Bacteriol, 2002 Aug, 184(16), 4536 - 43
Simultaneous coexpression of Borrelia burgdorferi Erp proteins occurs through a specific, erp locus-directed regulatory mechanism; El-Hage N et al.; An individual Borrelia burgdorferi bacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids . We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire . While it has been proposed that B . burgdorferi controls expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling . Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression . Furthermore, expression of loci physically adjacent to erp loci was observed to be independently regulated . It is concluded that Erp expression is regulated by a mechanism(s) directed at erp loci and not by a global, plasmid-wide mechanism.

J Bacteriol, 2002 Aug, 184(16), 4475 - 88
Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima; Nesbo CL et al.; Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species . Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization . The genome sequence of T . maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp . strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence) . Four hundred twenty-six RQ2 subtractive clones were sequenced . One hundred sixty-six had no DNA match in the MSB8 genome . These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database . From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8 . Assuming a similar genome size, this corresponds to 20% of the RQ2 genome . A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8 . Several clones encode proteins involved in the production of surface polysaccharides . RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase . Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage . Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.

Mol Microbiol, 2002 Aug, 45(3), 605 - 16
DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus; Quardokus EM et al.; Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division . Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus . Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells . New Z-rings can only form in the replicative stalked cell . Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle . Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell . Z-rings only assembled at the proper mid-cell location if DNA replication had initiated . Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome . Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid . These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.

Biochem J, 2002 Nov 1, 367(Pt 3), 781 - 9
Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis; Funa N et al.; RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus . The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate . The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA . Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction . Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction . The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain . Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

Biochemistry (Mosc), 2002 Jul, 67(7), 822 - 5
Antioxidative enzymes of sulfate-reducing bacterium desulfovibrio desulfuricans: superoxide dismutase and peroxidases; Davydova MN et al.; Extracts of Desulfovibrio desulfuricans B-1388 cells grown under anaerobic conditions displayed superoxide dismutase activity . The maximal activity was found during the stationary growth phase . The enzyme was virtually completely located in the periplasm fraction . D . desulfuricans B-1388 lacked catalase activity but contained active NADH- and NADPH-peroxidases . The activity of NADH-peroxidase depended on the physiological state of the culture . On changing the growth conditions (the presence of 5% CO in the gaseous phase), the activity of superoxide dismutase decreased.

Biochemistry (Mosc), 2002 Jul, 67(7), 765 - 9
Depletion of phosphatidylethanolamine--the major membrane phospholipid of Escherichia coli--depresses posttranslocational modification of alkaline phosphatase in the periplasm; Golovastov VV et al.; Unbalanced phospholipid composition due to depletion of the major phospholipid of Escherichia coli, phosphatidylethanolamine, was shown previously to significantly decrease the secretion and transcriptional expression of periplasmic alkaline phosphatase of this bacterium . The current work studies the effect of this unbalance on posttranslocational proteolytic modification of the enzyme, proceeding in the periplasm with the formation of its isoforms . It has been revealed that under phosphatidylethanolamine depletion the content of incompletely modified (intermediate) isoforms I and II increases . This increase does not depend on the level of enzyme synthesis and the mechanism of its regulation (expression of the chromosomal gene or the gene cloned in plasmid under the control of the endogenous promoter P(PHO) or exogenous promoter P(BAD)) . Depression of posttranslocational modification of alkaline phosphatase in phosphatidylethanolamine-depleted cells is supposed to be a result of the lower efficiency of secretion of modifying proteinase (product of the iap gene) localized, like alkaline phosphatase, in the periplasm of Escherichia coli.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(3), 337 - 340
The Photochemical Change in Photosynthetic Reaction Center of Purple Bacterium with Pheophytin Substitution; Zeng XH et al.; The reaction centers are isolated from chromatophores of Rhodobacter sphaeroides 601 by detergent LDAO, and purified by chromatography on a DEAE-52 cellulose column . In the presence of acetone and an access of free pheophytins (Phes), bacteriopheophytins (Bphes) in reaction centers are replaced by pheophytins at sites H(A) and H(B) when incubated under high temperature . The substituting amounts are about 50% and 71% Bphes in reaction centers with incubation of fifteen and sixty minutes respectively . In the absorption spectra of reaction centers containing Phes (Phe RC), the Q(X) 537 nm and Q(Y) 758 nm bands of Bphe disappeared, three distinct bands assigned to the Q(X 509/542 nm and QY) 674 nm bands of phe appeared . Compared to reaction centers in control, the photochemical activities of Phe RCs, with incubating time of fifteen and sixty minutes, drop to 78 and 71% of that in control respectively.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(3), 259 - 263
Mutagenesis of hupT Gene and H(2)-uptake Hydrogenase Expression in Photosynthetic Bacterium SDH2; Li Q et al.; HupT(-) insertion mutants, SDHT1 and SDHT2, were constructed by the homologous double exchange between the host, Rodobacter sp . SDH20, and the hupT gene fragment with a kan(R) gene inserted on pSE8, a suicide plasmid, which had been introduced into the host by using triparental conjugation . The HupT(-) mutants were verified by Southern hybridization . H(2)-uptake hydrogenase activity analysis revealed that the H(2)-uptake hydrogenase activities of HupT(-)mutants were two-fold as that of wild type strain SDH20 when grown anaerobically in MN medium and aerobically in MG or MN medium, and had no distinct improvement relative to wild type strain when grown anaerobically in MG medium . The expression analysis of hupS confirmed that the HupT- mutants exhibited two- to three-fold increase of the expression of hupS relative to wild type strain when grown anaerobically in MN medium and aerobically in MG or MN medium, but no obvious increase when grown anaerobically in MG medium . These results demonstrate that hupT regulates negatively the synthesis of H(2)-up-take hydrogenase in the photosynthetic bacterium strain SDH2.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10742 - 7 Epub 2002 Jul 22.
A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects; Daborn PJ et al.; Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis . This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects . Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system . Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction . However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear . Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect . The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

J Immunol, 2002 Aug 1, 169(3), 1433 - 43
The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo; Park MK et al.; Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation . The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice . We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice . Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3 . Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens . Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain . Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3 . In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells . Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.

J Immunol, 2002 Aug 1, 169(3), 1419 - 25
Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization; Li JS et al.; Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established . To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed . Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant . A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy . The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g . Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b . The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t(1/2) . Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

J Microbiol Methods, 2002 Oct, 51(2), 191 - 5
Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei; Yao Y et al.; RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA . Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality . The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus . In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis . Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA . About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A . mediterranei . The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.

Zhonghua Yi Xue Za Zhi, 2002 May 25, 82(10), 673 - 7
{Mutual interaction between hepatitis C virus core protein and translin, a recombination hotspot binding protein}; Li K et al.; OBJECTIVE: To study the role of the core protein of hepatitis C virus (HCV) in HCV pathogenesis and investigate the molecular mechanism of this protein in tumorigenesis . METHODS: By using the yeast two hybrid system 3, the bait plasmids of the gene of the core protein of HCV was constructed and transfected into AH109 yeast strain . Then this transfected AH109 yeast was mated with Y187 yeast containing liver cDNA library plasmids and cultured on quadropledropout (QDO) medium covered with x-alpha-gal and assayed for alpha-gal activity . The blue colonies growing on QOD were collected to extract the plasmids to transform Escherichia coli . Plasmids were extracted from the bacterium and sequenced . After bioinformatic analysis translin, a recombination hotspot binding protein interacting with HCV core protein was found . To further prove the interaction between translin protein and HCV core protein translation was performed by using reticulocyte lysate and immunoprecipitation in vitro . RESULTS: Among the 30 positive colonies, a colony was translin . The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro . CONCLUSION: The core protein of HCV can interact with translin protein, this can explain partly the tumorigenesis of HCV.

Mol Cells, 2002 Jun 30, 13(3), 369 - 76
Characterization of the conserved region of the mxaF gene that encodes the large subunit of methanol dehydrogenase from a marine methylotrophic bacterium; Jeong JH et al.; The highly conserved region of the mxaF gene that encodes the large subunit of methanol dehydrogenase (MDH) was cloned and sequenced from Methylophaga sp . strain MP cells . The calculated G + C content of the conserved region was found to be 44.9% . The nucleotide sequence homology of the region to those from methylotrophs was approximately 43.5%, while the identity of the deduced amino acid sequence to other MxaF peptides was approximately 76.8% . Analysis of the codon usage revealed that UUC and CGU codons seem to be used only for phenylalanine and arginine, respectively . The aligned amino acid sequences show that several key amino acids that are required for the MDH enzyme activity are located in the deduced MxaF peptide, together with tryptophan-docking motifs, called W4 and W5.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 67 - 72
Acid stress response in Helicobacter pylori; Toledo H et al.; To determine the existence of an acid stress response in Helicobacter pylori the global changes in the proteins synthesized by the bacterium when subjected to an acid stress were studied . H . pylori ATCC43504 previously adapted to pH 7 did not show an acid stress response as detected by the two-dimensional electrophoretic pattern of 35S-labeled proteins when incubated at pH 3 . This was probably due to the neutralization of the external medium by the action of urease . However, H . pylori DW504UreI-negative, a mutant strain unable to transport urea into the cell, showed a large number of proteins changed, as is typical in an acid stress response . Some of these proteins were identified by N-terminal sequencing.

Biochemistry (Mosc), 2002 Jun, 67(6), 689 - 95
Alpha-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes; Bakunina IY et al.; An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T . The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes . The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C . The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing . The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM . The molecular mass of the enzyme determined by gel filtration is 84 kD.

Biochemistry (Mosc), 2002 Jun, 67(6), 622 - 6
Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii; Bertsova YV et al.; A part of the gene encoding cbb3-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed . In contrast to the wild type strain, this one is unable to oxidize cytochromes c4 and c5 . Thus, the A . vinelandii respiratory chain is shown to contain cbb3-type cytochrome c oxidase . It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A . vinelandii at high oxygen concentrations.

Lasers Surg Med, 2002, 31(1), 45 - 52
Measurement of adhesive forces between S . epidermidis and fibronectin-coated surfaces using optical tweezers; Simpson KH et al.; BACKGROUND AND OBJECTIVES: Biomaterial-mediated infection, a common cause of medical device failure, is initiated by bacterial adhesion to an adsorbed protein layer on the implant surface . This adhesion is thought to be mediated by specific molecules present on the bacterial cell surface . Optical tweezers can be used to measure the adhesive force between a single bacterium and a protein-coated surface . STUDY DESIGN/MATERIALS AND METHODS: Using optical tweezers, a bacterium was trapped and brought in contact with a 10-microm diameter polystyrene microsphere coated with fibronectin . The minimum force required to detach the cell from the bead was determined over a range of fibronectin concentrations and contact times . RESULTS: The detachment forces were integer multiples of an 18-pN base value that was independent of contact time and coating concentration; we propose that the variation in force is related to the number of bonds formed . CONCLUSIONS: These experiments demonstrate that optical tweezers can be used to investigate the adhesion of individual bacteria to surfaces . The results suggest that S . epidermidis has surface proteins capable of binding fibronectin .

Oral Microbiol Immunol, 2002 Aug, 17(4), 245 - 51
Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin-producing strain of Actinobacillus actinomycetemcomitans; DiRienzo JM et al.; The periodontal pathogen Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT), a complex multicomponent toxin that arrests the growth of many types of eukaryotic cell . The kinetics of the effects of CDT-containing extracts, from an invasive strain of this bacterium, were examined on epithelial-like cells routinely used in invasion studies . Both KB and HEp-2 cells were exquisitely sensitive to the effects of the CDT with TD50 of 30 and 300 pg of total bacterial protein, respectively . Initial cell morphology changes were relatively rapid, occurring within the first 13 h of exposure . CDT-treated KB cells increased in size to 4-5 times the size of untreated controls . Cytotoxicity was irreversible when attached cells were incubated, for a minimum of 120 min, with nanogram quantities of CDT-containing extract . As cultures aged, the cells became more resistant to the effects of the CDT-containing extracts . These findings have important implications for understanding the ability of A . actinomycetemcomitans to invade and multiply in epithelial cells.

Oral Microbiol Immunol, 2002 Aug, 17(4), 231 - 8
Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitans isolates from geographically diverse populations; Fabris AS et al.; A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium . The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A . actinomycetemcomitans from Brazil, Kenya, Japan and Sweden . Thirty-nine of 40 cell lysates caused distension of Chinese hamster ovary cells . At least one of the cdt genes was detected in all strains examined . The three cdt genes were detected, by PCR, in 34 DNA samples . DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity . Restriction analysis was performed on every amplicon obtained . PCR-RFLP patterns revealed that the three cdt genes were conserved . These data provided evidence that the cdt genes are found and expressed in the majority of the A . actinomycetemcomitans isolates . Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found.

J Eukaryot Microbiol, 2002 May-Jun, 49(3), 209 - 19
Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles; Pimenta PF et al.; Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt . 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon . The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease: the latter appears not to be invasive . Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species . However, recent biochemical . immunological, and genetic studies provided convincing evidence that they belong to separate species . Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites . 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins . 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles . Loose vacuoles containing several bacteria were seen in E . histolytica whereas tight vacuoles containing a single bacterium were observed in E . dispar . Furthermore, bacteria were found only within vacuoles in E . histolytica; in E . dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.

Infect Immun, 2002 Aug, 70(8), 4564 - 70
Induction of a potential paracrine angiogenic loop between human THP-1 macrophages and human microvascular endothelial cells during Bartonella henselae infection; Resto-Ruiz SI et al.; Bartonella henselae is responsible for various disease syndromes that loosely correlate with the immune status of the host . In the immunocompromised individual, B . henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma . We hypothesize that B . henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells . Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1beta (IL-1beta), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to live B . henselae . Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1beta induction . IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells . In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential . These data suggest that Bartonella modulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis . B . henselae induction of VEGF, IL-1beta, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.

Infect Immun, 2002 Aug, 70(8), 4132 - 41
Roles of p38 mitogen-activated protein kinase, NF-kappaB, and protein kinase C in proinflammatory cytokine mRNA expression by human peripheral blood leukocytes, monocytes, and neutrophils in response to Anaplasma phagocytophila; Kim HY et al.; Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic ehrlichiosis . Within 2 h after addition of A . phagocytophila, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro . However, neutrophils generate only IL-1beta mRNA . In the present study, signaling pathways for induction of these three cytokines were examined . TNF-alpha and IL-6 mRNA expression by PBLs was inhibited with SB 203580 (a p38 mitogen-activated protein kinase {MAPK} inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an NF-kappaB inhibitor) . Activation of p38 MAPK and NF-kappaB mRNAs in monocytes was detectable within 15 to 30 min after addition of A . phagocytophila . Expression of these two cytokine mRNAs in PBLs and monocytes was also dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK) . IL-1beta mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in neutrophils incubated with A . phagocytophila . IL-1beta mRNA induction by PBLs, monocytes, and neutrophils was dependent on PKC and PKA . Neutrophil expression of IL-1beta mRNA was dependent on transglutaminase, phospholipase C, and PTK, all of which are also required for internalization of A . phagocytophila . However, monocyte expression of IL-1beta mRNA was less dependent on these enzymes . These results suggest that A . phagocytophila transduces different signals between its host neutrophils and monocytes for proinflammatory cytokine generation.

FEMS Microbiol Lett, 2002 Jul 2, 212(2), 193 - 202
Identification and characterization of a novel Chlamydia trachomatis reticulate body protein; Shaw AC et al.; The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis . As part of a large-scale proteome analysis of C . trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry . No homology of this protein was observed to proteins from other organisms . The protein was conserved in C . trachomatis but not found in Chlamydia pneumoniae . Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle . The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C . trachomatis development . We have termed the protein '7-kDa reticulate body protein'.

Proteomics, 2002 Jun, 2(6), 754 - 64
The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products; Ueberle B et al.; An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes . This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs) . The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry . The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin . A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis . The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.

J Mass Spectrom, 2002 May, 37(5), 481 - 8
Lipid A structure of Pseudoalteromonas haloplanktis TAC 125: use of electrospray ionization tandem mass spectrometry for the determination of fatty acid distribution; Corsaro MM et al.; The use of electrospray Ionization (ESI) tandem mass spectrometry (MS/MS) for the structural determination of the lipid A components of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 is reported . The lipid A contains the classical bisphosphorylated beta-(1' --> 6)-linked D-glucosamine disaccharide with 3-hydroxydodecanoyl residues (12 : 0 (3-OH)) linked both as esters and amides to 2', 3' (distal glucosamine) and 2, 3 positions (proximal glucosamine) of the sugar backbone . The hydroxyl of 12 : 0 (3-OH) fatty acid linked at the 3' position is esterified by a dodecanoyl residue (12 : 0) . In addition to the pentaacyl component, a minor tetraacyl lipid A, lacking the acyl residue at position 3, was also found in the lipid A fraction . The advantage of this MS technique for the investigation of the intra-ring fragmentation, which is useful for the determination of fatty acyl residue distribution on each glucosamine unit, is emphasized .

Stat Med, 2002 Jun 30, 21(12), 1773 - 85
The predictive value of microbiologic diagnostic tests if asymptomatic carriers are present; Gunnarsson RK et al.; If a proper gold standard is not available, then the predictive value of a test cannot be estimated . In this paper the concept of etiologic predictive value (EPV) is introduced . It is a quantity that will yield the predictive value of a test to predict presence of a specified disease in situations for which no proper gold standard is available . This is achieved by using information obtained from a healthy control population . This quantity requires that the marker in our test is present in all individuals having the specified disease, as in the case where the marker is the aetiologic factor for the specified disease . Furthermore this quantity requires that asymptomatic carriers are present . This means that not all individuals with the marker has the specified disease . EPV is developed with special reference to the evaluation of bacterial cultures, or rapid tests to detect a bacterium, but the quantity might be used in other circumstances as well . EPV is applied to an example in which conventional throat culture is evaluated . Further information concerning EPV can be found at

Pathology, 2002 Jun, 34(3), 270 - 4
The detection of Chlamydia pneumoniae in atherosclerotic plaques of Australian subjects; Cochrane M et al.; AIM: To determine whether the common respiratory pathogen, Chlamydia pneumoniae, was associated with atherosclerotic plaques in Australian subjects . METHODS: A total of 29 coronary atherosclerotic lesions and 18 normal coronary arterial samples were tested for the presence of C . pneumoniae by PCR and immunofluorescence methods . RESULTS: Chlamydia pneumoniae was detected in 15 of the atheromatous lesions as well as in three of the normal tissues; the immunofluorescence assay was more sensitive (P=0.028) than PCR (P=0.26) . CONCLUSIONS: These findings contradict previous Australian studies which did not detect C . pneumoniae in atherosclerotic plaques, thereby discounting the speculation that its absence was likely due to geographical variation . The detection of the bacterium in some of the normal tissues suggests that C . pneumoniae infection might be an initial trigger of atherosclerotic development.

J Bacteriol, 2002 Aug, 184(15), 4081 - 8
AmtB is necessary for NH(4)(+)-induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus; Yakunin AF et al.; Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY . In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants . Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine . The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH(4)(+) addition . ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.

Biochemistry, 2002 Jul 16, 41(28), 8868 - 75
The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center of Chromatium vinosum; Vermeglio A et al.; Under anaerobic conditions, intact cells of the purple sulfur bacterium Chromatium vinosum exhibit rapid photooxidation of the two low-potential hemes of the c-type cytochrome associated with the reaction center, after exposure to two short light flashes separated by a dark interval . Reduction of the photooxidized low-potential hemes is very slow under these conditions . On subsequent flashes, rapid photooxidation of a high-potential reaction center heme occurs and is followed by its rereduction on the millisecond time scale . Cells maintained under aerobic conditions exhibit the millisecond time scale reduction of the photooxidized high-potential heme after each flash . Cells grown autotrophically in the presence of Na(2)S and Na(2)S(2)O(3) appear to use the soluble {4Fe-4S}-containing protein, HiPIP, as the only direct electron donor to the reaction center heme under aerobic conditions . In contrast, cells grown in the presence of organic compounds, but in the absence of Na(2)S and Na(2)S(2)O(3), appear to use a soluble c-type cytochrome (most likely cytochrome c(8)) as the only electron donor to the reaction center heme under aerobic conditions . Cells grown autotrophically, in the presence of Na(2)S and Na(2)S(2)O(3), have a slightly higher ratio of HiPIP to cytochrome c(8) and a ratio of Rieske iron-sulfur protein to reaction center that is approximately one-half that of cells grown in the absence of Na(2)S and Na(2)S(2)O(3) but in the presence of organic compounds.

Mol Microbiol, 2002 Jul, 45(1), 45 - 57
The ramC gene is required for morphogenesis in Streptomyces coelicolor and expressed in a cell type-specific manner under the direct control of RamR; O'Connor TJ et al.; The bacterium Streptomyces coelicolor produces two cell types during the course of its life cycle: the aerial hyphae, which metamorphose into spores, and the substrate hyphae, which synthesize antibiotics . We show that the genes ramC and ramR are required for the production of the aerial hyphae but are dispensable for vegetative growth and antibiotic synthesis . We find that ramC is expressed in the substrate hyphae and shut off in the aerial hyphae by the time visible signs of sporulation-associated septation are evident . Production of RamC requires the developmental regulators bldD, cprA and ramR, but not bldM or bldN, and we show that the RamR protein interacts directly with DNA in the ramC promoter region suggesting that it is, at least in part, responsible for regulating ramC expression.

Nature, 2002 Jul 4, 418(6893), 76 - 9
A host parasite interaction rescues Drosophila oogenesis defects; Starr DJ et al.; The cytoplasmically inherited bacterium Wolbachia pipientis is a widespread parasite of arthropods that manipulates the reproductive biology of its hosts, often to their detriment, in order to foster its own transmission through egg cytoplasm . Here we report that infection by Wolbachia restores fertility to Drosophila melanogaster mutant females prevented from making eggs by protein-coding lesions in Sex-lethal (Sxl), the master regulator of sex determination . Suppression of sterility by Wolbachia discriminates markedly among similar germline-specific Sxl alleles, and is not observed for mutations in other genes that produce similar 'tumorous ovary' phenotypes, including one that blocks Sxl germline expression . This allele and gene specificity indicates that suppression probably results from a specific interaction with Sxl protein, rather than from a bypass of the normal germline requirement for this developmental regulator or from an effect on Sxl expression . The Sxl-Wolbachia interaction provides a rare opportunity to explore host-parasite relationships at the molecular level in a model insect . Furthermore, demonstration that a parasite infection can counteract the deleterious effects of mutations in host genes illustrates how hosts might become dependent on parasites.

Mol Cell Proteomics, 2002 Mar, 1(3), 179 - 85
The Rickettsia prowazekii invasion gene homolog (invA) encodes a Nudix hydrolase active on adenosine (5')-pentaphospho-(5')-adenosine; Gaywee J et al.; The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA) . The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family . To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli . The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives . The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules . At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1) . Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates . Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity . These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

Folia Microbiol (Praha), 2002, 47(3), 218 - 24
An evaluation of the outer membrane charge and softness of Thiobacillus ferrooxidans by the Ohshima's electrophoretic model of a "soft" particle; Skvarla J et al.; The surface charge of bacterial cells plays an important role in their interfacial physiology and adhesion to substrata mediated by the electrostatic double-layer interaction . The surface charge or potential of biological cells is generally calculated from the experimentally measurable electrophoretic velocity of these cells migrating in an external electric field, applying the well-known Smoluchowski equation which is valid for "hard" particles with a sharp interface . However, bacterial cells possessing a structured outer membrane of a finite thickness (dependent on the ionic strength and pH of the surrounding liquid medium) are expected to obey Ohshima's electrophoretic mobility equation derived recently for "soft" particles . The electrophoretic mobility of Thiobacillus ferrooxidans was measured here by the fully automated technique of electrophoretic light scattering, based on the proportionality between the mobility and the Doppler shift in the frequency of light scattered by electrophoresing cells . Agreement was obtained between the experimentally determined electrophoretic mobility expressed as a function of low ionic strength (60-6000 mumol/L) at different pH values and the best-fit theoretical predictions of the "soft" particle electrophoresis theory, which is better than in the case of applying the Smoluchowski formula . The best-fit surface-charge and softness parameters predict a rather rigid and low-charge outer membrane of the bacterium examined, as compared to the parameters obtained for other bacteria in media of high ionic strength.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1022 - 31
Purification, molecular cloning, and characterization of pyridoxine 4-oxidase from Microbacterium luteolum; Kaneda Y et al.; Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1) . The purified enzyme required FAD for its catalytic activity and stability . The enzyme was a monomeric protein with the subunit molecular mass of 53,000 +/- 1,000 Da . PN was the only substrate as the hydrogen donor . Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor . The gene (pno) encoding PN 4-oxidase was cloned . The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit . PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M . luteolum YK-1 . Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family . M . luteolum YK-1 has four plasmids . The pno gene was found on a chromosomal DNA . Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.

Rev Med Chir Soc Med Nat Iasi, 2001 Oct-Dec, 105(4), 682 - 4
{Spontaneous bacterial peritonitis- undervalued complication in liver cirrhosis}; Prelipcean CC; This complication described rather late and theoretically and practically under-evaluated occurs in long term evolution of ascitic hepatic cirrhosis . The positive diagnosis is indicated by the following elements: more than 250 polymorphonuclear count in ascitic fluid, under 1 g/dl protein concentration ascitic fluid, a positive culture for a unique bacterium . Treatment of SBP is made with third generation cephalosporins with immediate favorable evolution . Antibiotic prophylaxis appears to be effective in the prevention of the first SBP episode and long term norfloxacin administration appeared to be very effective in the prevention of the recurrence . The survivors of SBP episode should be recommended for a potential liver transplant.

J Consult Clin Psychol, 2002 Jun, 70(3), 739 - 50
Psychosocial factors in peptic ulcer and inflammatory bowel disease; Levenstein S; Over the past decade, while gastroenterologists' interest in mind-body interactions in organic disorders dwindled, stronger evidence has linked psychosocial factors with the incidence and recurrence of peptic ulcer and with the course of inflammatory bowel disease . Psychological-behavioral approaches to treatment continue to be disappointing . Psychosocial factors may affect ulcer by increasing duodenal acid load, altering local circulation or motility, intensifying Helicobacter pylori infection, stimulating corticosteroid secretion, and affecting health risk behaviors; possible mechanisms for inflammatory bowel disease include immune deregulation, gut permeability changes, and poor medication adherence . Both belong to the growing category of diseases thought to have an infectious component: for peptic ulcer the bacterium Helicobacter pylori, for inflammatory bowel disease an exaggerated immune response to gut bacteria . Peptic ulcer and inflammatory bowel disease, which present unique interactions among psychological, immunologic, endocrine, infectious, and behavioral factors, are splendid paradigms of the biopsychosocial model.

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