Vet J, 1997 Jan, 153(1), 75 - 9 Detection of an antigenic protein of Leptospira interrogans which shares epitopes with the equine cornea and lens; Parma AE et al.; A protein epitope which is involved in an antigenic relationship between equine ocular tissues and Leptospira interrogans was detected in homogenates of the bacterium . The antigenic determinant was harboured on a peptide structure which was shown to be sensitive to the action of denaturing and reducing agents by means of Western blotting . The outer surface of the leptospires appeared to be free of this epitope as was proved by dot-blot and electron microscopic studies. J Dairy Sci, 1997 Jan, 80(1), 167 - 75 Degradation of protein and utilization of the hydrolytic products by a predominant ruminal bacterium, Prevotella ruminicola B1(4); Griswold KE et al.; Degradation and utilization of protein by Prevotella ruminicola B1(4), a proteolytic bacterium that is prominent in the rumen, was examined . In preliminary experiments, proteinaceous N sources produced faster growth rates than did NH4Cl, based on changes in optical density over time . However, ammonium chloride produced a greater maximum cell density than did proteinaceous N sources . Of the proteinaceous N sources, an enzymatic hydrolysate of soybean protein with a relative peptide size of 3 AA residues produced a greater growth rate and maximum cell density compared with the other proteinaceous N sources . Further experiments revealed that P . ruminicola B1(4) grew faster and to a greater final dry weight with soybean protein than with casein . Degradation of both proteins was low as was indicated by the slow disappearance of soluble protein, low concentrations of free AA and peptides, and the decrease in ammonia concentrations over time . Patterns of degradation did differ between the two proteins, however . Accumulation of peptides and free AA from soybean protein peaked 2 h earlier than those from casein, and concentrations of free AA and peptides from soybean protein were lower on average than those from casein . Prevotella ruminicola B1(4) preferentially utilized Asp, Ile, Leu, Lys, and Arg from soybean protein compared with casein . The relative size of peptides that accumulated from both proteins, as determined by the ratio of ninhydrin reaction after HCl hydrolysis to ninhydrin reaction before HCl hydrolysis, suggested that part of the proteolytic activity of P . ruminicola B1(4) is a dipeptidase . Our findings suggest that P . ruminicola may have a greater impact on peptide degradation than on protein degradation in the rumen. J Periodontal Res, 1997 Jan, 32(1 Pt 1), 61 - 8 The potential role of alpha 2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis; Gron H et al.; Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis . The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K) . No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M) . Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated . All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3) . alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme . A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped . On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids. Carcinogenesis, 1997 Jan, 18(1), 233 - 6 Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice; Sipowicz MA et al.; Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas . This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection . A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes . Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months . The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration . DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes . Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual . Other genes may be targeted or H . hepaticus infection causes liver cancer through other pathways than direct damage to DNA. Vet Microbiol, 1997 Jan, 54(1), 47 - 62 Diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a Lawsonia intracellularis-specific multiplex PCR assay; Cooper DM et al.; Proliferative enteritis (PE) is an enteric disease that has been reported in a variety of animals . It is caused by an obligate intracellular bacterium identified in swine as Lawsonia intracellularis . The organism can be detected ante-mortem in swine with PE using molecular diagnostic methods . The disease can be diagnosed post-mortem in all species by gross examination of tissues and special histologic staining procedures . In this study we extracted total DNA from frozen or formalin-fixed, paraffin-embedded tissues from cases of pig, hamster, horse, deer and ostrich PE . The samples were subjected to a multiplex PCR reaction using primers specific for a swine isolate of L . intracellularis . Identical sized PCR products were detected in samples from all animals with PE and the specificity of the PCR reaction for L . intracellularis was demonstrated by Southern-blotting and hybridization using specific probes . These results suggest that the intracellular organism of PE in these species are all very closely related to the causative agent of PE in swine, L . intracellularis . In addition, this multiplex PCR assay can be used to detect the organism in frozen or archival tissues, facilitating retrospective diagnosis of PE. Mol Microbiol, 1997 Jan, 23(2), 399 - 407 A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E . coli K-12; McDaniel TK et al.; Attaching and effacing (AE) bacteria are a diverse group of gastrointestinal pathogens, comprising members of four genera, that cause the intestinal epithelial microvilli to be replaced with raised clusters of filamentous actin that conform to the surface of attached bacteria . We have cloned a 35.4 kb 'pathogenicity island' from the prototype AE bacterium, enteropathogenic Escherichia coli, containing all previously described AE genes . Transfer of this pathogenicity island to avirulent E . coli converts the recipients into strains that secrete virulence proteins, induce host signal-transduction pathways, and cause AE lesions on cultured epithelial cells . These results demonstrate that this pathogenicity island contains all pathogen-specific genes necessary for inducing AE lesions, and that the defining feature of this class of pathogens can be acquired by an avirulent bacterium in a single genetic step. Mol Microbiol, 1997 Jan, 23(2), 267 - 79 Isolation and analysis of a gene encoding alpha-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan; Ruile P et al.; This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans . A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8 . The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme . The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da . The alpha-glucuronidase of T . maritima has a novel primary structure with no significant similarity to any other known amino acid sequence . The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE . Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active . The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid . The K(m) for MeGlcAX2 was 0.95 mM . The pH optimum was 6.3 . Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date . When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days. Biochem Mol Biol Int, 1997 Jan, 41(1), 123 - 30 Spectroscopic characterization of the uptake of essential and xenobiotic metal cations in cells of the soil bacterium Azospirillum brasilense; Kamnev AA et al.; The results of flame (FAAS) or graphite furnace atomic absorption spectrometric (GFAAS) analyses are presented and discussed on the accumulation of essential metals (Mg, Ca, Mn and Fe contained in the cultivation medium) and traces of each one of the conventionally xenobiotic elements from the group V, Co, Ni, Cu, Zn or Pb, added to the medium in concentrations (0.2 mM) which do not essentially suppress growth of the bacterial culture, in cells of the plant root-associated nitrogen-fixing bacterium Azospirillum brasilense . Along with the essential cations assimilated by the bacterium, Zn and Cu were found to effectively accumulate in the biomass from the environment . The uptake of Co and Ni was significantly less pronounced, whereas Pb and V appeared to be present in cells in much lower concentrations than in the cultivation medium evidently showing no tendency to be assimilated by azospirilla . The effect of the above xenobiotics on the uptake level of the four essential elements provided evidence that they may compete for the formation of biologically active complexes with substances of both intracellular and extracellular localization . The analytical data obtained are compared with Fourier transform infrared (FTIR) spectra of intact vacuum-dried bacterial cells grown in a standard medium and under the conditions of an increased metal uptake. Biotechnol Prog, 1997 Jan-Feb, 13(1), 53 - 9 2,4-Dichlorophenol degradation using Streptomyces viridosporus T7A lignin peroxidase; Yee DC et al.; The Streptomyces viridosporus T7A bacterium produces the extracellular lignin peroxidase ALiP-P3 . The ALiP-P3-catalyzed oxidation of 2,4-dichlorophenol (DCP) was examined to understand its kinetic behavior . Initial rate data of the oxidation of DCP were obtained by a spectrophotometric peroxidase assay, and the kinetics were best modeled with a random-binding bireactant system, which differs from the ping-pong bireactant system that is typically used for horseradish peroxidase and lignin peroxidase from the fungus Phanerochaete chrysosporium, and suggests that either DCP or H2O2 may bind first to ALiP-P3 . Chloride ion measurements indicate that 16% of the reacted DCP was fully dechlorinated by ALiP-P3 . Chemical ionization mass spectrometry was also utilized to identify the DCP degradation product as a hydrophobic chlorinated dimer of mass 322. Yale J Biol Med, 1996 Jan-Feb, 69(1), 35 - 7 Helicobacter pylori: bacterial factors and interaction with the epithelial cells; Megraud F; Helicobacter pylori has been recognized as a major cause of most of the diseases of the stomach . These diseases are preceded by lesions of gastritis induced by H . pylori . This long-standing infection gives us a very good model of interaction between a bacterium and its host . We will review the direct and indirect effects of H . pylori. Microbiology, 1997 Jan, 143 ( Pt 1), 83 - 91 Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration; Pasternak C et al.; To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection . In situ and Southern hybridization analyses have demonstrated that the TrxA- mutation is lethal for R . sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions . In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R . sphaeroides . An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R . sphaeroides . The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy . The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable . These data suggest that the physiological function of R . sphaeroides thioredoxin is redox-dependent . Thioredoxin purified from R . sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins . This unexpected finding suggests that R . sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes . Thus, the fundamental role of R . sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo. Biophys J, 1997 Jan, 72(1), 24 - 36 Ultrafast absorption difference spectra of the Fenna-Matthews-Olson protein at 19 K: experiment and simulations; Buck DR et al.; We describe simulations of absorption difference spectra in strongly coupled photosynthetic antennas . In the presence of large resonance couplings, distinctive features arise from excited-state absorption transitions between one- and two-exciton levels . We first outline the theory for the heterodimer and for the general N-pigment system, and we demonstrate the transition between the strong and weak coupling regimes . The theory is applied to Fenna-Matthews-Olson (FMO) bacteriochlorophyll a protein trimers from the green photosynthetic bacterium Prosthecochloris aestuarii and then compared with experimental low-temperature absorption difference spectra of FMO trimers from the green bacterium Chlorobium tepidum. J Bacteriol, 1997 Jan, 179(2), 487 - 95 Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon; Lee WT et al.; The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons . The groESL1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes . The deduced amino acid sequences of GroEL1 (cpn60) and GroES1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K . C . Terlesky and F . R . Tabita, Biochemistry 30:8181-8186, 1991) . These sequences show a high degree of similarity to groESL genes isolated from other bacteria . Northern analysis indicated that the groESL1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock . Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min . The R . sphaeroides groESL1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes . Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop . In this region were also found putative -35 and -10 sequences that are conserved upstream from other bacterial heat shock genes . Transcription of the groESL1 genes was unexpectedly low under photoautotrophic growth conditions . Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth . A second operon (groESL2) was also cloned from R . sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions . A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain. J Bacteriol, 1997 Jan, 179(1), 194 - 201 Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate; Green LS et al.; The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined . The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position . A mutant strain of B . japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker . LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B . japonicum and that it is completely inactivated in the mutant . Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose . The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain . Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B . japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates . Our data support the idea that B . japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions. J Bacteriol, 1997 Jan, 179(1), 128 - 34 Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1; Gomelsky M et al.; The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M . Gomelsky and S . Kaplan, J . Bacteriol . 177:4609-4618, 1995) . To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression . We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression . Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene . Because ppsR gene expression in R . sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level . We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity . It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity . The profile of the isolated suppressor mutations in PpsR is in accord with this proposition . We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity. FEMS Immunol Med Microbiol, 1996 Dec 31, 16(3-4), 193 - 203 Comparison of T cell cytokines in resistant and susceptible mice infected with virulent Brucella abortus strain 2308; Fernandes DM et al.; C57BL/10 and BALB/c mice differ in their abilities to clear infections with the intracellular bacterium Brucella abortus strain 2308 . We have previously reported that in vivo of IL-10 in the susceptible BALB/c mice results in significantly fewer bacteria in their spleens 1 week after infection with 5 x 10(3) colony forming units (CFU) of 2308 . Here we extend those studies and report a similar effect when IL-4 is neutralized . In contrast, in the more resistant C57BL/10 mice infected with 5 x 10(3) CFU, neither neutralization of IL-4 significantly decreased the level of infection nor did it in either BALB/c or C57BL/10 mice infected with a 1000-fold higher dose of strain 2308 . While splenocytes from the later mentioned groups of mice produced IL-10 in response to stimulation with brucella antigen, they also produced higher levels of interferon (IFN)-gamma than those from BALB/c mice with the low challenge dose of 5 x 10(3) CFU . Results of in vivo neutralization of IFN-gamma by monoclonal antibodies (MAb) reported here and elsewhere indicated that IFN-gamma is important for control; thus, we postulate that the higher levels of IFN-gamma override the detrimental effects of Th2 cytokines . In vitro studies also showed that macrophages from the more resistant C57BL/10 mice were less susceptible to the ability of IL-10 to decrease anti-brucella activities than were BALB/c macrophages . CD4+ T cells were principally responsible for the production of IL-10 in BALB/c but not C57BL/10 splenocyte populations . C57BL/10 splenocytes produced more IFN-gamma than those from BALB/c mice in response to stimulation with brucella antigens . These differences between BALB/c and C57BL/10 mice may contribute to the superior capacity of C57BL/10 mice to control infections with B . abortus strain 2308. Genomics, 1996 Dec 15, 38(3), 405 - 17 The mouse region syntenic for human spinal muscular atrophy lies within the Lgn1 critical interval and contains multiple copies of Naip exon 5; Scharf JM et al.; Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13 . This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes . We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background . Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval for Lgn1, a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila . We have generated a mouse YAC contig across the Lgn1/Sma interval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig . In addition, we have localized the two SMA candidate genes, SMN and NAIP, to the Lgn1 critical region, making these two genes candidates for the Lgn1 phenotype . Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy of Naip exon 5 . Identification of the Lgn1 gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region . Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs. Gene, 1996 Dec 12, 183(1-2), 201 - 6 The Bradyrhizobium japonicum coxWXYZ gene cluster encodes a bb3-type ubiquinol oxidase; Surpin MA et al.; Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium, has a complex respiratory electron-transport chain, capable of functioning throughout a wide range of oxygen tensions . It does so by synthesizing a number of terminal oxidases, each appropriate for different environmental conditions . We have previously described the cloning of the large catalytic subunit, coxX, from one of the terminal oxidases from B . japonicum {Surpin, M.A., Moshiri, F., Murphy, A.M . and Maier, R.J . (1994) Genetic evidence for a fourth terminal oxidase from Bradyrhizobium japonicum . Gene 143, 73-77} . In this work, we describe the remaining subunits of this terminal oxidase complex, which is encoded by the coxWXYZ operon . The polypeptide encoded by coxW does not contain any amino acid residues that are known to bind the CuA atom of cytochrome c terminal oxidases, but contains residues thought to be involved in ubiquinol binding . Terminal oxidase cyanide inhibition titration pattern comparisons of the wild type with a coxWXYZ insertion mutant indicated the new oxidase is expressed microaerobically . However analysis of hemes extracted from microaerobically incubated cells revealed the absence of heme O in this strain (from both the wild type and the mutant) of B . japonicum . Therefore, coxWXYZ most likely encodes a microaerobically-expressed bb3-type ubiquinol oxidase. Gene, 1996 Dec 12, 183(1-2), 153 - 7 Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker; Ueki T et al.; Positive-negative KG cassettes were developed in order to create a number of independent deletion mutations on the bacterial chromosome using a single drug marker . These cassettes consist of a kanamycin-resistant (KmR) gene for positive screening and a galactokinase gene (galK) for negative screening . Both genes are in an operon driven by the native KmR promoter and are flanked by identical fragments of yeast chromosomal DNA approximately one kb in size . An internal region of a cloned target gene of a bacterium is replaced with a cassette, which is then transformed into the bacterium . The intact gene on the chromosome is replaced with the mutated gene by homologous recombination . From the KmR cells thus obtained, those cells which lose both KmR and galK genes by homologous recombination between the identical yeast DNA fragments are subsequently screened on plates containing 2-deoxygalactose, a non-metabolizable analogue of galactose . This method was applied to isolate a triple-deletion mutant of pkn3, pkn1, and pkn11 from Myxococcus xanthus. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14225 - 8 Symmetries in bacterial motility; Berg HC; Descriptions are given of three kinds of symmetries encountered in studies of bacterial locomotion, and of the ways in which they are circumvented or broken . A bacterium swims at very low Reynolds number: it cannot propel itself using reciprocal motion (by moving through a sequence of shapes, first forward and then in reverse); cyclic motion is required . A common solution is rotation of a helical filament, either right- or left-handed . The flagellar rotary motor that drives each filament generates the same torque whether spinning clockwise or counterclockwise . This symmetry is broken by coupling to the filament . Finally, bacterial populations, grown in a nutrient medium from an inoculum placed at a single point, usually move outward in symmetric circular rings . Under certain conditions, the cells excrete a chemoattractant, and the rings break up into discrete aggregates that can display remarkable geometric order. Biochemistry, 1996 Dec 3, 35(48), 15428 - 35 Electronic and vibrational structure of the radical cation of P840 in the putative homodimeric reaction center from Chlorobium tepidum as studied by FTIR spectroscopy; Noguchi T et al.; Light-induced FTIR difference spectra of P840 upon its oxidation (P840+/P840) have been measured with the reaction center complex from the green sulfur bacterium Chlorobium tepidum . A broad band centered near 2500 cm-1 was observed in P840+, which is comparable to the band near 2600 cm-1 previously observed in P870+ of purple bacteria and assigned to the electronic transition in the bacteriochlorophyll a (BChla) dimer (Breton et al . (1992) Biochemistry 31, 7503-7510} . The intensity of this electronic band found in P840+ was about the same as that in P870+ . The P840+ spectrum also showed several intensified vibrational modes, which are characteristic of the P870+ spectrum as well . These similar features of the electronic transition and the intensified lines indicate that P840+ is a BChla dimer whose electronic structure is similar to P870+ . Based on the previous theoretical works, the possibility that P840+ has an asymmetric structure as P870+ was suggested . Also, two strong positive bands at 1707 and 1694 cm-1 probably assigned to the keto C9 = O stretching modes of P840+ were observed in the P840+/P840 spectrum . Three different interpretations are possible for the presence of the two C9 = O bands: (i) P840+ is an asymmetric dimer cation . (ii) P840+ has a symmetric structure, and the time constant of positive charge exchange between the two BChla molecules coincides with that of IR spectroscopy (10-13 s) . (iii) The electric field produced by the positive charge on P840+ affects the C9 = O frequency of the neutral BChla in P840+ itself (when the charge exchange time is slower than the time scale of 10-13 s) or of a BChla in the close proximity of P840+ . The negative bands at 1734 and 1684 cm-1 were assigned to the ester C10 = O and the keto C9 = O of neutral P840, respectively, both of which are free from hydrogen bonding . These results and interpretations regarding the structural symmetry and the molecular interactions of P840 and P840+ are discussed in the framework of the "homodimeric" reaction center of green sulfur bacteria. Biochemistry, 1996 Dec 3, 35(48), 15411 - 7 Distant electrostatic interactions modulate the free energy level of QA- in the photosynthetic reaction center; Miksovska J et al.; In the reaction centers from the purple photosynthetic bacterium Rhodobacter capsulatus, we have determined that residue L212Glu, situated near the secondary quinone acceptor QB, modulates the free energy level of the reduced primary quinone molecule QA- at high pH . Even though the distance between L212Glu and QA is 17 A, our results indicate an apparent interaction energy between them of 30 +/- 18 meV . This interaction was measured by quantitating the stoichiometry of partial proton uptake upon formation of QA- as a function of pH in four mutant strains which lack L212Glu, in comparison with the wild type . These strains are the photosynthetically incompetent site-specific mutants L212Glu -->Gln and L212Glu-L213Asp-->Ala-Ala and the photocompetent strains L212Glu-->Ala and L212Ala-L213Ala-M43Asn-->Ala-Ala-Asp . Below pH 7.5, the stoichiometry of proton uptake from all strains is nearly superimposable with that of the wild type . However, at variance with the wild type, reaction centers from all strains that lack L212Glu fail to take up protons above pH 9 . The lack of a change in the free energy level of QA- at high pH in the L212Glu-modified strains is confirmed by the determination of the pH dependence of the rate (kAP) of P+QA- charge recombination in the reaction centers where the native QA is replaced by quinones having low redox potentials . Contrary to the wild-type reaction centers where kAP increases at high pH, almost no pH dependence could be detected in the strains that lack L212Glu . Our data show that the ionization state of L212Glu, either on its own or via interactions with closely associated ionizable groups, is mainly involved in the proton uptake at high pH by reaction centers in the PQA- state . This suggests that the formation of the QA- semiquinone state induces shifts in pKas of residues in the QB proteic environment . This long-distance influence of ionization states is a mechanism which would facilitate electron transfer from QA to QB on the first and second flashes . The functional communication between the two quinone protein pockets may involve the iron-ligand complex which spans the distance between them. Microbiology, 1996 Dec, 142 ( Pt 12), 3363 - 72 Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum; Dahl C; In purple sulphur bacteria of the family Chromatiaceae sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process . In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite:acceptor oxidoreductase has not been resolved . This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5'-phosphosulphate (APS) reductase from Chromatium vinosum strain D (DSM 180'), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin omega cartridge . The mutated genes were transferred to C . vinosum on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination . Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria . Enzymological studies demonstrated the absence of APS reductase from the mutants . Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions . In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite:acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in C . vinosum . Specific inactivation of selected genes as developed for C . vinosum in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria. Plant Mol Biol, 1996 Dec, 32(6), 1103 - 15 Regulation, unique gene organization, and unusual primary structure of carbon fixation genes from a marine phycoerythrin-containing cyanobacterium; Watson GM et al.; Marine phycoerythrin-containing cyanobacteria are major contributors to the overall productivity of the oceans . The present study indicates that the structural genes of the carbon assimilatory system are unusually arranged and possess a unique primary structure compared to previously studied cyanobacteria . Southern blot analyses of Synechococcus sp . strain WH7803 chromosomal DNA digests, using the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit gene from Synechococcus sp . strain PCC6301 as a heterologous probe, revealed the presence of a 6.4 kb HindIII fragment that was detectable at only low stringency . Three complete open reading frames (ORFs) were detected within this fragment . Two of these ORFs potentially encode the Synechococcus sp . strain WH7803 rbcL and rbcS genes . The third ORF, situated immediately upstream from rbcL, potentially encodes a homologue of the ccmK gene from Synechococcus sp . strain PCC7942 . The deduced amino acid sequences of each of these ORFs are more similar to homologues among the beta/gamma purple bacteria than to existing cyanobacterial homologues and phylogenetic analysis of the Rubisco large and small subunit sequences confirmed an unexpected relationship to sequences from among the beta/gamma purple bacteria . This is the first instance in which the possibility has been considered that an operon encoding three genes involved in carbon fixation may have been laterally transferred from a purple bacterium . Analysis of mRNA extracted from cells grown under diel conditions indicated that rbcL, rbcS and ccmK were regulated at the transcriptional level; specifically Rubisco transcripts were highest during the midday period, decreased at later times during the light period and eventually reached a level where they were all but undetectable during the dark period . Primer extension analysis indicated that the ccmK, rbcL and rbcS genes were co-transcribed. Am J Dermatopathol, 1996 Dec, 18(6), 571 - 9 Heterogeneity of Borrelia burgdorferi in the skin; Aberer E et al.; The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory . Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea . In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics . We conclude that the H9724-reactive structures represent various forms of B . burgdorferi rather than staining artifacts . These "atypical" forms of B . burgdorferi may represent in vivo morphologic variants of this bacterium. Genome Res, 1996 Dec, 6(12), 1160 - 9 The marine bacterium Pseudoalteromonas haloplanktis has a complex genome structure composed of two separate genetic units; Lanoil BD et al.; The genome size of Pseudoalteromonas haloplanktis, a ubiquitous and easily cultured marine bacterium, was measured as a step toward estimating the genome complexity of marine bacterioplankton . To determine total genome size, we digested P . haloplanktis DNA with the restriction endonucleases Notl and Sfil, separated the fragments using pulsed-field gel electrophoresis (PFGE), and summed the sizes of the fragments . The P . haloplanktis genome was 3512 +/- 112 kb by Notl digestion and 3468 +/- 54.1 kb by Sfil digestion . P . haloplanktis is also shown to have a complex genome structure, composed of two large replicons of approximately 2700 and 800 kb . Three pieces of evidence support this conclusion: (1) Two separate bands are always seen in PFGE of undigested P . haloplanktis DNA; (2) restriction digests of the larger band are missing a band of approximately 650 kb compared with restriction digests of total genomic DNA; and (3) a 16S rDNA probe hybridized to the larger replicon but not to the smaller . To our knowledge, P . haloplanktis is the first marine bacterium shown to have a complex genome structure. Eur J Biochem, 1996 Dec 1, 242(2), 327 - 31 Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate); Hensgens CM et al.; Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate) . We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies . Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K . Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates . 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D . gigas did not consist of polyphosphates . Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D . gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate). Nucleic Acids Res, 1996 Dec 1, 24(23), 4836 - 7 Improved large scale culture of Methylophilus methylotrophus for 13C/15N labeling and random fractional deuteration of ribonucleotides; Batey RT et al.; Isotopic labeling of RNA with 13C and 15N has become a routine procedure in structural studies by NMR spectroscopy . The methodology in this paper describes the random fractional deuteration of RNA using the obligate methylotropic bacterium, Methylophilus methylotrophus . This bacterium was grown using a non-deuterated carbon source in 52:48 D20/H20 and we have shown that all protons in the ribonucleotides except for the ribose H1 become 52% randomly fractionally deuterated . Improved growth conditions for this organism are also described that yield higher cell densities in liquid culture, which is applicable for all labeling procedures. Mol Microbiol, 1996 Dec, 22(5), 881 - 93 An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor; Nodwell JR et al.; Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium . We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation . Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK-mutant strain extracellularly complement bld261-mutant colonies, and are themselves extracellularly complemented by bldA- and bldH-mutant colonies . The bldK locus, which is located at 5 o'clock on the genetic map and within Asel fragment "N' on the physical map, consists of five adjacent open reading frames . These genes specify homologues of the subunits of the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters . Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer . We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild-type product of the bld261 gene . The BldK-imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action. Aliment Pharmacol Ther, 1996 Dec, 10(6), 985 - 95 Feasibility of analysing {13C}urea breath tests for Helicobacter pylori by gas chromatography-mass spectrometry in the selected ion monitoring mode; Kasho VN et al.; BACKGROUND: The {13C}urea breath test for Helicobacter pylori is nonradioactive, as well as noninvasive, but few clinical laboratories have the expensive isotope ratio mass spectrometer used for analysis . METHODS: To demonstrate the feasibility of analysing {13C}urea breath tests with a gas chromatograph-mass spectrometer routinely used for drug testing, 13CO2 standards for breath tests and breath samples from patients in a multiple-blind study were analysed . The breath samples were also analysed by isotope ratio mass spectrometry, and the diagnoses were compared with biopsy results . RESULTS: The precision of the enrichment measurements by gas chromatography-mass spectrometry was 1.1 parts per thousand, and the calculated differences in enrichment between standard gases equaled the certified values . The sensitivity (94%), specificity (94%), and percentage agreement (94%) for diagnosis of Helicobacter pylori (n = 34) were as high or higher than for analysis of replicate breath samples by isotope ratio mass spectrometry and comparable to the values reported for diagnosis of the bacterium by other currently accepted tests . CONCLUSIONS: The study demonstrates that a gas chromatograph-mass spectrometer can be used to analyse {13C}urea breath tests, thus potentially lowering the cost of the test and increasing the number of laboratories that can perform the test. Biophys J, 1996 Dec, 71(6), 3501 - 10 Torque generated by the bacterial flagellar motor close to stall; Berry RM et al.; In earlier work in which electrorotation was used to apply external torque to tethered cells of the bacterium Escherichia coli, it was found that the torque required to force flagellar motors backward was considerably larger than the torque required to stop them . That is, there appeared to be substantial barrier to backward rotation . Here, we show that in most, possibly all, cases this barrier is an artifact due to angular variation of the torque applied by electrorotation, of the motor torque, or both; the motor torque appears to be independent to speed or to vary linearly with speed up to speeds of tens of Hertz, in either direction . However, motors often break catastrophically when driven backward, so backward rotation is not equivalent to forward rotation . Also, cells can rotate backward while stalled, either in randomly timed jumps of 180 degrees or very slowly and smoothly . When cells rotate slowly and smoothly backward, the motor takes several seconds to recover after electrorotation is stopped, suggesting that some form of reversible damage has occurred . These findings do not affect the interpretation of electrorotation experiments in which motors are driven rapidly forward. FEMS Microbiol Lett, 1996 Dec 1, 145(2), 139 - 46 Genetics of Coxiella burnetii; Thompson HA et al.; Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies . Little is known about their mechanisms for natural genetic exchange . Many genes from the bacterium Coxiella burnetii, an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli . Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C . burnetii. J Bacteriol, 1996 Dec, 178(23), 6736 - 42 Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli; Pianzzola MJ et al.; In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs . This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris . Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype . rbo overexpression suppressed all deleterious effects of SOD deficiency in E . coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction . Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired . The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants . Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants . The physiological role of Rbo in sulfate-reducing bacteria is unknown . In E . coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters . It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage. Infect Immun, 1996 Dec, 64(12), 4940 - 5 Effect of Porphyromonas gingivalis on epithelial cell MMP-9 type IV collagenase production; Fravalo P et al.; Porphyromonas gingivalis is reportedly capable of stimulating the expression of host cell matrix metalloproteinases (MMP), contributing to tissue destruction . However, the impact of this bacterium on specific molecules remains to be determined . In this study, we evaluate the effect of P . gingivalis on regulation of MMP-9 expression in human gingival epithelial cells (HGEC) . Various inocula of P . gingivalis were added to cultures of HGEC . The effects of live bacteria, heat-killed bacteria, and outer membrane extract were analyzed . MMP-9 secretion by HGEC was evaluated by enzyme-linked immunosorbent assay . For inocula smaller than one bacterium per cell, the quantity of MMP-9 secreted by HGEC was increased in comparison to control conditions . For inocula from 2.5 to 250 bacteria per cell, an inhibition of MMP-9 secretion in a dose-response fashion was observed, with a maximum reduction (ranging from 80 to 95% in five experiments) at 50 bacteria per cell . Gelatin zymograms confirmed the decrease in MMP-9 secretion . A band of 83 kDa, corresponding to activated enzyme, was present for inocula of 0.5 to 50 bacteria . Inhibition took place without any alteration of epithelial cell viability . Heat-killed bacteria and outer membrane extract also provoked proenzyme activation but did not inhibit MMP-9 secretion . These results demonstrate a direct effect of P . gingivalis on HGEC, suggesting a specific action on the collagen renewal process at the interface between the epithelium and connective tissue. J Immunol, 1996 Dec 1, 157(11), 5042 - 8 Loss of either CD4+ or CD8+ T cells does not affect the magnitude of protective immunity to an intracellular pathogen, Francisella tularensis strain LVS; Yee D et al.; Normal mice readily survive a sublethal intradermal (i.d.) infection with Francisella tularensis live vaccine strain (LVS), a model intracellular bacterium, and are strongly protected against subsequent lethal challenge . However, athymic nu/nu mice, which lack mature alphabeta TCR+ T lymphocytes, succumb to i.d . infection within 30 days . Here we characterize the alphabeta T cell subpopulations necessary for both resolution of i.d . infection and generation of optimal protective immunity to LVS . BALB/cByJ mice treated with anti-CD4 or anti-CD8 Abs before i.d . infection survived and cleared bacteria, and anti-CD4- or anti-CD8-treated immune mice survived a very strong i.p . challenge of 10,000 LD50s . Among mutant mice with targeted gene disruptions (knockouts), CD4-, beta2-microglobulin-deficient (which are also CD8-), and gammadelta TCR- mice all resolved a large sublethal i.d . infection . All CD4- and beta2-microglobulin-deficient mice readily survived subsequent lethal i.p . challenge of 10,000 LD50s, even in the absence of specific IgG Abs, as did most (86%) gammadelta TCR- mice . In contrast, alphabeta TCR- mice or alphabeta + gammadelta TCR- mice died about 35 days after i.d . infection . Depletion of gammadelta+ T cells from alphabeta TCR- mice had no effect on mean time to death from i.d . LVS infection . Therefore alphabeta TCR+ cells are required for protection, but either CD4+ or CD8+ T cells are individually sufficient to resolve a large sublethal i.d . LVS infection and to protect against a maximal secondary lethal challenge . These results emphasize the remarkable plasticity of the alphabeta T cell response in protective immunity to intracellular bacteria. Gastroenterology, 1996 Dec, 111(6), 1674 - 8 Regression of duodenal mucosa-associated lymphoid tissue lymphoma after eradication of Helicobacter pylori; Nagashima R et al.; It is rare for low-grade lymphoma of mucosa-associated lymphoid tissue (MALT) to affect the duodenum, and no reports have mentioned any relationship between this disease and Helicobacter pylori infection . This case report describes a patient with multiple small erosions and diffuse erythema in the duodenal bulb diagnosed histopathologically as MALT lymphoma . Immunoglobulin (Ig) heavy-chain gene rearrangement was detected, and monotypic plasma cell proliferation (IgG kappa) was shown by immunohistochemistry . The lesion was localized to the duodenal bulb . Antibiotic therapy for H . pylori resulted in resolution of the morphological features of the lymphoma, as confirmed by endoscopic and pathological examination . Moreover, the gene rearrangement could not be detected after eradication of the bacterium . Although additional follow-up is needed, it is suggested that H . pylori eradication therapy may be effective for patients with MALT lymphoma in the duodenum as well as the stomach. Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13774 - 9 Ultrafast exciton relaxation in the B850 antenna complex of Rhodobacter sphaeroides; Nagarajan V et al.; Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacterium Rhodobacter sphaeroides . Absorption anisotropy decays also were measured . We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay . We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate. J Biol Chem, 1996 Nov 22, 271(47), 30158 - 66 Molecular characterization of a novel, developmentally regulated small embryonic chaperone from Caenorhabditis elegans; Linder B et al.; Low molecular weight chaperones inhibit protein aggregation and facilitate refolding of partially denatured polypeptides in cells subjected to physical and chemical stresses . The nematode Caenorhabditis elegans provides a system amenable for investigations on roles for chaperone proteins in normal homeostasis and development . We characterized a C . elegans gene and cDNAs that encode a novel, small embryonic chaperone-like protein (SEC-1) that is composed of 159 amino acids . The central core of SEC-1 (residues 45-126) is approximately 40% identical with a corresponding segment of mammalian Hsp27 and alphaB crystallin . Expression of SEC-1 in Escherichia coli confers thermotolerance on the bacterium . SEC-1 mRNA is evident only in C . elegans oocytes and developing embryos . Translation and accumulation of SEC-1 protein is temporally coupled with a prolonged burst of intense protein synthesis and rapid mitogenesis during early embryogenesis . As the rate of protein synthesis decreases during late embryogenesis, levels of SEC-1 and its cognate mRNA decline precipitously . Induction/deinduction of SEC-1 is precisely regulated by intrinsic developmental factors rather than extrinsic stresses . In vivo injection of C . elegans oocytes with antisense oligonucleotides that complement the 5'-end of SEC-1 mRNA arrests nematode development at an early stage after fertilization . Thus, SEC-1 appears to be adapted to perform essential functions in early embryogenesis. Gene, 1996 Nov 21, 180(1-2), 107 - 12 Cloning, expression and sequence analysis of the SphI restriction-modification system; Guthrie EP et al.; SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC . The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone . However, none of these clones contained the restriction endonuclease (ENase) gene . Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM . The complete sphIR was finally cloned using a two-step process . PCR was used to isolate the 3' end of sphIR from a library . The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E . coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM) . The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems . The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E . coli. Biochemistry, 1996 Nov 19, 35(46), 14634 - 42 Exploration of the relationship between tetrachlorohydroquinone dehalogenase and the glutathione S-transferase superfamily; McCarthy DL et al.; Tetrachlorohydroquinone dehalogenase is found in Sphingomonas chlorophenolica, a soil bacterium that degrades pentachlorophenol, a widely used wood preservative . This enzyme converts tetrachlorohydroquinone (TCHQ) to trichlorohydroquinone (TriCHQ) and TriCHQ to dichlorohydroquinone (DCHQ) (Xun et al . (1992) J . Bacteriol . 174, 8003-8007) . The reducing equivalents for each step are provided by two molecules of glutathione (Xun et al . (1992) Biochem . Biophys . Res . Commun . 182, 361-366) . In addition to the expected TriCHQ and DCHQ products, the enzyme also produces substantial amounts of 2,3,5-trichloro-6-S-glutathionylhydroquinone (GS-TriCHQ) and an unidentified isomer of dichloro-S-glutathionylhydroquinone (GS-DCHQ) . Treatment of the purified enzyme with dithiothreitol dramatically decreases the formation of GS-TriCHQ and GS-DCHQ . Furthermore, enzyme in freshly-prepared crude extracts forms only very small amounts of GS-TriCHQ and GS-DCHQ . We conclude that GS-TriCHQ and GS-DCHQ are produced by enzyme that has undergone some type of oxidative damage and are therefore not physiologically relevant products . The fact that the oxidative damage can be repaired by DTT suggests that a cysteine or methionine residue may be involved . We have created the C13S and C156S mutants of the enzyme . The C13S mutant converts TCHQ to GS-TriCHQ and GS-DCHQ, rather than to DCHQ . Thus, Cys13 is required for the reductive dehalogenation of TCHQ . A mechanism for the reaction which involves Cys13 is proposed. Nucleic Acids Res, 1996 Nov 15, 24(22), 4420 - 49 Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae; Himmelreich R et al.; The entire genome of the bacterium Mycoplasma pneumoniae M129 has been sequenced . It has a size of 816,394 base pairs with an average G+C content of 40.0 mol% . We predict 677 open reading frames (ORFs) and 39 genes coding for various RNA species . Of the predicted ORFs, 75.9% showed significant similarity to genes/proteins of other organisms while only 9.9% did not reveal any significant similarity to gene sequences in databases . This permitted us tentatively to assign a functional classification to a large number of ORFs and to deduce the biochemical and physiological properties of this bacterium . The reduction of the genome size of M . pneumoniae during its reductive evolution from ancestral bacteria can be explained by the loss of complete anabolic (e.g . no amino acid synthesis) and metabolic pathways . Therefore, M . pneumoniae depends in nature on an obligate parasitic lifestyle which requires the provision of exogenous essential metabolites . All the major classes of cellular processes and metabolic pathways are briefly described . For a number of activities/functions present in M . pneumoniae according to experimental evidence, the corresponding genes could not be identified by similarity search . For instance we failed to identify genes/proteins involved in motility, chemotaxis and management of oxidative stress. Structure, 1996 Nov 15, 4(11), 1291 - 301 Small structural changes account for the high thermostability of 1{4Fe-4S} ferredoxin from the hyperthermophilic bacterium Thermotoga maritima; Macedo-Ribeiro S et al.; BACKGROUND: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest . Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities . The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases . Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified . RESULTS: The 1.75 A crystal structure of the extremely thermostable 1{4Fe-4S} ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability . CONCLUSIONS: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure . Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins . These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds . Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone . The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm. J Gastroenterol, 1996 Nov, 31 Suppl 9, 77 - 82 Atopic dermatitis successfully treated by eradication of Helicobacter pylori; Murakami K et al.; A relationship between allergic diseases and Helicobacter pylori infection has recently been noted . We report a case of atopic dermatitis and H . pylori infection in a 14-year-old girl . She had had widespread diffuse skin erythema with erosions and pigmentation since the age of 3 years . Endoscopically, there was chronic antral gastritis with H . pylori infection and histological eosinophilic infiltration . A high titer of H . pylori-specific IgG was present in serum . She was treated with a proton pump inhibitor (lansoprazole 60 mg), an antibiotic (clarithromycin 800 mg), and plaunotol (a mucosal protective agent, 480 mg) for 2 weeks to eliminate the infection . After 10 days of treatment, erythema and itching were more widespread and vesicle formation was seen on the foot . Generalized skin lesions abated a few days later . After eradication of the bacterium by the treatment, eosinophils decreased from 38.8% to 19.0%, and the clinical signs of atopic dermatitis almost disappeared . Serum gastrin level and the pepsinogen I/II ratio were normalized and histological findings of gastric mucosa showed improvement . H . pylori-specific IgE antibody, analyzed by the Western blot method, gradually decreased with the eradication treatment. J Invertebr Pathol, 1996 Nov, 68(3), 286 - 92 Bacterial Infections of Hemocytes Associated with the Maternally Inherited Male-Killing Trait in British Populations of the Two Spot Ladybird, Adalia bipunctata Hurst GDD, Walker LE, Majerus MEN. Adalia bipunctata, the two spot ladybird, carries a vertically transmitted bacterial agent which kills male progeny during embryogenesis . Some matrilines of A . bipunctata give rise to strongly female-biased sex ratios . 16S rDNA sequence analysis revealed a bacterium of the genus Rickettsia associated with this trait, a conclusion which is corroborated here . Using light microscopy, an association between a bacterium located in A . bipunctata hemocyte cytoplasm and matrilines which show the sex ratio trait was found . This element was not found in hemocytes taken from females from normal sex ratio lines, nor in hemocytes taken from males . The association is confirmed by study of the inheritance of the sex ratio trait . Only daughters of sex ratio crosses that bear this cytoplasmic bacterium also show the sex ratio trait, with other daughters being normal with respect to sex ratio . Transmission electron microscopy of hemocytes revealed a walled bacterium, bearing features of members of the genus Rickettsia, free in the cytoplasm of hemocytes taken from infected lines, but not in those taken from uninfected lines. Can J Microbiol, 1996 Nov, 42(11), 1081 - 6 Assimilation of oxalate, acetate, and CO2 by Oxalobacter formigenes; Cornick NA et al.; Oxalobacter formigenes is the only well-documented oxalate-degrading bacterium isolated from the gastrointestinal tract of animals . The production of ATP by Oxalobacter formigenes is centered around oxalate metabolism and oxalate is required for growth . A small amount of acetate (0.5 mM) is also required . Oxalate is decarboxylated to formate plus CO2 in nearly equimolar amounts . Experiments were conducted to determine which potential carbon sources (oxalate, acetate, formate, CO2) were assimilated by Oxalobacter formigenes and which metabolic pathways were operative in carbon assimilation . Measurements of the specific activities of total cell carbon after growth with different 14C-labeled precursors indicated that at least 54% of the total cell carbon was derived from oxalate and at least 7% was derived from acetate . Carbonate was also assimilated, but formate was not a significant source of cell carbon . Labeling patterns in amino acids from cells grown in {14C}oxalate or 14CO3 were different; however, in both cases 14C was widely distributed into most cellular amino acids . Carbon from {14C}acetate was less widely distributed and detected mainly in those amino acids known to be derived from alpha-ketoglutarate, oxaloacetate, and pyruvate . Cell-free extracts contained citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities . The labeling observed in amino acids derived from acetate is in agreement with the function of these enzymes in biosynthesis and indicates that the majority of acetate carbon entered into amino acid biosynthesis via well-known pathways. Plant Physiol, 1996 Nov, 112(3), 1219 - 27 Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli; Nast G et al.; The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate . We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.) . RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers . The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form) . Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium . Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction . A protein of identical size was also detected in isolated potato tuber mitochondria . Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells. FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 141 - 4 Purification and characterization of malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB; van Kuijk BL et al.; Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold . The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa . The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 degrees C . The Ka for oxaloacetate was 50 microM and for NADH 30 microM . The Km values for L-malate and NAD were 4 and 1.1 mM, respectively . Substrate inhibition was found at oxaloacetate concentrations higher than 250 microM . The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms. Appl Environ Microbiol, 1996 Nov, 62(11), 4086 - 94 Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR; Bourne DG et al.; An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin . Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes . The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products . The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond . Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH . The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide . These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg . Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase. J Clin Microbiol, 1996 Nov, 34(11), 2722 - 7 Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R . conorii in circulating endothelial cells: a 6-year follow-up; La Scola B et al.; Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever . We correlated the results of 205 R . conorii blood and skin cultures for 157 patients and the results of 48 detections of R . conorii in circulating endothelial cells (CEC) for 41 patients with relevant serological, clinical, and therapeutic data . R . conorii was cultured from 40% of patients and 29.8% of samples . R . conorii was detected in CEC in 50% of samples, representing 46.2% of patients . When these calculations were limited to the samples from untreated patients prior to their seroconversion to R . conorii, the sensitivity of culture was 59%, whereas it remained at 50% for detection in CEC . We also performed PCRs for the detection of R . conorii on eight shell vial supernatants from positive cultures and on 43 blood samples . Only nonfrozen supernatants from fresh cultures were positive . The methods described in this report are suitable for use in all laboratories . Our findings suggest that for samples to be suitable for culture they must be collected prior to the initiation of an antibiotic regimen, as early as possible in the course of the disease, and be inoculated onto shell vials with minimal delay, if R . conorii is to be successfully isolated . For patients who have been treated or who have a delayed diagnosis, detection of R . conorii in CEC remains helpful. J Mol Biol, 1996 Oct 18, 263(1), 53 - 69 Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 A resolution; Schneider F et al.; The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain . The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide . At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water . The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A . The crystals diffract beyond 1.8 A with synchrotron radiation . The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques . The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections . The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center . The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule . Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion . The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away . It might be involved in electron shuttling from the protein surface to the molybdenum center. J Mol Biol, 1996 Oct 18, 263(1), 40 - 52 Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus; Knablein J et al.; The operon encoding the periplasmic enzymes dimethyl sulfoxide reductase (DMSOR) and trimethylamine N-oxide reductase (TMAOR) from the purple, non-sulfur, photosynthetic bacterium Rhodobacter capsulatus was isolated, cloned and sequenced, and its chromosomal location determined . It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes . Degenerate primers were derived from short peptide sequences generated by automated Edman degradation and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library . Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/ TMAOR operon . The promoter consisted of an A + T-rich region followed by a -35 region, a putative ribosome binding site, and a leader sequence of 13 amino acid residues . The transcription terminator was a G + C-rich dyad sequence capable of forming a hairpin structure, which may act rho-independently . An optimized protein purification of the wild-type enzyme is also described, giving high yields (5 mg protein per liter of culture) and a specific activity of 30 units/mg . The molecular mass was determined by electrospray mass spectrometry to be 85,034 Da; from the deduced amino acid sequence the molecular mass of the apoenzyme was 85,033 Da. Gene, 1996 Oct 17, 176(1-2), 269 - 70 A novel Escherichia coli vector for oxygen-inducible high level expression of foreign genes; Gao B et al.; By utilizing the oxygen-sensitive Escherichia coli Mn-superoxide dismutase (Mn-SOD) promoter, we have developed a vector system that expresses high levels of cloned foreign genes . The promoter for the bacterial Mn-SOD, as well as both 5'-untranslated and transcriptional termination sequences were ligated to a synthetic linker containing two restriction enzyme cloning sites . The vector also contained the gene for beta-lactamase, which confers ampicillin resistance to the host bacterium and provides a selectable marker . After screening and selection, high level of expression was achieved by exposure to the superoxide-generating agent paraquat (methyl viologen) as the inducer . To test the vector, both native and mutated human Mn-SOD cDNAs were cloned and expressed, respectively . To determine the optimal concentration of inducer necessary for maximal expression, recombinant bacteria were exposed to increasing concentrations of paraquat and subsequently assayed for superoxide dismutase (SOD) activity . The highest expression was induced by 20 microM paraquat, and approached 50% of total soluble protein. Gene, 1996 Oct 17, 176(1-2), 97 - 101 Choosing highly specific primers for the polymerase chain reaction using the octomer frequency disparity method: application to Chlamydia trachomatis; Chenal V et al.; The eight-nucleotide sequence (octomer) at the 3' end of PCR primers is important to PCR specificity . We describe a correlation between the specificity of PCR primers used with human DNA and the frequency of the 3' octomer in a human database . We therefore applied a methodology (OFD) based on octomer frequency disparity to identify 16 PCR targets in the chromosome of the intracellular bacterium, Chlamydia trachomatis (Ct) . In addition, the 16 sets of primers were tested with a standard procedure . All the primer pairs were highly specific for Ct and did not lead to non-specific amplification when used with human DNA . This work shows that the choice of specific PCR primers is possible using a method based on the statistical representativeness of octomers in genomes. Oral Microbiol Immunol, 1996 Oct, 11(5), 326 - 31 Laminin binding to a heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans; Alugupalli KR et al.; The interaction of Actinoabacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a 125I-labeled protein-binding assay . The binding of laminin increased by lowering the pH . The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium . Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell-envelope and outer membrane of A . actinomycetemcomitans displayed a 125I-laminin-reactive protein band with a molecular weight of 29 k . The laminin-binding protein was the previously described outer membrane protein A of A . actinomycetemcomitans . It was identified by its heat-modifiable property, detergent-solubility profile and reactivity with outer membrane protein A-specific polyclonal antiserum . At acidic pH, 25I laminin bound to several cell-envelope components of A . actinomycetemcomitans, but at neutral pH, laminin bound only to the heat-modifiable protein . Despite the existence of the laminin-binding protein, cells grown in blood-containing media did not bind laminin . Several mammalian proteins interfered with laminin-bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin-bacterial interaction. Ir J Med Sci, 1996 Oct-Dec, 165 Suppl 5, 1 - 11 Guidelines for the management of Helicobacter pylori-related upper gastrointestinal diseases . Irish Helicobacter Pylori Study Group; Buckley M et al.; The discovery of H . pylori is the most important development in gastroduodenal disease this century . It has completely changed our understanding of conditions that occur in the upper gastrointestinal tract . Eradication of the bacterium will cure peptic ulcer disease, decrease the symptoms of non-ulcer dyspepsia and may prevent gastric cancer . There has been an exponential increase in the amount of research in this area in recent years and, consequently, there is considerable confusion as to how H . pylori-related conditions should be managed . It is hoped that these guidelines will clarify some of these issues. Am J Trop Med Hyg, 1996 Oct, 55(4), 449 - 51 Enterotoxigenic Escherichia coli diarrhea in children less than five years of age in central Java; Orndorff GR et al.; A community-based prospective study was performed from December 1993 through March 31, 1994 in Indonesia in children less than five years of age . Enterotoxigenic Escherichia coli (ETEC) was identified in diarrheic stool by colony hybridization assay, using toxin probes, and this bacterium was isolated from 19% of 340 episodes of diarrhea . Sixty-one percent of ETEC produced heat-labile toxin (LT) only, 325 LT and heat-stable toxin (ST), and 75 ST only . The age-specific incidence rates of diarrhea among children 0-1 and 2-3 years of age were 77% and 61%, respectively, during the study period; ETEC was isolated from 26% of children 0-1 years of age versus 53% for children 2-3 years of age . As many as seven episodes of diarrhea were repeatedly experienced by a single child during the four-month study period; however, only two children had more than one episode of known ETEC-associated diarrheal disease during the period of observation. Eur J Epidemiol, 1996 Oct, 12(5), 509 - 13 High incidence of Coxiella burnetii markers in a rural population in France; Thibon M et al.; Since Coxiella burnetii, the causative agent of Q fever, is often transmitted from goats and sheep to humans through aerosols, we examined the sera from 168 persons involved in goat breeding in the Centre region of France and 40 members of veterinary and medical staff from the same region for the presence of antibodies against C . burnetii . An immunofluorescence assay was used to detect the presence of antibodies of the IgG isotope against epitopes from phase II of C . burnetii, which are the first antibodies to appear in infected people, and from phase I, which reflect more chronic stages of the infection . Our serological survey showed that most of the tested sera were positive for C . burnetii markers, indicating at least an encounter with the bacterium . In the overall population of 208 subjects, 71% of the sera had antibodies against phase II epitopes (titres > or = 1:40) . Among the goat farmers and their immediate families, 78% had antibodies against phase II and 33% against phase I (titres > or = 1:40) . Considering only high titres (> or = 1:320), though, only 37% of the farmers had antibodies against phase II and 15% against phase I . Only 3 out of 12 veterinarians working in the field had high titres of antibodies against phase II and phase I, while none of 28 members of veterinary and medical laboratories had significant levels of antibodies . These results emphasize the need for closer surveillance of populations at risk for Q fever, to prevent the infection by C . burnetii from reaching chronic stages of the disease. Eur J Biochem, 1996 Oct 1, 241(1), 1 - 5 Cloning and expression of the gene for serine-glyoxylate aminotransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2; Hagishita T et al.; The gene encoding serine-glyoxylate aminotransferase, one of key enzymes for the assimilation of one-carbon compounds in methylotrophs, and its flanking regions were isolated from an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2 . Nucleotide sequencing of the recombinant plasmids revealed that the serine-glyoxylate aminotransferase gene encodes a 405-amino-acid protein with a calculated molecular mass of 43880 Da . The amino acid sequence of the enzyme showed identity to the sequences of serine-glyoxylate aminotransferase of Methylobacterium extorquens AM1 (57%), aspartate aminotransferase of Methanobacterium thermoformicicum (31%), human peroxisomal alanine-glyoxylate aminotransferase (27%), and serine-pyruvate aminotransferase of rat liver mitochondria (33%) . The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101 . The recombinant enzyme was purified from transformed E . coli cells and analyzed by immunological and enzymological methods . The overexpressed enzyme was indistinguishable from the wild-type enzyme isolated from H . methylovorum GM2. Pediatr Infect Dis J, 1996 Oct, 15(10), 866 - 71 Nasopharyngeal carriage of pneumococci in Gambian children and in their families; Lloyd-Evans N et al.; BACKGROUND: Nasopharyngeal carriage of pneumococci is prevalent among children in developing countries but little is known about the relationship of nasopharyngeal carriage to invasive disease or about the way in which pneumococci spread within households . OBJECTIVES: To determine the prevalence of nasopharyngeal carriage in healthy and sick Gambian children and to investigate transmission within households . METHODS: Nasopharyngeal swabs were obtained by the per nasal route and cultured for pneumococci on selective media . Pneumococci were serotyped with the use of latex particles coated with type-specific antisera . RESULTS: Pneumococci were isolated from the nasopharynx of 73 (90.1%) of 81 children with invasive pneumococcal disease, 86 (76.1%) of 113 healthy, age-matched control children and 911 (85.1%) of 1071 sick children . Pneumococci belonging to serotypes 1, 14 and 12 were isolated significantly more frequently from cases than from matched controls . In 43 (76.8%) of 56 children with invasive disease, pneumococci isolated from the nasopharynx and from the blood or other sterile site belonged to the same serotype . Pneumococci of the same serotype as the bacterium responsible for invasive disease in a child were obtained from 72 (8.5%) of 843 family members, most frequently from young siblings of the case patients . CONCLUSION: Nasopharyngeal carriage of pneumococci is more prevalent among young Gambian children than among adults and invasive infections are probably acquired more frequently from siblings than from parents . However, further studies are needed to confirm this hypothesis with more discriminating markers than polysaccharide serotyping. Hum Pathol, 1996 Oct, 27(10), 1085 - 8 Analysis of chronic endometritis for Chlamydia trachomatis by polymerase chain reaction; Stern RA et al.; Chronic endometritis is characterized histologically by plasma cells infiltrating endometrial stroma . Although it has been speculated that many instances of chronic endometritis are infectious, the origin of most cases is not apparent by routine histopathologic evaluation . Chlamydia trachomatis, an obligate intracellular bacterium, is known to cause chronic endometritis in the setting of pelvic inflammatory disease . The authors analyzed 43 specimens of histopathologically diagnosed chronic endometritis from 38 patients for C trachomatis by PCR using primers for the single-copy major outer membrane protein (MOMP) gene . C trachomatis was detected in only one such case in which dense plasma cell infiltrates were present and concurrent C trachomatis infection of the cervix was documented . Using serially diluted DNA from formalin-fixed, paraffin-embedded McCoy cells, the sensitivity of this method was shown to be equivalent to a single infected cell in a paraffin section . In conjunction with the results of other studies, these data indicate a limited role, if any, of C trachomatis in the origin of mild or moderate chronic endometritis. Microbiology, 1996 Oct, 142 ( Pt 10), 2959 - 67 The urea cycle of Helicobacter pylori; Mendz GL et al.; The presence and activities of the enzymes of the urea cycle in the bacterium Helicobacter pylori were investigated employing one- and two-dimensional NMR spectroscopy and radioactive tracer analysis . Cell suspensions, lysates and membrane preparations generated L-ornithine and ammonium at high rates in incubations with L-arginine, indicating the presence of arginase activity . Anabolic ornithine transcarbamoylase (OTCase) activity was identified by the formation of heat-stable products in incubations of cell-free extracts with ornithine and radiolabelled carbamoyl phosphate . The heat-labile product that resulted from incubations of cell-free extracts with citrulline radiolabelled in the guanidino moiety revealed the presence of catabolic OTCase activity . Argininosuccinate formation and catalysis indicated the presence of argininosuccinate synthetase and argininosuccinase activities . The findings suggested that H . pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells. Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S145 - 9 Pertussis antigens that abrogate bacterial adherence and elicit immunity; Brennan MJ et al.; Infectious disease processes follow the initial steps of adherence of the organism to host tissues and subsequent colonization of the target tissues that can occur through specific adhesion-receptor systems . Bordetella pertussis, the human pathogen that causes whooping cough, has evolved a genetically controlled system whereby adhesins are expressed when they enter the human host . Two adhesins, filamentous hemagglutinin (FHA) and pertactin, mediate the adherence of the bacterium to eukaryotic cells through varied attachment mechanisms, including lectin-like binding sites that interact with sulfated sugars on cell surface glycoconjugates and the ARG-GLY-ASP binding sequence, which recognizes a family of integrins found on the cell surface . The differential expression of relevant receptors by various eukaryotic cells likely plays a role in the pathogenesis and immune response to the bacterium by the host, directing the organism to specific cell types and to specific tissue sites . Substantial evidence exists that the B . pertussis adhesins, FHA and pertactin, elicit immune responses that are protective in animal models for the disease, including serum antibody production and local immune responses in the respiratory tract following nasal administration of encapsulated antigens . Both of these adhesins are components of new acellular pertussis vaccines that have proven safe and highly effective for prevention of serious disease in infants. Int J Syst Bacteriol, 1996 Oct, 46(4), 1183 - 4 Ureaplasma gallorale, an isolate from chickens, is most closely related to the human isolate, U . urealyticum; Stemke GW et al.; Ureaplasma gallorale is a urease-containing mycoplasma (a member of the Mollicutes) which is pathogenic for chickens, from which it was originally isolated . We amplified the 16S rRNA gene of this bacterium and then cloned and sequenced the amplicon . A phylogenetic analysis based on an alignment of the 16S rRNA sequences of U . gallorale and several other Ureaplasma species revealed that U . gallorale is more closely related to Ureaplasma urealyticum than to other Ureaplasma species. Int J Syst Bacteriol, 1996 Oct, 46(4), 960 - 6 Phylogeny of Prosthecobacter, the fusiform caulobacters: members of a recently discovered division of the bacteria; Hedlund BP et al.; Prosthecobacter fusiformis is morphologically similar to caulobacters; however, it lacks a dimorphic life cycle . To determine the relatedness of the genus Prosthecobacter to dimorphic caulobacters and other prosthecate members of the alpha subgroup of the Proteobacteria (alpha-Proteobacteria), we isolated and sequenced 16S rRNA genes from four Prosthecobacter strains . Surprisingly, the results of phylogenetic analyses placed the fusiform caulobacters in a deeply rooted division of the Bacteria that was most closely affiliated with the Planctomyces-Chlamydia group and only distantly related to the alpha-Proteobacteria . The genus Prosthecobacter shares a common lineage in this division with Verrucomicrobium spinosum, a polyprosthecate, heterotrophic bacterium . Consistent with this phylogenetic placement, menaquinones were isolated from Prosthecobacter strains and menaquinones have been isolated from Verrucomicrobium strains and planctomycetes but not from members of the alpha-Proteobacteria . Thus, the genus Prosthecobacter is a second genus in the recently described order Verrucomicrobiales . Members of the genus Prosthecobacter are susceptible to beta-lactam antibiotics and contain mesodiaminopimelic acid, indicating that they, unlike members of the Planctomycetales or Chlamydiales, have peptidoglycan cell walls . This major phenotypic difference, together with the phylogenetic independence of the verrucomicrobia, indicates that these bacteria and the sources of related 16S ribosomal DNAs obtained from soils, freshwater, and the marine pelagic environment represent an unrecognized division of the Bacteria. Am J Gastroenterol, 1996 Oct, 91(10), 2135 - 8 Helicobacter pylori infection and blood group antigens: lack of clinical association; Umlauft F et al.; OBJECTIVES: Blood group antigens traditionally have been associated with a risk of developing peptic ulcer and gastric cancer . Helicobacter pylori is a bacterium associated with chronic active gastritis and ulcer disease, and its attachment to gastric mucosa was recently shown in vitro to be mediated by blood group Lewisb and H antigens . This study was designed to test the clinical relevance of this laboratory observation in patients undergoing endoscopy and gastric biopsy . METHODS: Blood group phenotypes and gastric biopsies for H . pylori and histology were determined and correlated in 384 patients undergoing upper endoscopy . Blood from healthy blood donors was tested for the same blood group antigens and used as a control group . RESULTS: The distribution of blood groups ABO, Lewis, Rhesus, and MN was similar among the patients undergoing endoscopy and a control group of 2369 healthy blood donors from the same geographic area . There was no correlation between H . pylori infection or the H . pylori-associated diseases, peptic ulcer or chronic active gastritis, with any blood group phenotype, including Lewisb, blood group O, or both . CONCLUSION: No in vivo correlation between H . pylori infection or disease and Lewisb or H antigen could be demonstrated . Moreover, patients with H . pylori infection and disease have a distribution of blood group antigens similar to a control population. Appl Environ Microbiol, 1996 Oct, 62(10), 3885 - 6 Enrichment and isolation of a nitropropanol-metabolizing bacterium from the rumen; Anderson RC et al.; A bacterium capable of metabolizing nitropropanol, nitropropionate, and nitrate has been isolated from a mixed ruminal population enriched for enhanced rates of nitropropanol metabolism . The numbers of nitropropanol-metabolizing bacteria in mixed populations increased > 10,000-fold during enrichment; the rates of nitropropanol metabolism increased 8-fold . Hydrogen and phytone were important nutrients for nitropropanol metabolism. J Bacteriol, 1996 Oct, 178(20), 5938 - 45 Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489; Liu SY et al.; An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined . Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases . The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa . Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate . The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined . Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa . The biochemical properties of the cloned enzyme were the same as those of the native enzyme . Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI . In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar . Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T . saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively) . The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively) . The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T . saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference. J Bacteriol, 1996 Oct, 178(20), 5877 - 83 Differential carotenoid composition of the B875 and B800-850 photosynthetic antenna complexes in Rhodobacter sphaeroides 2.4.1: involvement of spheroidene and spheroidenone in adaptation to changes in light intensity and oxygen availability; Yeliseev AA et al.; Rhodobacter sphaeroides 2.4.1 is a member of the nonsulfur purple facultative photosynthetic proteobacteria, capable of growth under a variety of cultivation conditions . In addition to the structural polypeptides and bacteriochlorophyll, the two major antenna complexes, B875 and B800-850, contain a variety of carotenoids which are an important structural and functional component of the membrane-bound photosynthetic complexes of this bacterium . Two major carotenoids, spheroidene and its keto derivative, spheroidenone, are differentially synthesized by R . sphaeroides, depending on the growth conditions . Spheroidene prevails during growth under anaerobic conditions and low light intensities, whereas spheroidenone is predominant in semiaerobically grown cells or during anaerobic growth at high light intensities . In this study, we demonstrate that in wild-type cells, spheroidene is predominantly associated with the B800-850 photosynthetic antenna complex and spheroidenone is more abundant in the B875 complex . Exploiting mutants defective in the biosynthesis of either the B875 or B800-850 light-harvesting complex, we demonstrate an association between the formation of either the B875 or B800-850 complex, on the one hand, and the accumulation of spheroidenone or spheroidene, on the other . The possible involvement of the conversion of spheroidene to spheroidenone as a significant control mechanism involved in the adaptation of R . sphaeroides to changes in light intensity and oxygen tension is discussed. J Bacteriol, 1996 Oct, 178(19), 5748 - 54 Global changes in protein composition of N2-fixing-Azoarcus sp . strain BH72 upon diazosome formation; Karg T et al.; The strictly respiratory, diazotrophic bacterium Azoarcus sp . strain BH72 fixes nitrogen under microaerobic conditions . In empirically optimized batch cultures at nanomolar O2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N2 fixation are formed . Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp . strain BH72 with an ascomycete originating from the same host plant, Kallar grass . To initiate studies on function of diazosomes and regulation of their formation, diazosome-containing bacteria were compared with respect to composition or total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions . In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions . N-terminal amino acid sequence analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P(II), an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism . According to its electrophoretic mobility, it might be uridylylated in diazosome-free cells but not in diazosome-containing cells, or it may represent a second, not identical P(II) protein . Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp . strain BH72 in Southern hybridizations. J Bacteriol, 1996 Oct, 178(19), 5579 - 85 Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima; Lee PJ et al.; As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima . The genes have been designated HpkA and drrA, respectively . The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins . A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus . The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins . Recombinant T . maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli . Partial purification of T . maritima proteins was achieved by heat denaturation of E . coli host proteins . In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP . Thus, the N-terminal hydrophobic region is not required for kinase activity . Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins . The phosphorylation reactions were strongly temperature dependent . The results indicate that the recombinant T . maritima two-component proteins overexpressed in E . coli are stable as well as enzymatically active at elevated temperatures. Mol Gen Genet, 1996 Sep 25, 252(4), 379 - 85 Development of gene transfer methods for Rubrivivax gelatinosus S1: construction, characterization and complementation of a puf operon deletion strain; Ouchane S et al.; Gene transfer systems were developed in Rubrivivax (Rx.) gelatinosus S1 . First, a system for conjugative transfer of mobilizable plasmids from Escherichia coli to Rx . gelatinosus S1 was established . Secondly, optimal conditions for the transformation of Rx . gelatinosus S1 by electroporation were determined . A delta puf strain was constructed . Complementation with the puf operon from a wild-type strain cloned in a replicative plasmid restored photosynthetic growth . Two insertion strains were also selected . All the strains constructed were green, due to a change in carotenoid content . Characterization of these strains provides genetic evidence for a "superoperon" organization in this bacterium. Carbohydr Res, 1996 Sep 2, 290(2), 175 - 81 Structure determination of a novel uronic acid residue isolated from the exopolysaccharide produced by a bacterium originating from deep sea hydrothermal vents; Dubreucq G et al.; The exopolysaccharide produced by the bacterium Alteromonas sp . strain 1644 originating from deep sea hydrothermal vents was shown to contain a novel glucuronic acid derivatives: 3-O-{(R)-1-carboxyethyl}-D-glucuronic acid . The structure of this compound was established on the basis of mass spectrometric data, methylation analysis, preparation of derivatives, and chemical synthesis of references compounds. Riv Eur Sci Med Farmacol, 1996 Sep-Dec, 18(5-6), 183 - 6 The mouth-stomach crossing of Helicobacter pylori; Pustorino R et al.; In order to confirm the mouth-stomach crossing of Helicobacter pylori (H . pylori) we performed a study to isolate the bacterium both from dental plaque and gastric mucosa of 83 subjects . Out of the 83 subjects, 62 were affected by gastroduodenal pathologies usually related to H . pylori whereas, the other 21 subjects were healthy . Each patient underwent one plaque sampling and three gastric samplings . The samples were cultured and an analytical technique, isoelectrofocusing (IEF), was used to show if dental and gastric strains isolated from the same subject had the same proteic profile . The H . pylori was isolated from the dental plaque of five subjects . Out of these, four subjects were suffering from gastric pathologies and the H . pylori was besides isolated from their gastric mucosa . The dental and gastric strains isolated from the same patient showed the same proteic profile . The hypothesis of H . pylori crossing from mouth to stomach seems to be confirmed. Appl Microbiol Biotechnol, 1996 Sep, 46(2), 132 - 7 A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae; Meijer A et al.; We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads . Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption . The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120 x 10(6) and 225 x 10(6) bacteria/10(6) HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively) . However, up to 64% savings in labour time and 27% saving in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method . The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24 - 1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection of PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35 degrees C or 37 degrees C) . The yield of microcarrier cultures was the same at an incubation temperature of 35 degrees C or 37 degrees C in contrast to an increased production at 35 degrees C in the microtitre-plate culture method using centrifugation-assisted infection . In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically. Minerva Gastroenterol Dietol, 1996 Sep, 42(3), 145 - 51 {Helicobacter pylori, chronic gastritis and precancerous gastric manifestations}; Testino G et al.; Helicobacter Pylori (HP) has been noticed in about 80% of cases of superficial chronic gastritis . In about 75% of HP positive biopsies degenerative lesions of superficial gastric epithelium, represented by irregularities of the superficial profile, micropapillar-transformation and erosions have been observed . Furthermore, it is possible the observation, in areas of high bacterial colonization, of vacuoles which are the result of a direct action of HP strains producing vacuolating cythotoxin . In correspondence with the glandular necks active inflammation is present in about 90% of cases . Hp is responsible, by means of direct cytotoxicity and inflammatory cell aggression, of most superficial gastritis, it may help the evolution towards atrophic gastritis and may superimpose on an already noted gastritis situation, promoting their inflammatory exacerbation . The presence of HP infection decreases with the increase histological damage: superficial gastritis (SG) 89%, atrophic gastritis (AG) 58%, intestinal metaplasia (IM) 51% and dysplasia (D) 47% . The founding of the bacterium in conditions such as AG or in surrounding zones IM or D is the demonstration of a possible role of the bacterium in the development of the phases subsequent to AG or of phenomena like IM and D, and not confined to the already verified passage SG/AG . The continuous rearrangement in correspondence with the glandular necks induced by active inflammation HP induced and the subsequent hyperproliferation may help mytotic error, giving rise to metaplastic or dysplastic cellular lines . Therefore, HP in the progression towards IM and D should act as promoter by means of the increase of cellular kinetics. Prim Care, 1996 Sep, 23(3), 443 - 54 The infectious etiology of peptic ulcer disease . Diagnosis and implications for therapy; Brooks MJ et al.; During the past decade, peptic ulcer disease has become recognized as multifactorial in etiology, with a major component thought to be infection of the gastric mucosa with a spiral-shaped bacterium known as Helicobacter pylori . This organism has been found to cause most cases of chronic gastritis and is clearly pathogenic in most cases of duodenal and gastric ulceration . Biologic characteristics, epidemiology, and methods of detection (invasive and noninvasive) of H . pylori are discussed from a clinical perspective . Finally, eradication of H . pylori infection is difficult because of bacterial resistance and patient noncompliance. Biochem Mol Biol Int, 1996 Sep, 40(1), 209 - 15 Susceptibility of growth factors to degradation by Helicobacter pylori protease: effect of ebrotidine and sucralfate; Slomiany BL et al.; In this study, we investigated the susceptibility of the growth factors (EGF, bFGF, TGF beta and PDGF) to degradation by the protease elaborated by H . pylori and assessed the eff