Microorganisms

Soc Sci Med, 1996 Jan, 42(1), 141 - 52
Prostitutes, prostitution and STD/HIV transmission in mainland China; Gil VE et al.; China's opening to the world has enabled massive social and economic transformations and the liberalization of many policies, but also the rise of coincident social problems and diseases . A revival of wide-scale female prostitution since the 1980s has now accelerated to a nationwide dilemma . Prostitutes have long been considered to be reservoirs, if not 'vectors' for the transmission of sexual diseases . A well established STD epidemic in the last decade, plus the presence of growing HIV infections in China now, underscore the need to evaluate the prostitute's role in STD and HIV propagation . This report examines unobtrusive data on female prostitutes in the People's Republic of China through an analysis of prison records from eight sexually segregated prisons (six in Sichuan Province and two in Guizhou Province), two female re-education institutions, and arrest records for convicted prostitutes from four counties in Sichuan Province and Chengdu City (also in Sichuan) . Collectively, these data represent 2057 female prostitution cases, and span the years 1988-1990 . Demographics are examined to enable a profile of the prostitute as based on data reviewed, and this is contrasted to the stereotype of the prostitute as described in government propaganda against prostitution . STD prevalence rates in the samples are examined and contrasted to two other studies on STDs in nonprostitution populations made available to the authors . Prostitute arrest records reveal a majority had active STD infection{s} at the time of their apprehension, with gonorrhea being the most common bacterium; in many instances, prostitutes also had a history of other sexually transmitted diseases . Thus, data examined support the notion of prostitution as an agent in STD transmission . Presence of HIV infection in prostitutes could not be corroborated through sampled records, nor could prostitution itself be confirmed as an agent in HIV transmission at present . However, given the type of clients serviced by prostitutes in China, a prostitute's own risk of HIV infection is discussed . Control measures by the Chinese government to curb prostitution are examined at both national and provincial levels . Questions are raised as to the effectiveness of present tactics as adjunctive strategies in reducing STD infection and HIV risk in the prostitution population.

Crit Rev Microbiol, 1996, 22(1), 27 - 56
Ecology, metabolism, and genetics of ruminal selenomonads; Ricke SC et al.; Selenomonas ruminantium is one of the more prominent and functionally diverse bacteria present in the rumen and can survive under a wide range of nutritional fluctuations . Selenomonas is not a degrader of complex polysaccharides associated with dietary plant cell wall components, but is important in the utilization of soluble carbohydrates released from initial hydrolysis of these polymers by other ruminal bacteria . Selenomonads have multiple carbon flow routes for carbohydrate catabolism and ATP generation, and subspecies differ in their ability to use lactate . Some soluble carbohydrates (glucose, sucrose) appear to be transported via the phosphoenolpyruvate phosphotransferase system, while arabinose and xylose are transported by proton symport . High cell yields and the presence of electron transport components in Selenomonas strains has been documented repeatedly and this may partially account for the energy partitioning observed between energy consumed for growth and maintenance functions . Most strains can utilize ammonia, protein, and/or amino acids as a nitrogen source . Some strains can hydrolyze urea and/or reduce nitrate and use the ammonia for the biosynthesis of amino acids . Experimental evidence suggests that ammonia assimilatory enzymes in some strains may possess unique properties with respect to other presumably similar bacteria . Little is known about the genetics of ruminal selenomonads . Plasmid DNA has been isolated from some strains, but it is unknown what physiological functions may be encoded on these extrachromosomal elements . Due to the predominance of S . ruminantium in the rumen, it is an ideal candidate for genetic manipulation . Once the genetics of this bacterium are better understood, it may be possible to amplify its role in the rumen.

Scand J Gastroenterol Suppl, 1996, 215, 48 - 51
Helicobacter pylori and gastric cancer; Forman D; The International Agency for Research on Cancer, sponsored by the World Health Organization, has recently categorized Helicobacter pylori infection as a class I carcinogen, based on evidence that this infection increases the risk of gastric cancer . The classification was intentionally qualitative in nature and not associated with any public health recommendations . In addition, no specific causal mechanism was proposed to explain the relationship between H . pylori and gastric cancer . In this paper, the magnitude of the risk, implications of the relationship for the prevention of gastric cancer and nature of the causal mechanisms are considered . Relative risk of gastric cancer may be substantial; even with conservative assumptions, the proportion of new cases of gastric cancer worldwide attributable to H . pylori infection is approximately one third of a million annually . This figure is likely to increase with changes in the age structure of the population, and the eradication of H . pylori as a means of prevention of gastric cancer should be considered . A strategy of screening populations in middle age and treating those infected could be relatively inexpensive to administer, but the efficacy is totally unknown and requires evaluation in a randomized controlled trial . Studies designed to address this issue in the general population would need to be large and long-term if gastric cancer is used as an end-point . With respect to carcinogenic mechanisms, a number of constitutive properties of H . pylori may be of relevance to cancer without being specifically carcinogenic . Thus ammonia, which is produced in abundance as a result of urease activity, may promote cell division . Other relevant properties result from the immune response of the host to the bacterium . For example, the excessive production of reactive oxygen metabolites can lead to extensive DNA damage and molecular mutations.

Scand J Gastroenterol Suppl, 1996, 215, 32 - 7
Helicobacter pylori and disturbance of gastric function associated with duodenal ulcer disease and gastric cancer; McColl KE et al.; Helicobacter pylori is now recognized as the major acquired factor in the pathogenesis of duodenal ulcer disease (DU) . There is also an association between H . pylori infection and the subsequent development of gastric cancer . The mechanisms by which such infection predisposes the host to these diseases are incompletely understood, but disorders induced by the bacterium in gastric function play a pivotal role . In most patients, H . pylori infection stimulates acid secretion, leading to a predisposition to DU development . However, in some patients, the infection is associated with a significant decrease in acid secretion, a predisposition to gastric cancer . These divergent effects of H . pylori on gastric acid secretion explain the early conflicting reports on changes in acid secretion associated with the infection . The reason why H . pylori infection produces divergent effects on gastric acid secretion is unclear, but may be related to differences in bacterial strains or genetic, dietary or other environmental factors.

Farmaco, 1996 Jan, 51(1), 49 - 52
3-Diazopyrroles . Part 6 . Mutagenic activity of 3-diazopyrroles in Streptomyces coelicolor A3(2) during various phases of growth; Cirrincione G et al.; 3-Diazopyrroles, a class of compounds particularly interesting from a chemical and biological point of view, were assayed for their ability to induce gene mutations employing back mutation (his+ reversion) test in the philamentous bacterium Streptomyces coelicolor at various time during life cycle . Our results suggest that in evaluating the mutagenicity and toxicity of chemicals in Streptomyces system it is important to consider factors such as growth phase . Furthermore in this series of diazopyrroles a relationship between toxicity, mutagenicity and chemical structure was found . The observed mutagenic activity can be the molecular basis for the appearance of antitumor activity.

J Biomol NMR, 1996 Jan, 7(1), 35 - 47
1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis; Donaire A et al.; The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H NMR spectroscopy . TOCSY and NOESY experiments in H2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H2O and D2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected . It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure . Secondary structural elements identified in Tl Fd are a three-stranded antiparallel beta-strand involving the termini of the protein, a double beta-strand (also antiparallel), two alpha-helices and four turns . The existence of a disulfide bridge between the nonligated cysteines is also proposed . Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the 'diamagnetic' protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas {Kissinger et al . (1991) J . Mol . Biol., 219, 693-723} . The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins . Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.

Ann Ital Chir, 1996 Jan-Feb, 67(1), 27 - 33; discussion 34
{B-cell lymphoma (MALToma) of the stomach and Helicobacter pylori infection}; Rodolico A et al.; The authors, after illustrating physiopathologic aspects of gastric MALTomas, examine the H.P . infections and the possible relations between this kind of bacterium and gastric MALTomas . They hypothesize that H.P . infection represents an important predisposition to gastric lymphoma, due to anatomopathological modifications over gastric mucosa . They conclude that a decreasing of gastric lymphoma could be correlatable an opportune eradication of H.P . despite of at moment it's impossible hypothesize a marked decreasing of lymphoma by an eradication of H.P.

Lett Appl Microbiol, 1996 Jan, 22(1), 52 - 6
Genetic transfer of lactate-utilizing ability in the rumen bacterium Selenomonas ruminantium; Gilmour M et al.; Matings between the lactate-utilizing, tetracycline-sensitive Selenomonas ruminantium strains 5521C1 and 5934e and the lactate-non-utilizing, tetracycline-resistant strain FB322 resulted in putative recombinant strains capable of growth on lactate . Analysis of total protein by SDS-PAGE and chromosomal DNA by hybridization, indicated that the recombinants were derived from strain FB322 . DNA hybridization produced no evidence that plasmid transfer occurred, leaving chromosomal DNA transfer as the most likely mechanism for the altered phenotype . Analysis of strains 5934e, FB322 and the resulting recombinant TC3 indicated that all three strains contained D-nLDH and L-nLDH activities . In addition strains 5934e and TC3 possessed D-iLDH activity when grown on DL-lactate . The ability of strain FB322 to grow on pyruvate but not lactate suggested that the lactate-utilizing recombinant had acquired the ability to synthesize D-iLDH.

Int J Syst Bacteriol, 1996 Jan, 46(1), 94 - 7
Phylogenetic relationships of the filamentous sulfur bacterium Thiothrix ramosa based on 16S rRNA sequence analysis; Polz MF et al.; The phylogeny of Thiothrix ramosa based on 16S rRNA sequences was determined . This species is the first species in this genus that has been shown to be capable of autotrophic growth with reduced sulfur compounds as sole energy sources . T . ramosa forms a monophyletic clade with Thiothrix nivea, as determined by distance, parsimony, and maximum-likelihood methods . Both of these species clearly belong to the gamma subdivision of the Proteobacteria, where they are loosely associated with other sulfur-oxidizing chemoautotrophic organisms.

Appl Environ Microbiol, 1996 Jan, 62(1), 67 - 73
Description of a new polymer-secreting bacterium from a deep-sea hydrothermal vent, Alteromonas macleodii subsp . fijiensis, and preliminary characterization of the polymer; Raguenes G et al.; A deep-sea, aerobic, mesophilic, heterotrophic bacterium was isolated from fluid collected near an active hydrothermal vent . On the basis of phenotypic and phylogenetic analyses and DNA-DNA relatedness, strain ST716 could be assigned to the species Alteromonas macleodii as a new subspecies . This bacterium secreted an unusual high-molecular-weight polysaccharide in the presence of glucose in batch cultures . The viscosity of this exopolysaccharide is of the same order of magnitude as that of xanthan, another bacterial polysaccharide of industrial interest . This polysaccharide, produced during the stationary phase, contained glucose, mannose, pyruvated mannose, and galactose along with galacturonic acid and glucuronic acid.

J Bacteriol, 1996 Jan, 178(2), 484 - 9
Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima; Swanson RV et al.; An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C . W . Kim, P . Markiewicz, J . J . Lee, C . F . Schierle, and J . H . Miller, J . Mol . Biol . 231: 960-981, 1993) . Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T . maritima lambda library by using PCR . Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced . The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY . In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function . Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors . Upon induction, both proteins were synthesized at high levels . T . maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E . coli protein by heat treatment at 80 degrees C for 10 min . CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with {32P}acetyl phosphate . In E . coli, CheW mediates the physical coupling of the receptors to the kinase CheA . The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.

Gastroenterology, 1996 Jan, 110(1), 21 - 9
Extract of Helicobacter pylori induces neutrophils to injure endothelial cells and contains antielastase activity; Takemura T et al.; BACKGROUND & AIMS: Previous studies indicate that a water extract of Helicobacter pylori promotes leukocyte adhesion and emigration as well as endothelial barrier disruption (increased vascular protein leakage) in rat mesenteric venules . The aims of this study were to assess whether H . pylori extract-activated neutrophils disrupt endothelial cell monolayers and to identify the mechanisms involved in this process . METHODS: Human neutrophils were incubated with monolayers of human umbilical vein endothelial cells (HUVECs) in the presence or absence of H . pylori extract . RESULTS: H . pylori extract-activated human neutrophils produced endothelial cell detachment from HUVEC monolayers, the severity of which was dependent on the duration of exposure . Endothelial cell detachment was prevented by a monoclonal antibody directed against CD11/CD18 on neutrophils or a monoclonal antibody against intercellular adhesion molecule 1 on endothelial cells . HUVEC monolayer disruption was also prevented by superoxide dismutase, catalase, and a monoclonal antibody against elastase . Further studies indicated that H . pylori extract was capable of inhibiting human neutrophil elastase . The antielastase activity was not diminished by oxidants . CONCLUSIONS: These studies indicate that H . pylori extract-activated human neutrophils can disrupt HUVEC monolayers only when human neutrophils are allowed to adhere to HUVECs and may provide an explanation for the H . pylori extract-induced, neutrophil-dependent vascular protein leakage observed in vivo . The possibility that H . pylori releases antiproteases may explain, in part, why this bacterium is so virulent.

Structure, 1995 Dec 15, 3(12), 1295 - 306
2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability; Hennig M et al.; BACKGROUND: Recent efforts to understand the basis of protein stability have focused attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms . Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain . Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains . In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90 degrees C . RESULTS: The 2.0 A crystal structure of sIGPS was determined and compared with the known 2.0 A structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS) . sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity . Both are single-domain (beta/alpha)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first beta strand . The thermostable sIGPS has many more salt bridges than eIGPS . Several salt bridges crosslink adjacent alpha helices or participate in triple or quadruple salt-bridge clusters . The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS . CONCLUSIONS: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions . These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Biochemistry, 1995 Dec 12, 34(49), 15997 - 6003
Ion pair formation between basic residues at 144 of the Cyt b polypeptide and the ubiquinones at the Qo site of the Cyt bc1 complex; Ding H et al.; Loci of spontaneous Qo site inhibitor resistant mutants in the cyt bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus are M140, F144, G152, G158, and T163 of the cyt b polypeptide . In this report, we have studied the effects of arginine (R) substitution at these positions with a view to test for specific interactions with the {2Fe-2S} cluster, cyt bL with Qo site ubiquinone (Q), or hydroquinone (QH2) . All the arginine mutants displayed severely or completely impeded photosynthetic growth resulting from dysfunctional cyt bc1 complexes . The source of dysfunction in G158R and T163R was identified by a > 1000-fold decrease in the Qo site affinity for QH2 and Q, sufficient to empty the site in the presence of the 30 mM ubiquinone pool of the chromatophore membrane; they appear similar to the class of mutants described in the preceding paper {Ding, H., Moser, C . C., Robertson, D . E., Tokito, M., Daldal, F., & Dutton, P . L (1995) Biochemistry 34, 15979-15996} . The source(s) of dysfunction of M140R and G152R is not so apparent since they possess Qo sites with normal QH2/Q affinity; they appear to be members to the class of mutants identified and characterized in the following paper {Saribas, S., Ding, H., Dutton, P . L., & Daldal, F . (1995) Biochemistry 34, 16004-16012} . The present paper focuses on the unique affects of F144R . Redox potential and EPR spectral properties of the Qo site of F144R showed that arginine forms an ion pair with the head group of an anionic ubiquinone, tentatively suggested to be a ubihydroquinone anion (QH-) in the Qos domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11829 - 33
Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation; Peterson SN et al.; We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism . One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced . At least four others were partially sequenced . The complete element is a composite of six regions . Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon . The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family . Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene . However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence . Thus, these repetitive elements do not appear to be directly expressible protein coding sequences . The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M . genitalium strains . Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates . Therefore, we propose that the repetitive elements of M . genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.

FEMS Microbiol Rev, 1995 Dec, 17(4), 401 - 13
Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans; Yamanaka T et al.; The enzymes and redox proteins, which participate in the oxidation of ferrous ion by the acidophilic iron-oxiding bacterium Thiobacillus ferrooxidans, have been isolated and characterized . They are Fe(II)-cytochrome c oxidoreductase, cytochromes c-552(s), c-552(m) and c-550(m), rusticyanin, and cytochrome c oxidase . On the basis of the interactions of these components, an electron transfer system has been proposed which seems to function in the oxidation of ferrous ion by the bacterium.

Int J Biol Macromol, 1995 Dec, 17(6), 353 - 5
The cellulolytic system of Streptomyces reticuli; Schrempf H et al.; The bacterium Streptomyces reticuli produces an unusual mycelia-associated cellulase (Avicelase, Cel1) which is solely sufficient to degrade crystalline cellulose to cellobiose . The enzyme consists of a binding domain, one adjoining region with unknown function, and a catalytic domain belonging to the cellulase family E . During cultivation, the strain produces a specific protease which processes the Avicelase to a truncated enzyme lacking the binding domain . The cellulase synthesis is regulated by induction (Avicel) and repression (metabolizable sugars and glycerol).

J Mol Evol, 1995 Dec, 41(6), 803 - 12
Arrangement and nucleotide sequence of the gene (fus) encoding elongation factor G (EF-G) from the hyperthermophilic bacterium Aquifex pyrophilus: phylogenetic depth of hyperthermophilic bacteria inferred from analysis of the EF-G/fus sequences; Bocchetta M et al.; The gene fus (for EF-G) of the hyperthermophilic bacterium Aquifex pyrophilus was cloned and sequenced . Unlike the other bacteria, which display the streptomycin-operon arrangement of EF genes (5'-rps12-rps7-fus-tuf-3'), the Aquifex fus gene (700 codons) is not preceded by the two small ribosomal subunit genes although it is still followed by a tuf gene (for EF-Tu) . The opposite strand upstream from the EF-G coding locus revealed an open reading frame (ORF) encoding a polypeptide having 52.5% identity with an E . coli protein (the pdxJ gene product) involved in pyridoxine condensation . The Aquifex EF-G was aligned with available homologs representative of Deinococci, high G+C Gram positives, Proteobacteria, cyanobacteria, and several Archaea . Outgroup-rooted phylogenies were constructed from both the amino acid and the DNA sequences using first and second codon positions in the alignments except sites containing synonymous changes . Both datasets and alternative tree-making methods gave a consistent topology, with Aquifex and Thermotoga maritima (a hyperthermophile) as the first and the second deepest offshoots, respectively . However, the robustness of the inferred phylogenies is not impressive . The branching of Aquifex more deeply than Thermotoga and the branching of Thermotoga more deeply than the other taxa examined are given at bootstrap values between 65 and 70% in the fus-based phylogenies, while the EF-G(2)-based phylogenies do not provide a statistically significant level of support (< or = 50% bootstrap confirmation) for the emergence of Thermotoga between Aquifex and the successive offshoot (Thermus genus) . At present, therefore, the placement of Aquifex at the root of the bacterial tree, albeit reproducible, can be asserted only with reservation, while the emergence of Thermotoga between the Aquificales and the Deinococci remains (statistically) indeterminate.

Protein Sci, 1995 Dec, 4(12), 2619 - 20
Crystallization and preliminary X-ray analysis of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10; Ridder IS et al.; Haloacid dehalogenases are enzymes that cleave carbon-chlorine or carbon-bromine bonds of 2-haloalkanoates . X-ray-quality crystals of L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8-7.0, using macroseeding techniques . The crystals, which diffract in the X-ray beam up to 2.0 A resolution, belong to the spacegroup C2221 . Cell parameters are a = 58.8 A, b = 93.1 A, c = 84.2 A . A native data set to 2.3 A has been collected, with a completeness of 97% and an Rsym of 6.0%.

Microbiology, 1995 Dec, 141 ( Pt 12), 3113 - 8
Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth; Burns BP et al.; A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized . The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE . A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex . The protein was isolated from the soluble fraction of the cell . Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pretreated with excess biotin . The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy . The activity of the enzyme was 0.29 mumol min-1 (mg protein)-1 . The Vmax for bicarbonate was calculated at 0.73 mumol min-1 (mg protein)-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent Km of 16.6 mM . These data provide the first evidence of a possible physiological role for the requirement of high levels of CO2 for growth in vitro of this bacterium.

Plant Mol Biol, 1995 Dec, 29(5), 897 - 907
Transformation of Synechococcus with a gene for choline oxidase enhances tolerance to salt stress; Deshnium P et al.; Choline oxidase, isolated from the soil bacterium Arthrobacter globiformis, converts choline to glycinebetaine (N-trimethylglycine) without a requirement for any cofactors . The gene for this enzyme, designated codA, was cloned and introduced into the cyanobacterium Synechococcus sp . PCC 7942 . The codA gene was expressed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at intracellular levels of 60-80 mM . Consequently the cells acquired tolerance to salt stress, as evaluated in terms of growth, accumulation of chlorophyll and photosynthetic activity.

Gaoxiong Yi Xue Ke Xue Za Zhi, 1995 Dec, 11(12), 650 - 3
Evaluation of the severity of Helicobacter pylori infection with urease test: its correlation with histopathology and bacterial density; Jan CM et al.; In 69 patients, the severity of Helicobactor pylori (H . pylori) infection was evaluated by bacterial density of tissue implants and inflammatory responses by histology . The specimens were taken from gastric angle and antrum (greater and lesser curvature sides) by gastroduodenal endoscopy . In urease test, the severity was measured in 3 grades according to color change of the agar: those change are within 30 minutes (grade 3), 30 minutes to 3 hours (grade 2), and 3 to 6 hours (grade 1), respectively; while the grade 0 indicated no color change occurring 6 hours after tissue inoculation . The severity of infection was assessed according to the bacterial density under high power microscopic fields (Gram's stain) . Grade 0 indicated no bacterium seen; grade 1, only 1 to 10 bacteria at all fields; grade 2, 1 to 3 bacteria in each high power field; and grade 3 was 4 bacteria or more on average in each high power field . The degree of inflammatory response was evaluated by inflammatory cell infiltration (H & E stain) and classified into grade 0, 1 and 2, which indicated the inflammatory cell infiltration below 50%, between 50% and 75%, and above 75%, respectively . There are no positive relationships among urease test reaction time, bacterial density grading and degrees of inflammatory cell infiltration . Clinically, the reaction time of urease test cannot reflect the severity of H . pylori infection semi-quantitatively, either in terms of bacterial density or cellular inflammatory response.

Appl Environ Microbiol, 1995 Dec, 61(12), 4329 - 33
Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens; Li J et al.; Two yellow pigments were isolated for the first time from the entomopathogenic bacterium Photorhabdus luminescens in liquid culture and were identified as the anthraquinone derivatives 3,8-dimethoxy-1-hydroxy-9,10-anthraquinone (minor) and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (major) . A known antibiotic, 3,5-dihydroxy-4-isopropylstilbene, was also detected and for the first time showed strong fungicidal activity against several fungi of medical and agricultural importance.

Curr Microbiol, 1995 Dec, 31(6), 332 - 5
Legionella pneumophila has two 60-kilodalton heat-shock proteins; Lema MW et al.; Legionella pneumophila is a thermotolerant bacterium . To learn more about the thermal adaptation of this organism, we studied the properties of the Legionella 60-kDa heat-shock protein (MopA, GroEL-analog, HtpB, Lp-Hsp60) in L . pneumophila and in an Escherichia coli strain containing the cloned gene . Lp-Hsp60 was found in both cytosol and membrane fractions; however, Lp-Hsp60 in the membrane fraction of L . pneumophila was slightly larger than Lp-Hsp60 in the cytosol . In contrast, both membrane-associated and cytosolic Lp-Hsp60 in the E . coli clone were similar in size to the smaller cytosolic Lp-Hsp60 of L . pneumophila . While peptide mapping suggests there are differences between the two proteins, the larger membrane-associated Lp-Hsp60 and the smaller cytosolic LP-Hsp60 shared Legionella-specific and E . coli GroEL cross-reacting epitopes, and the sequence of their first 20 N-terminal amino acids was identical . Further, Southern blot analysis of EcoRI-digested chromosomal DNA from several strains of L . pneumophila showed two fragments reacting with an htpAB-operon probe . In summary, L . pneumophila contains two Hsp60 proteins, and possibly two hsp60 genes.

Infect Immun, 1995 Dec, 63(12), 4877 - 82
Effect of free and vesicle-bound cysteine proteinases of Porphyromonas gingivalis on plasma clot formation: implications for bleeding tendency at periodontitis sites; Imamura T et al.; Infection by Porphyromonas gingivalis is strongly associated with adult periodontitis, with proteinases from this bacterium now considered to be important virulence factors . In order to investigate possible pathological functions of these enzymes, we examined the effect of both free and vesicle-bound forms of the two major cysteine proteinases (gingipains) of P . gingivalis on plasma clot formation by using thrombin time (TT) measurements . Both Lys-gingipain (gingipain-K) and Arg-gingipain (gingipain-R) prolonged plasma TT in a dose- and time-dependent manner, and this was also found with vesicles which are the biological carriers of P . gingivalis proteinases . The increase in plasma TT by vesicles could be completely reversed by treatment with nonspecific cysteine proteinase inhibitors but only partially by compounds selective for either gingipain-K or gingipain-R . Preincubation of vesicles with a gingipain-K-specific inhibitor (z-FK-ck) reduced plasma TT more than a gingipain-R-specific inhibitor (leupeptin), suggesting that under physiological conditions gingipain-K was more effective in fibrinogen destruction . Each purified enzyme also markedly increased fibrinogen TT, gingipain-R being fourfold more potent than gingipain-K . However, in plasma, gingipain-R was ineffective because of the inhibitory effect of albumin . These results imply that cysteine proteinases, especially gingipain-K, abrogate the clotting potential of fibrinogen and, therefore, may contribute to the bleeding tendency and to persistent inflammation in periodontitis sites infected with P . gingivalis.

Infect Immun, 1995 Dec, 63(12), 4606 - 12
Escherichia coli-induced activation of neutrophil NADPH-oxidase: lipopolysaccharide and formylated peptides act synergistically to induce release of reactive oxygen metabolites; Karlsson A et al.; The prevailing view of neutrophil NADPH-oxidase activation during interaction with bacteria is that the production of toxic oxygen metabolites should be directed into the phagosome containing the engulfed prey . However, in this report we show that a common Escherichia coli strain, HB101, may induce a release of neutrophil oxygen metabolites to the extracellular milieu . This phenomenon is dependent on three factors: (i) the mobilization (upregulation) of neutrophil secretory vesicles prior to interaction with the bacteria, (ii) soluble bacterial factors binding to the formylmethionyl-leucyl-phenylalanine receptor and tentatively identified as formylated peptides, and (iii) a bacterium-associated priming factor identified as lipopolysaccharide.

Biochemistry, 1995 Nov 21, 34(46), 15259 - 66
CP-MAS 13C-NMR dipolar correlation spectroscopy of 13C-enriched chlorosomes and isolated bacteriochlorophyll c aggregates of Chlorobium tepidum: the self-organization of pigments is the main structural feature of chlorosomes; Balaban TS et al.; Magic angle spinning (MAS) NMR dipolar correlation spectroscopy was applied for the first time to a biologically intact system, the light-harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum . The MAS spectra provide evidence that the self-organization of many thousands of bacteriochlorophyll c (BChl c) molecules is the predominant structural feature of the chlorosome . 13C-Enriched chlorosomes were prepared from nonuniformly labeled cultures grown with NaH13CO3 as the main carbon source and from a uniformly 13C-labeled culture grown with NaH13CO3 as the sole carbon source . For the nonuniformly labeled samples, the positions of the chlorin macrocycle originating from C-4 and C-5 of 5-aminolevulinic acid contained > 95% 13C while the remaining positions, which could have originated also from unlabeled acetate, were labeled to approximately 60% with 13C . The 1-D and 2-D MAS data of the labeled chlorosomes, when compared with data on the isolated labeled BChl c aggregated in n-hexane, show that the major component of the MAS signals in the chlorosomes is from BChl c, and only minor signal contributions arise from lipids and proteins . The 13C MAS signals of the BChl c aggregates were fully assigned by MAS 2-D dipolar correlation spectroscopy, using data on monomeric BChl c in CDCl3/CD3OD as reference . The 2(1)-, 3-, 3(2-), 5-, 12(1)-, 13-, and 13(1)-carbons are shifted by 2.5 ppm or more upfield with respect to the solution data.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 21, 34(46), 15235 - 47
Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides . 1 . PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions; Barz WP et al.; The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides . In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX . Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated . We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division . Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo . We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells . Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions . During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth . These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.

Biochemistry, 1995 Nov 21, 34(46), 15230 - 4
The asymmetry of P+ in bacterial reaction centers revealed by circular dichroism spectroscopy; Olson JM et al.; The circular dichroism anisotropy, (AL-AR)/A, has been measured for the far-red absorption band of P+ in reaction centers of two purple bacteria (Rhodopseudomonas viridis and Rhodobacter sphaerides) and one green sulfur bacterium (Chlorobium tepidum) . The anisotropy values for P960+ (Rps . virdis) at 1310 nm was found to be +(13 +/- 2) x 10(-4) . The corresponding for P870+ (Rb . sphaeroides) at 1250 nm was +(11 +/- 1) x 10(-4), but for P840+ (C . tepdium) at 1160 nm the value was negative: -(27 +/- 2) x 10(-4) . These results show that the configuration of the special pair in P840 is significantly different from the configuration in P870 and P960.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 293 - 6
Sulfate reduction by a syntrophic propionate-oxidizing bacterium; Van Kuijk BL et al.; The syntrophic propionate-oxidizing bacterium MPOB was able to grow in the absence of methanogens by coupling the oxidation of propionate to the reduction of sulfate . Growth on propionate plus sulfate was very slow (mu = 0.024 day-1) . An average growth yield was found of 1.5 g (dry weight) per mol of propionate . MPOB grew even slower than other sulfate-reducing syntrophic propionate-oxidizing bacteria . The growth rates and yields of strict sulfate-reducing bacteria (Desulfobulbus sp.) grown on propionate plus sulfate are considerably higher.

Mol Microbiol, 1995 Nov, 18(4), 779 - 89
Functional and regulatory analysis of the OmpF-like porin, OpnP, of the symbiotic bacterium Xenorhabdus nematophilus; Forst S et al.; The function and novel regulation of OpnP of the symbiotic/pathogenic bacterium, Xenorhabdus nematophilus was studied . In vitro pore-function analysis of purified OpnP indicated that the single-channel-conductance values were similar to that measured for the porin protein, OmpF, of Esherichia coli . Nucleotide sequence analysis revealed that the mature OpnP protein contained 348 amino acid residues and shared 55% amino acid sequence identity with OmpF . Similar to ompF, opnP mapped between asnS and aspC . The 16 transmembrane beta-sheet structures and the internal loop 3 were highly conserved, while the remaining external loop domains were more divergent . Primer extension analysis identified the start site of transcription of opnP . A sigma 70-type promoter, a perfect 20 bp OmpR-binding site, and a binding site for the antisense molecule, micF RNA, were found in the upstream region of opnP . While the overall sequence identity of the asn-opnP-aspC region was high, the intergenic region between asnS and opnP had diverged markedly . The asnS-opnP region was 313 bp shorter than the intergenic region between asnS and ompF and lacked the OmpR-binding site that is required for ompF repression by high osmolarity in E . coli . Results from osmolarity-shift experiments indicated that OpnP was not repressed by high osmolarity . It was also found that OpnP was thermally regulated.

Fogorv Sz, 1995 Nov, 88(11), 355 - 64
{Current trends in antibiotic therapy in dentistry}; Gaspar L et al.; The place and role of surgical, dental physio- and pharmacotherapy in oral and head and neck diseases has been debated for decades . In addition the price and reimbursement system had been changed lately in our country that also underlines the relevance of this issue . The overconsumption (1.7 packages per inhabitant per year) of antibiotics is also proven in Hungary . Beside surgical and dental interventions the treatment of dental inflammations caused by pathogenics requires the administration of different kinds of antibiotics . The first choice antibiotic is determined on the base of clinical features and general resistance of oral flora in the given period . When the first choice antibiotic proves to be ineffective, treatment has to be followed by antibiotics determined on individual resistance . In stomatology antibiotics are administered for prevention or treatment of dental inflammations . The length of therapy has a great importance which is usually 4 days in prevention and 5 days in treatment of dental inflammations . The modern antibiotics have to comply with the following requirements: efficacy, safety and cost-effectiveness . The typical mistakes in the administration of antibiotics can be avoided by the application of therapeutical schedules . The macrolide and cephalosporin derivates have become more and more popular . The mixed bacterium flora has been changing in time and also influenced by geographical factors depending on the habit of prescriptions . Therefore the experiences obtained in other countries are not applicable.

Gen Pharmacol, 1995 Nov, 26(7), 1553 - 8
Inhibition of gastric mucosal mucin receptor by H . pylori lipopolysaccharide: effect of ebrotidine; Slomiany BL et al.; 1 . H . pylori infection causes the loss of mucus coat continuity and its patchy appearance . Here, we present evidence that the bacterium, through its cell wall lipopolysaccharide, disrupts the interaction between mucin and its mucosal receptor, and that ebrotidine is capable of counteracting this process . 2 . The receptor for mucin, isolated from the solubilized gastric epithelial cell membrane by affinity chromatography on Sepharose-bound wheat germ agglutinin, displayed a molecular weight of 97 kDa and exhibited specific affinity towards mucin-coated surface . 3 . The mucin binding to the receptor was susceptible to the inhibitin by H . pylori lipopolysaccharide and reached a maximum of 91% . This effect of the lipopolysaccharide was counteracted by ebrotidine, which caused a dose-dependent relief of the lipopolysaccharide inhibitory effect with maximum restoration in mucin-receptor binding of 51% at 60 microliters/ml ebrotidine . 4 . The results show that H . pylori, through its lipopolysaccharide, is capable of disrupting the integrity of mucus perimeter of gastric mucosal defense and that antiulcer agent, ebrotidine, counteracts this untoward effect.

Zentralbl Veterinarmed B, 1995 Nov, 42(9), 533 - 42
{Electron microscopic detection of spirochetes in dermatitis digitalis of cattle}; Grund S et al.; In typical Dermatitis digitalis (D.d.) lesions, spirochaete-like bacteria with variations in spiralization were revealed by electron microscopy . While, in the early stages of the disease, these are found to be associated with fibrillar material of keratocytes, they occur massively in vacuoles at more advanced stages . The spirochaetes carry one pair of endoflagella, originating with a hook from the poles of the bacteria . These flagellae are composed of coiled flagellating fibrils in the pole region, merging towards the centre of the bacterium . A coat of fibrils was found in association with the cytoplasmatic membrane . The winding of this coat follows and may influence the coiling of the protoplast, and is probably involved in the rapid motility of this spirochaetes, together with the flagella . Immuno-electronmicroscopy revealed an antigenic relationship with Borrelia burgdorferi, at least with regard to the regions of flagella and undulating membrane . The paper discusses: 1 . The possible classification of these spirochaetes with the genus Treponema; 2 . The layer of peptidoglycan occurring on the outer membrane; and 3 . The keratolytic activity of spirochaetes in D.d.

Res Vet Sci, 1995 Nov, 59(3), 255 - 60
Entry of the bacterium ileal symbiont intracellularis into cultured enterocytes and its subsequent release; McOrist S et al.; Separate suspensions of two strains of ileal symbiont (IS) intracellularis, an obligate intracellular bacterium and the causative agent of porcine proliferative enteropathy, were added to 40 or 80 per cent confluent monolayers of established cultures of rat (IEC-18) or pig enterocytes (IPEC-J2) . Peak numbers of intracellular organisms were detected within the enterocytes six days later, but no cytopathic effects were evident . After an initial close association with the cell membrane of the enterocytes, single bacteria were internalised after three hours within membranes-bound vacuoles . The formation of an electron-dense projection between cell membranes and external bacteria was only evident if the bacterial suspensions were centrifuged on to the monolayers . The release of internalised bacteria into the cytoplasm, with the breakdown and loss of membrane-bound vacuoles, was also evident three hours after infection . Internalised bacteria were associated with, but not observed within, coated membrane pits . Mitochondria were closely associated with internalised vacuoles and with released bacteria . Two to six days after infection, multiplication of the bacteria free in the cytoplasm was frequently observed . In infected cells six days after the inoculation of monolayers, groups of bacteria were found within large, balloon-like, cytoplasmic protrusions, and the subsequent release of bacteria from the monolayer provided a means of bacterial exit from the cells . Many events in the in vitro culture model closely resembled events observed at the cellular level in animals infected with IS intracellularis and the model provides a useful basis for investigating the pathogenetic mechanisms of this bacterium.

Arch Microbiol, 1995 Nov, 164(5), 337 - 45
Metabolism of cyclohexane carboxylic acid by the photosynthetic bacterium Rhodopseudomonas palustris; Kuver J et al.; Cyclohexane carboxylate supported relatively rapid growth (doubling times 7-8 h) of Rhodopseudomonas palustris under oxic or photosynthetic conditions, but did not serve as a substrate for either of the known aromatic CoA ligases . A CoA ligase that thioesterifies cyclohexane carboxylate was partially purified and did not cross react immunologically with the two CoA ligases purified previously from this bacterium . Crude extracts of R . palustris cells grown with a range of aromatic or alicyclic acids contained a dehydrogenase that reacted with cyclohexane carboxyl-CoA or cyclohex-1-ene carboxyl-CoA, using 2,6-dichlorophenolindophenol or ferricenium ion as electron carrier . This activity was not detected in extracts of adipate-, glutamate-, or succinate-grown cells . No oxidation or reduction of nonesterified cyclohexane carboxylate or cyclohexene carbocylate was detected in extracts of cells grown with aromatic or aliphatic substrates, neither aerobically nor anaerobically . A constitutively expressed thioesterase that hydrolyzed cyclohexane carboxyl-CoA and also some alicyclic and aliphatic CoA derivatives was purified and characterized . The enzyme had little or no activity on benzoyl-CoA or 4-hydroxybenzoyl-CoA . The presence of a thioesterase that effectively hydrolyzes cyclohexane carboxyl-CoA suggests that transient production of cyclohexane carboxylate is a physiological response to temporary excess of reductant during metabolism of aromatic compounds.

Semin Pediatr Surg, 1995 Nov, 4(4), 221 - 7
The significance of Helicobacter pylori colonization of the stomach; Cilley RE et al.; Helicobacter pylori (Hp) was discovered in 1982 by the Australians Robin Warren and Barry Marshall . Initially rejected by a skeptical scientific community, it has since gained worldwide recognition as a clinically significant bacterium . The incidence of colonization with Hp increases with age, affecting approximately one third of the world's population . Hp is uniquely capable of surviving in the acid environment of the stomach, and has properties of adherence to epithelial cells that resist parastalsis . Strains of Hp associated with human disease produce specific cytotoxic proteins . After ingestion, there is a period of intense proliferation and ensuing gastric inflammation that may result in chronic gastritis . Hp infection in children may produce symptomatic antral gastritis or duodenal ulceration . The diagnosis of Hp infection is confirmed by gastric biopsy and culture, where the organism is recognized by its characteristic histological appearance . Treatment for Hp includes combinations of bismuth amoxicillin and metronidazole administered for several weeks . In adults, chronic infection with Hp is associated with chronic gastritis, achlorhydria, and gastric cancer . An organism that was unheard of 15 years ago is now recognized as a clinically significant pathological entity . The ultimate significance of Hp as an agent of disease remains to be seen.

Appl Environ Microbiol, 1995 Nov, 61(11), 3884 - 8
Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1; Spiess E et al.; Xanthobacter flavus 14p1 was isolated from sludge of the river Mulde by selective enrichment with 1,4-dichlorobenzene as the sole source of carbon and energy . The bacterium did not use other aromatic or chloroaromatic compounds as growth substrates . During growth on 1,4-dichlorobenzene, stoichiometric amounts of chloride ions were released . Degradation products of 1,4-dichlorobenzene were identified by gas chromatography-mass spectrometry analysis . 3,6-Dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene and 3,6-dichlorocatechol were isolated from culture fluid . 2,5-Dichloromuconic acid and 2-chloromaleylacetic acid as well as the decarboxylation product 2-chloroacetoacrylic acid were identified after enzymatic conversion of 3,6-dichlorocatechol by cell extract . 1,4-Dichlorobenzene dioxygenase, dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase activity were induced in cells grown on 1,4-dichlorobenzene . The results demonstrate that 1,4-dichlorobenzene degradation is initiated by dioxygenation and that ring opening proceeds via ortho cleavage.

Eur J Biochem, 1995 Nov 1, 233(3), 873 - 9
Molecular properties of the dissimilatory sulfite reductase from Desulfovibrio desulfuricans (Essex) and comparison with the enzyme from Desulfovibrio vulgaris (Hildenborough); Steuber J et al.; The dissimilatory sulfite reductase desulfoviridin was purified from the membrane (mSiR) and the soluble fraction (sSiR) of the sulfate-reducing bacterium Desulfovibrio desulfuricans (Essex) . Molecular and spectroscopic properties were determined and compared with the properties of the soluble desulfoviridin from Desulfovibrio vulgaris (Hildenborough) . The enzymes were isolated as alpha 2 beta 2 gamma n (n = 1-3) multimers with a relative molecular mass of 200 +/- 10 (gel filtration) . Both mSiR and sSiR from D . desulfuricans contained 24 +/- 3 Fe and 18 +/- 3 labile sulfide/200 kDa, respectively, and showed identical EPR spectra . Quantification of the high-spin Fe(III) heme resonances at g of approximately 6 indicated that close to 80% of the siroheme moiety in the enzyme from D . desulfuricans was demetallated . D . desulfuricans sulfite reductase showed S = 9/2 EPR signals with the highest apparent g value at g = 17 as reported for SiR from D . vulgaris . Antibodies raised against the alpha, beta and gamma subunit of the D . vulgaris enzyme exhibited cross-reactivity with the subunits of mSiR and sSiR from D . desulfuricans . N-terminal sequences of alpha, beta and gamma subunits of both mSiR and sSiR from D . desulfuricans were identical and showed a high degree of similarity with the sequences of the corresponding subunits obtained from the D . vulgaris enzyme . During gel filtration of sSiR from D . desulfuricans, under non-denaturing conditions, a small protein (molecular mass approximately 11 kDa) was separated . This 11-kDa protein exhibited cross-reactivity with the antibody raised against the gamma subunit of D . vulgaris sulfite reductase . In the case of D . desulfuricans sulfite reductase, the 11-kDa gamma subunit seems not to be an integral part of the protein and can be obtained from the soluble fraction and during purification of the soluble enzyme.

J Bacteriol, 1995 Nov, 177(22), 6630 - 7
Flagellar structure and hyperthermophily: analysis of a single flagellin gene and its product in Aquifex pyrophilus; Behammer W et al.; The polytrichously inserted flagella of Aquifex pyrophilus, a marine hyperthermophilic bacterium growing at 85 degrees C, were isolated and purified . Electron micrographs of the 19-nm-diameter flagellar filaments show prominent helical arrays of subunits . The primary structure of these 54-kDa flagellin monomers determining the helical shape and heat stability of filaments was of particular interest . The genomic region encoding the flagellin subunit (flaA gene) and an upstream open reading frame (orf1) were cloned and sequenced . The 1,503-bp flaA and 696-bp orf1 are preceded by separate sigma 28-like promoters and ribosome-binding motifs and succeeded by palindromic transcription terminators . Both genes are actively transcribed, but the nature and function of the orf1-encoded 231-residue polypeptide remain unknown . The deduced primary structure of the 501-amino-acid flagellin encoded by flaA consists of conserved N- and C-terminal regions and a variable 246-residue central domain . In comparison to mesophilic flagellins, the thermostable A . pyrophilus flagellin is characterized by increases in aromatic residues and prolines as well as by a 7.9% +/- 3.2% increase in all hydrophobic residues that is balanced by a respective decrease in hydrophilic residues . This composition is thought to form more compact flagellin monomers and stable interface contacts between neighboring subunits in the polymer.

J Bacteriol, 1995 Nov, 177(21), 6316 - 8
Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus; Peak MJ et al.; Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C . It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure . We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P . furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli . At 100 degrees C, DNA in P . furiosus cells is about 20 times more resistant to thermal breakage than that in E . coli cells, and six times fewer breaks were found in P . furiosus DNA after exposure to 110 degrees C for 30 min than in E . coli DNA at 95 degrees C . Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.

J Bacteriol, 1995 Nov, 177(21), 6170 - 5
Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2; Small FJ et al.; Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented . In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone . In the presence of CO2, no acetone was produced . Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2 . The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation . The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation . With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed . Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate . No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source . The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth . We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.

Infect Immun, 1995 Nov, 63(11), 4345 - 9
Cloning and characterization of hemolytic genes from Helicobacter pylori; Drazek ES et al.; Strains of Helicobacter pylori, the bacterium associated with gastritis, peptic ulcer disease, and gastric cancer in humans, express different degrees of hemolysis on agar containing erythrocytes (RBC) . Here we report the isolation and characterization of six recombinant clones from a genomic library of H . pylori ATCC 49503 that confer on Escherichia coli the ability to lyse sheep RBC . DNA hybridizations indicated no sequence homology among these hemolytic clones . Hybridization mapping of them to an ordered H . pylori cosmid library identified their separate chromosomal locations . One clone hybridized to two regions separated by approximately 200 kb . The specificities of the hemolytic activities of these clones were tested with RBC from humans, monkeys, cattle, horses, guinea pigs, rabbits, and chickens as well as with RBC from sheep . One clone conferred the ability to lyse RBC from five species, a second clone allowed the lysis of RBC from four of these species, three other clones allowed the lysis of RBC from three of these species, and the sixth clone allowed the lysis of RBC from just two species . We propose that some or all of the genes that confer these various hemolytic activities contribute to pathogen-host tissue interactions and that the different specificities seen here are important for H . pylori infections of humans of different genotypes or disease states.

J Biol Chem, 1995 Oct 27, 270(43), 25792 - 7
Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides; Rossak M et al.; The sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol is found in the photosynthetic membranes of all plants and most photosynthetic bacteria . Progress toward the elucidation of the pathway for sulfolipid biosynthesis has been slow in the past . However, the recent isolation of three genes of the photosynthetic bacterium Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities . Two of the genes flank an open reading Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities . Two of the genes flank an open reading frame predicted to encode a protein with amino acid sequence similarity to sugar nucleotide-dependent glycosyltransferases . The UDP-sulfoquinovose:diacylglcerol sulfoquinovosyltransferase thought to catalyze the last step of sulfolipid biosynthesis belongs to this group of glycosyltransferases . To test whether this open reading frame encodes the sulfoquinovosyltransferase of R . sphaeroides, it was inactivated by gene replacement avoiding polar mutagenesis . The resulting sulfolipid-deficient mutant defines a new gene, designated sqdD . Mutant cells grown in the presence of {35S}sulfate accumulate a water-soluble 35S-labeled compound . The purified compound was tentatively identified by co-chromatography with standards and enzymatic conversion as UDP-sulfoquinovose, the final precursor of sulfolipid biosynthesis . This result strongly suggests that the inactivation of sqdD causes a metabolic block in the last step of sulfolipid biosynthesis.

Biochem Biophys Res Commun, 1995 Oct 24, 215(3), 855 - 60
Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp . strain RW1; Bertini I et al.; The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp . strain RW1, are reported . In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution . Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines . These signals show strong sensitivity to the presence of the substrate . The oxidized form of the enzyme shows no hyperfine shifted signals . It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines . This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

FEBS Lett, 1995 Oct 23, 374(1), 130 - 4
A new mutation in the pufL gene responsible for the terbutryn resistance phenotype in Rubrivivax gelatinosus; Ouchane S et al.; Rubrivivax gelatinosus is a facultative phototrophic non-sulfur bacterium belonging to the beta subclass of the purple bacteria . A terbutryn-resistant mutant of R . gelatinosus has been isolated and characterized . Increased resistance levels to terbutryn (300-fold), atrazine (6-fold) and o-phenanthroline (3-fold) were observed for the mutant compared with wild type . Sequence analysis of the mutant revealed a new mutation in the pufL gene coding for the L subunit of the reaction centre (RC) at codon 192 leading to an amino-acid substitution from Gly in the wild type to Asp in the mutant . This substitution is located in the D helix of the L subunit, suggesting an interaction between terbutryn and this part of the polypeptide in the RC of R . gelatinosus . This is the first report of a mutation leading to herbicide resistance and affecting the D helix in purple bacteria . Furthermore R . gelatinosus wild type is highly sensitive to o-phenanthroline compared with other purple bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides) . Sequence comparison of the L subunit from six purple bacteria in which o-phenanthroline sensitivity was measured suggests that SerL226 might be responsible for this phenotype.

Biochim Biophys Acta, 1995 Oct 10, 1231(3), 220 - 2
Molecular cloning and sequencing of cytochrome c' from the phototrophic purple sulfur bacterium Chromatium vinosum; Even MT et al.; The gene for cytochrome c' from Chromatium vinosum was cloned from a HindIII-SalI digest of genomic DNA . A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method . The cytochrome c' gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein . The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space . An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Biochemistry, 1995 Oct 10, 34(40), 13091 - 7
Highly purified photosynthetic reaction center (PscA/cytochrome c551)2 complex of the green sulfur bacterium Chlorobium limicola; Oh-oka H et al.; The photosynthetic reaction center (RC) complex that forms a homodimer of core and cytochrome c subunits was isolated from Chlorobium limicola f . thiosulfatophilum, strain Larsen . The complex showed only two subunit bands at 68 (PscA core) and 21 kDa (cytochrome c551) on SDS-PAGE analysis, indicating the complete deletion of the light-harvesting bacteriochlorophyll a (BChl a) protein as well as the iron-sulfur protein . It contained 27 +/- 3 molecules of BChl a, 7 +/- 1 Chl-670, 3 +/- 1 carotenoids, and 1.6 +/- 0.1 c-type hemes per the primary electron donor P840 . The complex showed a light-induced charge separation and recombination between P840 and the acceptor Chl-670 at 77 K as follows: P840*Chl-670-->P840+Chl-670(-)-->P840TChl-670-->P84 0 Chl-670 . Pigment compositions and their function in the (PscA/cytochrome c551)2 complex were studied by absorption, circular dichroism, and fluorescence spectroscopy.

J Theor Biol, 1995 Oct 7, 176(3), 411 - 30
A Monte Carlo simulation of the Escherichia coli cell cycle; Keasling JD et al.; A Monte-Carlo simulation of the division cycle of an individual bacterium has been developed to test theories concerning the control of cell cycle events and for the analysis of cell cycle data . The model is based on the work of Cooper and Helmstetter and other theories concerning the control of DNA replication initiation, chromosome segregation and cell division . Variability in the molecular events that initiate a particular cell cycle event is incorporated using a Monte Carlo approach . The model results are compared with experimental data from a number of different sources and collected using a number of different analytical techniques . This model is able to accurately represent the cell size distributions of an exponentially growing population as determined by a Coulter Counter and the DNA distributions as determined by flow cytometry . The model is also able to accurately simulate the cell age distribution and chromosome replication and segregation patterns as determined by the membrane-elution technique . Results from parameter sensitivity analysis indicate that variability in the symmetry of cell division and in the cell size at initiation of chromosome replication have the most significant impact on the cell size, age and DNA distributions . Results from the membrane-elution simulation support the hypothesis that total cell protein is synthesized exponentially during the division cycle and that cell size control is a plausible explanation for control of DNA replication . The simulation results for chromosome segregation agree with experimental data and support chromosome strand segregation models . In the future, this simulation should be useful in testing complex theories about molecular control of cell cycle events against experimental data.

J Biol Chem, 1995 Oct 6, 270(40), 23875 - 82
Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides; Fowler GJ et al.; One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies . In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna . The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb . sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting . Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex . This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps . acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.

Biochemistry, 1995 Oct 3, 34(39), 12830 - 41
Structure of the tetraheme cytochrome from Desulfovibrio desulfuricans ATCC 27774: X-ray diffraction and electron paramagnetic resonance studies; Morais J et al.; The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement {Frazao et al . (1994) Acta Crystallogr . D50, 233-236} and refined at 1.75 A to an R-factor of 17.8% . When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept {heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues} . The three-dimensional structure of D . desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes {Palma et al . (1994) Biochemistry 33, 6394-6407} . This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling . The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations . Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

Mol Microbiol, 1995 Oct, 18(2), 225 - 36
Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp . BH72; Hurek T et al.; We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp . BH72, which was isolated as an endophyte from Kallar grass . Nitrogenase is derepressed under microaerobic conditions at O2 concentrations in the micromolar range . With increasing O2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O2 . Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions . The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies . Membrane deficiency in NifH/K- mutants, a deletion mutant in the nifK gene and the character of NH+4-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation . We propose the term 'diazosomes' for them . Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions . Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation . Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.

New Microbiol, 1995 Oct, 18(4), 441 - 4
Gastrospirillum hominis and human chronic gastritis; Monno R et al.; Gastrospirillum hominis, a new spiral bacterium, was found in the gastric mucosa of two patients with antral chronic gastritis . These 2 cases originated from a series of 2781 consecutive gastric biopsies observed over a period of five years, with a prevalence of 0.072% . Dogs and cats may be responsible for transmission to humans but in our experience no contact with pets was documented . Detection of these organisms might provide new insight into the pathogenesis of human gastritis.

J Bacteriol, 1995 Oct, 177(20), 5865 - 71
Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6; Heiss G et al.; An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity . DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp . This is the smallest gene encoding an extradiol dioxygenase found until now . Expression of the gene in a T7 expression vector enabled purification of the enzyme . Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa . The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3- and 4-chlorocatechol, and 3- and 4-methylcatechol . Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail . The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain . This indicates that strain BN6 carries at least two different extradiol dioxygenases.

Eur J Biochem, 1995 Oct 1, 233(1), 238 - 48
A cluster of structural and regulatory genes for light-induced carotenogenesis in Myxococcus xanthus; Botella JA et al.; In the bacterium Myxococcus xanthus, several genes for carotenoid synthesis lie together at the carA-carB chromosomal locus and are co-ordinately activated by blue light . A 12-kb DNA stretch from wild-type M . xanthus has been sequenced that includes the entire carA-carB gene cluster . According to sequence analysis, the cluster contains 11 different genes . Intergenic distances are very short or nil (implying translational coupling), giving further support to previous evidence indicating that most (or all) of the genes in the cluster form a single operon . At the promoter region, a potential -35 site for the binding of sigma factors is found . However, the -10 region shows little similarity with analogous sites in other bacterial promoters . Five (possibly six) genes in the carA-carB operon code for enzymes acting on early or late steps of the pathway for carotenoid synthesis . Other genes in the operon show no overall similarity with previously known genes . However, peptide stretches in the predicted products of two genes exhibit strong similarity with the DNA binding domain of the MerR family of transcriptional regulators . At least one of the predicted DNA-binding domains is altered in a mutant strain affected in light-regulation of the car genes.

Microbiology, 1995 Oct, 141 ( Pt 10), 2619 - 28
Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography; Modak HV et al.; Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme . The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-1 . In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-1 . The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140,000 Da and was absolutely dependent on acetyl-CoA for activity . Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation . The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration . The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined . An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.

Curr Opin Biotechnol, 1995 Oct, 6(5), 501 - 6
Gene fusion expression systems in Escherichia coli; LaVallie ER et al.; In recent years, Escherichia coli gene fusion expression systems have circumvented many of the problems inherent in the use of this bacterium for the production of recombinant proteins . These systems also provide a powerful means for identifying peptides or proteins with desired binding specificities . Gene fusion technology continues to expand with the introduction of new fusion partners, purification and detection tags, cleavage reagents and ways to display peptides on the surface of bacteria.

J Bacteriol, 1995 Oct, 177(19), 5644 - 52
Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction; Clemens DL et al.; Mycobacterium tuberculosis urease (urea amidohydrolase {EC 3.5.1.5}) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da . The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases . M . tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine . The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea . The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate . Urease activity was readily detectable in M . tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium . The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria . The nucleotide sequence of the M . tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.

J Bacteriol, 1995 Oct, 177(19), 5495 - 505
Interchromosomal recombination in the extremely radioresistant bacterium Deinococcus radiodurans; Daly MJ et al.; Deinococcus radiodurans and other members of the genus Deinococcus are remarkable for their extreme resistance to ionizing radiation and many other agents that damage DNA . We have recently shown that recombinational processes participate in interplasmidic repair following in vivo irradiation . We now present direct studies on interchromosomal recombination among chromosomes irradiated in vivo during stationary phase (four chromosomes per cell) . Following an exposure to 1.75 Mrad (the dose required to achieve a survival of 37%, which degrades the cells' four chromosomes into about 500 fragments), we determined that there may be as many as 175 crossovers per chromosome (700 crossovers per nucleoid) undergoing repair . In addition, these studies suggest that many of the crossovers occurring during repair are nonreciprocal.

J Bacteriol, 1995 Oct, 177(19), 5480 - 4
Physical map of the genome of the green phototrophic bacterium Chlorobium tepidum; Naterstad K et al.; A physical restriction map of the chromosome of the green sulfur bacterium Chlorobium tepidum was generated by determining the order of the fragments obtained after digestion with the restriction endonucleases XbaI and PacI and subsequent separation of the fragments by pulsed-field gel electrophoresis . The size of the chromosome is estimated to be 2.1 Mb . Fifteen genes and operons, mainly encoding proteins involved in photosynthesis, have been placed on this map by hybridization to fragments obtained after single- and double-restriction digestions.

Int J Syst Bacteriol, 1995 Oct, 45(4), 820 - 5
Characterization of Lawsonia intracellularis gen . nov., sp . nov., the obligately intracellular bacterium of porcine proliferative enteropathy; McOrist S et al.; A novel obligately intracellular bacterium, ileal symbiont intracellularis, which was obtained from the intestines of pigs with proliferative enteropathy disease, was grown in pure cocultures with tissue cultures of rat cells . An examination of the 16S ribosomal DNA gene sequence revealed that the isolates which we obtained are members of the delta subdivision of the Proteobacteria and that the sequences of these organisms exhibit a level of similarly of 91% with the sequence of Desulfovibrio desulfuricans ATCC 27774 . These isolates were homogeneous and differed in cellular morphology, acid fastness, phenotype, electrophoretic protein profile, and habitat from Desulfovibrio species . On the basis of the results of an integrated study of the phenotype and genotype of a consistent morphological entity found in particular porcine cells and associated with a well-defined clinical condition, we concluded that these bacteria belong to a previously undescribed genus and species, for which we propose the name Lawsonia intracellularis gen . nov., sp . nov . A species-specific recombinant DNA probe was cloned previously, and this probe was used to identify the bacterium in tissue culture cells and in the ileal epithelia of pigs with proliferative enteropathy disease . Coculture of the organism with a rat enterocyte cell line allowed us to designate strain NCTC 12656 the type strain and to describe the new genus and species . The organism which we cultured is pathogenic for pigs and causes proliferative enteropathy lesions in their ilea and colons, and Koch's postulates were fulfilled for this organism.

Trends Biotechnol, 1995 Oct, 13(10), 418 - 21
Engineering gut flora of ruminant livestock to reduce forage toxicity: progress and problems; Gregg K; The rumen bacterium Butyrivibrio fibrosolvens has been genetically modified to detoxify fluoroacetate (a poisonous component of trees and shrubs in Australia, Africa and Central America) and has been shown to persist when it is returned to the rumen . Such bacteria may save animals from poisoning and, therefore, reduce economic losses for livestock industries in those countries . The ability to make genetic changes to rumen bacteria raises important questions about their practicality, and about the environmental factors that must be considered before releasing modified strains . The fluoroacetate-detoxifying bacterium provides an important model by which these issues can be examined.

Recenti Prog Med, 1995 Oct, 86(10), 382 - 5
{Influence of Helicobacter pylori on gastric secretion . Study on variously associated gastric body, fundus and antrum chronic gastritis}; Testino G et al.; Among the various themes related to Helicobacter pylori (HP) which is still a subject of discussion, there is the possible influence of this bacterium on gastric secretory physiology . In the present study, an evaluation has been carried out of stimulated gastrinemia, stimulated acid secretion and total peptic activity in gastric juice in the course of a paradigmatic condition, as autonomous chronic gastritis, in order to reveal possible modifications induced by the HP infection . In cases of HP positive chronic superficial antral gastritis associated either with normal body-fundic mucosa or with superficial gastritis, there is a significant increase of stimulated gastrinemia in comparison to HP negative groups and controls . In the course of body-fundic atrophic and preatrophic chronic gastritis associated either with antral superficial chronic gastritis or with antral atrophic gastritis, there are no statistically significant differences between HP positive and HP negative subjects . As regards acid and pepsin secretion no significant differences emerge in any group between HP positive and HP negative subjects . In the HP positive subjects with antral superficial gastritis and higher gastrin values the study of acid and pepsin secretion has yielded no significant variations . From the results of this study it emerges how gastric secretory parameters vary exclusively according to the histologic state of gastric mucosa . Therefore, the lesion action of HP may mainly be attributed to a direct action, rather than to substantial gastric secretory changes.

J Biol Chem, 1995 Sep 29, 270(39), 22801 - 6
Codon pair utilization biases influence translational elongation step times; Irwin B et al.; Two independent assays capable of measuring the relative in vivo translational step times across a selected codon pair in a growing polypeptide in the bacterium Escherichia coli have been employed to demonstrate that codon pairs observed in protein coding sequences more frequently than predicted (over-represented codon pairs) are translated slower than pairs observed less frequently than expected (under-represented codon pairs) . These results are consistent with the findings that translational step times are influenced by codon context and that these context effects are related to the compatabilities of adjacent tRNA isoacceptor molecules on the surface of a translating ribosome . These results also support our previous suggestion that the frequency of one codon next to another has co-evolved with the structure and abundance of tRNA isoacceptors in order to control the rates of translational step times without imposing additional constraints on amino acid sequences or protein structures.

Gene, 1995 Sep 22, 163(1), 115 - 9
The citrate synthase-encoding gene of Rickettsia prowazekii is controlled by two promoters; Cai J et al.; The transcripts of the citrate synthase-encoding gene (gltA) in Rickettsia prowazekii (Rp), an obligate intracellular parasitic bacterium, were analyzed by RNase protection (RP), primer extension (PE) and in vitro transcription assays . Analysis of the 5' end of the gltA mRNA by RP and PE assays revealed that there were two gltA mRNAs with the 5' ends located at 16 bp and 307 bp upstream from the gltA coding region . Since these two mRNAs might represent two species of mRNA transcribed from two different promoters or a single transcript that was processed to give two mRNAs, an in vitro transcription analysis with purified Rp RNA polymerase (RNAP) was performed to distinguish these two possibilities . Purified Rp RNAP catalyzed the formation of two transcripts initiated from the same nucleotides indicated by RP and PE . Sequence analysis identified Escherichia coli (Ec) promoter-like sequences immediately upstream from both transcription start points (tsp) . The first promoter (promoter P1) had the core sequence TTCTAA-N17-TATACT, was 6 bp upstream from the tsp (base A) and was centered at 37 bp upstream from the coding region . The second promoter (promoter P2) had the core sequence ATGAAA-N17-TAAAGT, was 7 bp upstream from the tsp (base T) and was centered at 329 bp upstream from the coding region . This is the first demonstration of multiple promoters in this obligate intracellular parasite which has implications concerning transcriptional regulation.

J Mol Biol, 1995 Sep 15, 252(2), 235 - 47
Refined crystal structure of ferrocytochrome c2 from Rhodopseudomonas viridis at 1.6 A resolution; Sogabe S et al.; The three-dimensional structure of ferrocytochrome c2 from the purple photosynthetic bacterium Rhodopseudomonas viridis has been refined to a final R-factor of 18.2% for 15,014 unique reflections collected by synchrotron radiation between 6.0 and 1.6 A resolution . The refined model includes 107 amino acid residues, one heme prosthetic group and 125 water molecules . The root-mean-square deviations from the ideal bond lengths and angles were 0.014 A and 3.0 degrees, respectively . The atomic coordinate error was estimated to be less than 0.3 A . A structure comparison of this cytochrome c2 with those of the other c-type cytochromes demonstrated that these cytochromes exhibit a high degree of structural similarity with the exception of the surface loop and the terminal region of the polypeptide chain . The deletion of an intrahelical amino residue distorted the conformation of the alpha-helix and it divided into two pieces . The C-terminal extension of the polypeptide chain caused significant conformational changes of the contact residues compared with the other c-type cytochromes . Of the water molecules conserved in various c-type cytochromes, two are located internally in the vicinity of the heme group . One of these water molecules found in this cytochrome c2 is evolutionarily conserved among eukaryotic cytochromes c . This water molecule is located in the heme proximate environment in a position similar to that of eukaryotic cytochromes c . The position of this water molecule is associated with the oxidation state of the heme iron in electron transfer.

FEMS Microbiol Lett, 1995 Sep 15, 131(3), 307 - 12
The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein; Yu JS et al.; The phosphoglycerate kinase (pgk), triosephosphate isomerase (tpi), and enolase (eno) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E . coli pgk- strain . In T . neapolitana, the pgk and tpi genes appear to be fused and eno is near those genes . Like T . maritima, T . neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity . Unlike T . maritima, which forms only a 70-kDa fusion protein, T . neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein . These isozymes are present in both T . neapolitana cells and in E . coli cells expressing T . neapolitana genes.

EMBO J, 1995 Sep 15, 14(18), 4395 - 402
(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima; Sterner R et al.; To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima . The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli . The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon . Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively . Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated . Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms . Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.

Biol Pharm Bull, 1995 Sep, 18(9), 1184 - 8
Purification and characterization of beta-glucuronidase from Escherichia coli HGU-3, a human intestinal bacterium; Kim DH et al.; beta-Glucuronidase was purified 360-fold from Escherichia coli HGU-3, an human intestinal bacterium . The specific activity of the purified enzyme was 17.78 units/mg protein . The enzyme (M.W . 290000) is composed of four subunits (M.W . 72000) with a pI and optimal pH of 4.8 and 6-7, respectively . The apparent Km for p-nitrophenyl-beta-D-glucuronide was found to be 0.22 mM . The enzyme was inhibited by saccharic acid 1,4-lactone, glycyrrhizin, N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonic acid (PCMS) . Using the bile containing bilirubin diglucuronide as a substrate, the purified beta-glucuronidase was able to hydrolyze it to bilirubin . This hydrolyzed bilirubin formed calcium bilirubinate with a reaction mixture containing CaCl2.

Mol Microbiol, 1995 Sep, 17(5), 961 - 9
Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides, a bacterium with a unidirectional, stop-start flagellum; Shah DS et al.; Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation stops and restarts . Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli, a bacterium with bidirectional flagella . In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5 . DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E . coli . MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation . Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R . sphaeroides and E . coli MotA sequences, along with a highly charged cytoplasmic linker region . Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB . Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a sigma 54-type promoter, including a potential enhancer binding site . The overall similarities between the R . sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of the R . sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.

Mol Microbiol, 1995 Sep, 17(5), 843 - 53
Gene-regulatory modules in Escherichia coli: nucleoprotein complexes formed by cAMP-CRP and CytR at the nupG promoter; Pedersen H et al.; Repression by CytR depends on the formation of nucleoprotein complexes in which the CytR repressor and the cAMP-CRP activator complex bind co-operatively to the DNA . Transcription initiation from CytR-regulated promoters requires cAMP-CRP; therefore, the cAMP-CRP complex functions both as an activator and as a co-repressor in these promoters . Another interesting aspect of the CytR regulon is that each promoter appears to have individual features . Therefore, structural and functional rules governing the formation of repression and activation complexes in one promoter may not be valid for other promoters of the CytR regulon . Here we show that the Escherichia coli nupG gene contains one CytR- and four CRP-binding sites in the control region . Notably, the architecture of the CytR binding site is different from previously described targets . In addition, the CytR repressor triggers a DNA repositioning of a cAMP-CRP complex in the -35 region upon binding to its operator . Thus, formation of the repression and activation complexes at the nupG promoter involves different subsets of CRP-binding sites . These findings show that the bacterium uses positive and negative regulatory modules to differentially control the expression of CytR- and cAMP-CRP-regulated genes.

Baillieres Clin Gastroenterol, 1995 Sep, 9(3), 563 - 82
Helicobacter pylori as a risk factor for cancer; Webb PM et al.; In 1985, gastric cancer was the second most common cause of cancer death in the world . The rapid decline in gastric cancer rates over the last few decades has been attributed to a decline in the prevalence of environmental risk factors for gastric cancer and/or an increase in the prevalence of protective factors . One such risk factor could be the bacterium Helicobacter pylori . Epidemiological studies have shown that areas with high gastric cancer rates often have a correspondingly high prevalence of H . pylori and prospective studies have shown that subjects with serological evidence of H . pylori infection were significantly more likely to go on to develop gastric cancer than those who did not . Helicobacter pylori itself does not appear to be either genotoxic or mutagenic . Infection is, however, associated with increased cell turnover, a chronic immune response accompanied by increased levels of reactive oxygen metabolites and a reduction in gastric levels of ascorbic acid, all conditions that could favour the development of cancer . Nonetheless, the majority of those who are infected with H . pylori do not go on to develop gastric cancer and other factors, such as the strain of the infecting organism or consumption of dietary antioxidants including vitamin C, could also affect the risk of cancer . Finally, it has been estimated that more than one third, and possibly as many as 90% of gastric cancers might be attributable to infection with H . pylori . Prevention and treatment of infection are, therefore, possible approaches to reducing gastric cancer rates . It is, however, unclear what, if any, effect eradication of the infection would have on an individual's risk of gastric cancer and, to date, anti-Helicobacter therapy has only been shown to be of potential benefit in the treatment of low grade gastric MALT lymphomas.

Baillieres Clin Gastroenterol, 1995 Sep, 9(3), 549 - 62
Nature of non-ulcer dyspepsia and related conditions; Porro GB et al.; To date, the precise role of Helicobacter pylori in the pathogenesis of NUD remains uncertain . There is some evidence to suggest that the organism is implicated in specific subgroups (mostly the ulcer-like form), but it is not enough for any firm conclusions to be drawn as to the importance of the bacterium as a cause of dyspeptic symptoms or as to the efficacy of anti-infective regimens in the treatment of NUD . Large, well-designed prospective studies with a long-term follow-up are needed to establish which subgroups of dyspeptic patients may benefit most from eradication of H . pylori.

Biokhimiia, 1995 Sep, 60(9), 1429 - 34
{Analysis of amino acid sequences of Rhodobacter sphaeroides glutamine synthetase}; Zinchenko VV et al.; A comparative analysis of the amino acid sequence of glutamine synthetase (GS) of the photosynthetic purple bacterium Rhodobacter sphaeroides revealed that the enzyme is typical for first type procaryotic GSs and structurally resembles GSs of enteric bacteria . The data obtained indicate that the complex phenotype of purple bacterial mutants at the glnA gene coding for GS may be conditioned by specific regulation of nitrogen metabolism in bacterial cells rather than by structural-and-functional peculiarities of GS.

Indian J Med Res, 1995 Sep, 102, 104 - 13
Propagation of Mycobacterium lepraemurium on supplemented minimal medium and its experimental pathogenesis; Banerjee P et al.; The splenic tissue of a mouse experimentally infected with M . lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M . lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc . This could be maintained, by serial passages in vitro with good growth . Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible . The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.

Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1666 - 9
A hyperthermophilic sulfur-reducing archaebacterium, Thermococcus sp . DT1331, isolated from a deep-sea hydrothermal vent; Kwak YS et al.; A hyperthermophilic archaebacterium was isolated from a deep-sea black smoker chimney (depth, 760 m) at the Minami-ensei Knoll (28 degrees 23'N, 127 degrees 38'E) . The strain, designated DT1331, was a coccoid shaped bacterium about 0.5 to 1.0 microns in diameter . The cells were surrounded by a cell envelope . The temperature for growth was between 55 degrees C and 93 degrees C with an optimum 80 degrees C . The growth occurred from pH 4.5 to 8.5 and the optimum pH was 6.0 . DT1331 required 1% to 5% NaCl for growth and cell lysis was observed below 1% NaCl concentration . The strain was an anaerobic chemoorganotroph requiring elemental sulfur obligately . Organic substrates used included tryptone, peptone, soytone, casein, gelatin, and yeast extract . Under the optimal conditions, DT1331 had a generation time of 50 min and could reach densities of about 1.5 x 10(8) cells/ml . DT1331 was resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, streptomycin, and tetracycline, which was one of the common characteristics of archaebacteria . The G+C content of DT1331 was 52.3 mol% . Analysis of the 16S rRNA gene by restriction enzymes coincided with those of Thermococcus celer, indicating that this strain belonged to the genus Thermococcus.

Biophys J, 1995 Sep, 69(3), 1117 - 29
Nonlinear annihilation of excitations in photosynthetic systems; Valkunas L et al.; The theory of the singlet-singlet annihilation in quasi-homogeneous photosynthetic antenna systems is developed further . In the new model, the following important contributions are taken into account: 1) the finite excitation pulse duration, 2) the occupation of higher excited states during the annihilation, 3) excitation correlation effects, and 4) the effect of local heating . The main emphasis is concentrated on the analysis of pump-probe kinetic measurements demonstrating the first two above possible contributions . The difference with the results obtained from low-intensity fluorescence kinetic measurements is highlighted . The experimental data with picosecond time resolution obtained for the photosynthetic bacterium Rhodospirillum rubrum at room temperature are discussed on the basis of this theory.

Am J Gastroenterol, 1995 Sep, 90(9), 1424 - 7
Triple therapy with sucralfate, tetracycline, and metronidazole for Helicobacter pylori-associated duodenal ulcers; Sung JJ et al.; Triple therapy with bismuth, metronidazole, and tetracycline or amoxicillin is effective for the treatment of Helicobacter pylori, but side effects are common . Sucralfate inhibits H . pylori hemagglutinin, protease, and lipase and thus might affect colonization of the bacterium in the stomach . OBJECTIVE: We compared the efficacy and side effects of triple therapy with sucralfate versus triple therapy with bismuth plus omeprazole in the treatment of H . pylori-associated duodenal ulcer (DU) . METHODS: One hundred and fifty DU patients were recruited in this study; 71 cases were randomized to receive bismuth 120 mg q.i.d., metronidazole 400 mg q.i.d., and tetracycline 500 mg q.i.d . (BMT) for 1 wk, and 79 cases were randomized to receive sucralfate 1 g q.i.d., metronidazole 400 mg q.i.d., and tetracycline 500 mg q.i.d . (SMT) for 1 wk . For the ulcer treatment, BMT patients were also given omeprazole 20 mg daily for 4 wk, and SMT patients received sucralfate for 4 wk from day of randomization . RESULTS: Fifty-three patients in the BMT group and 60 in the SMT group finished the treatment and follow-up at 8 wk . H . pylori was eradicated in 49 out of 53 (92%) patients in the BMT group and in 45 out of 60 (75%) patients in the SMT group (p = 0.0057) . Forty-nine (92%) patients who received omeprazole and BMT and 53 (88%) patients who received SMT had healed DU at 8 wk (p = 0.34) . Side effects related to medication were reported in 38 (71.7%) patients in the BMT group and in 42 (70%) patients in the SMT group . On an intention-to-treat basis, there was no difference in ulcer healing between the BMT group (93.1%) and the SMT group (89.7%) . H . pylori eradication was achieved in 84.4 and 66.2% in the BMT and SMT groups, respectively (p = 0.018) . CONCLUSION: Therapy of sucralfate, tetracycline, and metronidazole for 1 wk has a satisfactory but lower success rate in eradication of H . pylori when compared with the conventional triple therapy plus omeprazole . Side effects of this therapy are no fewer than the conventional triple therapy.

J Biol Chem, 1995 Sep 1, 270(35), 20621 - 8
The leucyl/phenylalanyl-tRNA-protein transferase . Overexpression and characterization of substrate recognition, domain structure, and secondary structure; Abramochkin G et al.; Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation . We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) . This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins . We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase . The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component . We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme . Limited digestion of the L/F-transferase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical . Finally, a region of sequence conservation between the L/F-transferase and the NH2-terminal protein acetylases has been identified.

Infect Immun, 1995 Sep, 63(9), 3739 - 44
Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells; McKee ML et al.; In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M . S . Donnenberg, S . Tzipori, M . L . McKee, A . D . O'Brien, J . Alroy, and J . B . Kaper, J . Clin . Invest . 92:1418-1424, 1993) . However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated . To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo . We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells . The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.

Infect Immun, 1995 Sep, 63(9), 3527 - 30
Murine cytotoxic T lymphocytes induced following Chlamydia trachomatis intraperitoneal or genital tract infection respond to cells infected with multiple serovars; Starnbach MN et al.; The obligate intracellular bacterium Chlamydia trachomatis is associated with human diseases ranging from blinding trachoma to sexually acquired genital infections and the systemic disease lymphogranuloma venereum (LGV) . We have previously reported the isolation and culture of protective murine cytotoxic T lymphocytes (CTL) following intraperitoneal infection with C . trachomatis serovar L2, a serotype associated with human LGV . In this report, we now demonstrate that CTL can also be primed following introduction of C . trachomatis serovar L2 into the uterus or ovarian bursa of mice . We also describe Chlamydia-specific CTL lines isolated following murine infection with a typical human urogenital isolate of C . trachomatis (serovar D) and show that such CTL can be primed by intraperitoneal, intrauterine, or intrabursal infection . Last, we demonstrate that these murine CTL lines respond to multiple serovars, recognizing and lysing cells infected with C . trachomatis serovars B, C, D, F, J, K, L2, and L3, representative of organisms causing blinding trachoma, genital infection, and LGV.

Infect Immun, 1995 Sep, 63(9), 3401 - 10
Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells; Uitto VJ et al.; The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied . In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T . denticola . Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles . Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died . Blebbing could also be induced by a purified chymotrypsin-like proteinase of T . denticola . Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T . denticola and the isolated proteinase . Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T . denticola than migrating isolated cells . In multilayer epithelial cultures, adhesion of T . denticola and membrane blebbing were observed infrequently . There was no evidence of invasion of T . denticola into epithelial multilayers . However, immunogold electron microscopy showed rapid transport of T . denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers . These vacuoles were lined by membranes studded with ribosomes . T . denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability . Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T . denticola has the potential to contribute to the initiation of periodontal disease.

Br J Surg, 1995 Sep, 82(9), 1204 - 6
Gastric juice epidermal growth factor concentration and Helicobacter pylori in patients with duodenal ulcer; Tunio AM et al.; The level of epidermal growth factor (EGF) was measured in the basal and maximally stimulated gastric juice of 20 control subjects and 20 patients each with duodenal ulcer and non-ulcer dyspepsia . Basal gastric juice was analysed for ammonia and urea concentrations, and the {ammonia}3/{urea} ratio was used to show Helicobacter pylori status, as was the {13C}urea breath test in nine controls . There was complete concordance in the nine controls between the two methods for determining H . pylori status . Twenty-five subjects were H . pylori positive (seven with duodenal ulcer, nine with non-ulcer dyspepsia, nine controls) and 35 H . pylori negative (13, 11 and 11 respectively) . In H . pylori-positive subjects, the median EGF concentrations in the stimulated secretion of patients with duodenal ulcer and without (non-ulcer dyspepsia and controls combined) were 46.7 and 18.0 ng/ml (P < 0.001), and in H . pylori-negative subjects were 40.0 and 26.5 ng/ml respectively (P < 0.01) . There was no difference in EGF concentration between controls and subjects with non-ulcer dyspepsia irrespective of H . pylori status . Lack of EGF is unlikely to be a cause of duodenal ulcer . The increased EGF concentration in patients with ulcer bore no relationship to the H . pylori status of the individual . If this bacterium causes duodenal ulcer, it is not via a reduction in EGF concentration.

J Clin Microbiol, 1995 Sep, 33(9), 2445 - 50
Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers; Chomel BB et al.; The isolation of Bartonella henselae, the agent of cat scratch disease, from the blood of naturally infected domestic cats and the demonstration that cats remain bacteremic for several months suggest that cats play a major role as a reservoir for this bacterium . A convenience sample of 205 cats from northern California was selected between 1992 and 1994 to evaluate the B . henselae antibody and bacteremia prevalences and to determine the risk factors and associations between bacteremia and antibody titers . B . henselae was isolated from the blood of 81 cats (39.5%) . Forty-two (52%) of these bacteremic cats were found to be infected with > or = 1,000 CFU/ml of blood . Impounded or former stray cats were 2.86 (95% confidence interval {CI} = 1.94, 4.22) times more likely to be bacteremic than the pet cats . Young cats ( < 1 year old) were more likely than adult cats to be bacteremic (relative risk = 1.64; (95% CI = 1.19, 2.28) . Bacteremic cats were more likely than nonbacteremic cats to be infested with fleas (relative risk = 1.64; 95% CI = 1.38, 1.96) . No association between B . henselae infection and feline immunodeficiency virus antibody prevalence was observed . Eighty-one percent of the cats (166 of 205) tested positive for B . henselae antibodies, and titers were higher in bacteremic than in nonbacteremic cats . Multiple logistic regression analysis indicated that younger age and seropositivity for B . henselae antibodies were associated with bacteremia . Serological screening for Bartonella antibodies may not be useful for the identification of bacteremic cats (positive predictive value = 46.4%), but the lack of antibodies to B . henselae was highly predictive of the absence of bacteremia (negative predictive value = 89.7%) . Seronegative cats may be more appropriate pets for immunocompromised individuals who are at increased risk for developing severe B . henselae disease.

Chemosphere, 1995 Sep, 31(5), 3273 - 89
A modified SOS-Chromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction; Dutka BJ et al.; A modified SOS-Chromotest bioassay using a chromogenic pad (pad procedure) was developed to test for genotoxicity in sediments directly without extraction . This test is based on the de novo synthesis of beta-galactosidase enzyme by a genetically-engineered E . coli strain PQ37 . In the bioassay, an exponential growth phase antibiotic-containing culture of the test bacterium is introduced into a series of tubes with the first tube containing 0.1 gram of sediment . Serial dilutions are then made and the tubes of sediment plus bacterial culture are incubated at 37 degrees C for four hours, followed by placing a drop of each mixture on a chromogenic pad and additional incubation for 20 hours at 37 degrees C . The solid particulates are then washed off with tap water and positive (genotoxic) activity is noted by the presence of a distinctive blue colour on the pad . The SOS-Chromotest pad procedure may be best used as a relative measure of genotoxicity by comparing results to a reference sample . In addition it can also determine sediment cytotoxicity by comparing samples spiked with a genotoxic standard (i.e., 4-nitroquinoline-N-oxide) . Preliminary results suggest that this new bioassay is highly sensitive, consistent and discriminating.

Acta Pharm Hung, 1995 Sep, 65(5), 163 - 6
{Interaction between Bordetella pertussis vaccine and chlorpromazine in mice}; Antmann K et al.; In acute toxicity experiments authors observed increased mortality on the first day following 75 mg/kg chlorpromazine (CPZ) treatment in mice pretreated with Bordetella pertussis vaccine (9 x 10(9) killed bacterium/mouse) compared to control animals treated with CPZ alone . Initially, the increased drug sensitivity observed after combined treatment was attributed to summation of the toxic effects . However, the cumulation of mortality did not cease on the following days; furthermore, an increase of bacterial translocation (translocation index of P-CPZ group: 4.5) was observed on days 6 and 7, i.e . when the lymphocytosis, splenic hypertrophy and shrinkage of thymus--changes consequent to the vaccination--were at their maximum levels . On the basis of all these and on literary data it is supposed that the early cumulation of deaths after combined treatment may be in connection with an interaction between the two agents and that the side-effects following vaccination of humans may be induced by undesirable pharmacological interactions.

J Biol Chem, 1995 Aug 25, 270(34), 19823 - 7
A mutant Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase with an altered metal requirement functions in situ for tetrapyrrole synthesis in soybean root nodules; Chauhan S et al.; The tetrapyrrole synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase requires Mg2+ for catalytic activity in photosynthetic organisms and in Bradyrhizobium japonicum, a bacterium that can reside symbiotically within plant cells of soybean root nodules or as a free-living organism . ALA dehydratase from animals and other non-photosynthetic organisms is a Zn(2+)-dependent enzyme . A modified B . japonicum ALA dehydratase, ALAD*, was constructed by site-directed mutagenesis of hemB in which three proximal amino acids conserved in plant dehydratases were changed to cysteine residues as is found in the Zn(2+)-dependent enzyme of animals . These substitutions resulted in an enzyme that required Zn2+ rather than Mg2+ for catalytic activity, and therefore a region of the ALA dehydratase from B . japonicum, and probably from plants, was identified that is involved in Mg2+ dependence . In addition, the data show that a change in only a few residues is sufficient to change a Mg(2+)-dependent ALA dehydratase to a Zn(2+)-dependent one . B . japonicum strains were constructed that contained a single copy of either hemB or the altered gene hemB* integrated into the genome of a hemB- mutant . Cultures of the hemB* strain KPZn3 had Zn(2+)-dependent ALA dehydratase activity that functioned in vivo as discerned by its heme prototrophy and expression of wild type levels of cellular hemes . Strain KPZn3 elicited root nodules on soybean that contained viable bacteria and exhibited traits of normally developed nodules, and the symbiotic bacteria expressed nearly wild type levels of cellular hemes . We conclude that the Zn(2+)-dependent ALAD* can function and support bacterial tetrapyrrole synthesis within the plant milieu of root nodules.

Neuroreport, 1995 Aug 21, 6(12), 1629 - 32
Nitric oxide modulates blood-brain barrier permeability during infections with an inactivated bacterium; Shukla A et al.; The objective of the present investigation was to study the involvement of NO in regulating the permeability of the blood-brain barrier (BBB) during infections, since NOS is known to be induced following infections . The administration of inactivated Escherichia coli (a source of lipopolysaccharide) or poly (I:C), an interferon inducer, to rats increased the permeability of BBB significantly . This increase was found to be potentiated in the presence of L-arginine, a substrate for NOS, while D-arginine had no such effect . N-nitro L-arginine methyl ester, an inhibitor of NOS, and dexamethasone, an inhibitor of NOS induction, blocked the E . coli-induced effects . These results suggest that during infections, NOS inductions causes the release of large quantities of NO, resulting in increased BBB permeability.

Mol Microbiol, 1995 Aug, 17(4), 643 - 52
Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus; Tabatabai N et al.; In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR . Modulation of the levels of OmpR-phosphate controls the differential expression of ompF and ompC . While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood . We have used a comparative approach to study the sensing function of EnvZ . During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ . Nucleotide sequence analysis revealed that EnvZ of X . nematophilus (EnvZX.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E . coli (EnvZE.c.) . Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ molecules shared 57% sequence identity . In contrast, the large hydrophilic periplasmic domain of EnvZE.c . was absent in EnvZX.n., and was replaced by a shorter hydrophobic region . Although the periplasmic domains had diverged extensively, envZX.n . was able to complement a delta envZ strain of E . coli . OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain . Further genetic analysis established that heterologous phosphorylation between EnvZX.n . and OmpR of E . coli (OmpRE.c.) accounted for the complementation of the delta envZ strain . In addition we show that the OmpR molecules of X . nematophilus and E . coli share 78% amino acid sequence identity . These results indicate that the EnvZ protein of X . nematophilus was able to sense these changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E . coli.

Mamm Genome, 1995 Aug, 6(8), 540 - 5
Natural resistance to infection with Legionella pneumophila: chromosomal localization of the Lgn1 susceptibility gene; Beckers MC et al.; Legionella pneumophila is a strict intracellular pathogen that replicates in the professional phagocytes of the human and guinea pig host . Although murine macrophages from most inbred strains are non-permissive to intracellular replication of L . pneumophila, inflammatory macrophages from the mouse strain A/J are completely permissive to intracellular replication of this bacterium . This genetic difference is controlled by the expression of a single autosomal gene designated Lgn1, with non-permissiveness behaving as completely dominant over permissiveness . We have used a total of 25 AXB/BXA recombinant inbred mouse strains and 182 (A/J x C57BL/6J) x A/J segregating backcross progeny (A/J, permissive; C57BL/6J, non-permissive) to map the Lgn1 gene . Animals were individually typed for tolerance to intracellular replication by in vitro infection of their inflammatory macrophages with L . pneumophila . All animals segregated into two nonoverlapping groups . Examination of the strain distribution pattern of the AXB/BXA strains for Lgn1 initially identified linkage to Chromosome (Chr) 13 markers . Genotyping of the 25 AXB/BXA strains and the 182 backcross progeny for 11 Chr 13 markers established that Lgn1 mapped to Chr 13, with the gene order and intergene distance D13Mit231-(5.5 +/- 1.5)-D13Mit193-(2.2 +/- 0.9)-D13Mit194- (1.1 +/- 0.6)-D13Mit128-(2.6 +/- 1.0)-Lgn1-(2.2 +/- 0.9)-D13Mit70-(3.9 +/- 1.3)- D13Mit73-(7.2 +/- 1.7)-D13Mit53-(0.7 +/- 0.5)-D13Mit32-(0.7 +/- 0.5)-D13Mit77- (0.7 +/- 0.5)-D13Mit78 . This portion of Chr 13 is homologous to the distal portion of human Chr 5, 5q11-5q13, suggesting a possible location of a human LGN1 homolog . Understanding the molecular basis of the high permissiveness of A/J macrophage to L . pneumophila may shed light on the survival strategy of this bacterium in highly permissive human phagocytes . This may be achieved by positional cloning of Lgn1, and the identification of the Lgn1 subchromosomal region reported here is a first step towards that goal.

Cytokine, 1995 Aug, 7(6), 534 - 41
Relative cytokine-stimulating activities of surface components of the oral periodontopathogenic bacterium Actinobacillus actinomycetemcomitans; Reddi K et al.; The purpose of this study was to determine whether bacterial surface components other than lipopolysaccharide (LPS) could stimulate pro-inflammatory cytokine synthesis by mesenchymal and myelomonocytic cells in vitro . LPS, lipid A-associated proteins (LAP) and saline-extractable surface-associated material (SAM) were isolated from the periodontopathogenic bacterium Actinobacillus actinomycetemcomitans and added to cultures of human gingival fibroblasts (HGFs), human PBMCs and the human myelomonocytic MonoMac-6 cell line . Pro-inflammatory cytokine release into culture supernatants was determined by two-site ELISAs . Contrary to expectation, the highly purified LPS extracted from this bacterium was significantly less potent than the other surface extracts in stimulating release of IL-1 beta, IL-6 and TNF-alpha by all three cell types . The SAM was the most potent cytokine-stimulating agent showing equivalent activity to highly purified E . coli LPS in stimulating IL-6 release by PBMCs . LAP also had cytokine-stimulating activity although it was generally significantly less potent than the SAM . Thus in the case of this organism, which is involved in the pathology of chronic inflammatory diseases the LPS does not appear to be the major cytokine-stimulating component.

FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 137 - 43
Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector; Kobayashi Y et al.; A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced . The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats . The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved . The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved . The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest . When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E . coli and B . fibrisolvens by electroporation, and was stably maintained in both hosts . Both ORF1 and ORF2 were required for successful transformation of B . fibrisolvens.

J Bacteriol, 1995 Aug, 177(16), 4609 - 18
appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1; Gomelsky M et al.; A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified . The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system . The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions . Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes . When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors . The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type . To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds . The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant . This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se . The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.

J Bacteriol, 1995 Aug, 177(15), 4575 - 8
Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT; Gak ER et al.; The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT . Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M . flagellatum KT . However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation . A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M . flagellatum KT.

J Bacteriol, 1995 Aug, 177(15), 4537 - 9
Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp . strain F199; Stillwell LC et al.; A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp . strain F199, and a physical map was generated . Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid . Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.

Microbiology, 1995 Aug, 141 ( Pt 8), 1857 - 63
Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase; Schauder S et al.; A sorbitol dehydrogenase (SDH; L-iditol:NAD+ 2-oxidoreductase; EC 1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R . sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene . SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl-Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200 . The relative molecular mass (M(r)) of the native SDH was 61000 as calculated from its Stokes' radius (rs = 3.5 nm) and sedimentation coefficient (S20,w = 4.23S) . SDS-PAGE resulted in one single band representing a polypeptide with a M(r) of 29,000, indicating that the native protein is a dimer . The isoelectric point of SDH was determined to be pH 4.8 . The enzyme was specific for NAD+ and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D-tagatose and of L-iditol . The apparent Km values were NAD+, 0.06 mM; D-glucitol, 6.2 mM; galactitol, 1.5 mM; NADH, 0.13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM . The pH-optimum of substrate oxidation was 11.0 and that of substrate reduction 6.0-7.2 . It was demonstrated that SDH is expressed in the wild-type strain R . sphaeroides Si4 together with MDH during growth on D-glucitol . Forty-four amino acids of the SDH N terminus were sequenced . This sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family.

Microbiology, 1995 Aug, 141 ( Pt 8), 1805 - 19
Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase; Gomelsky M et al.; The photosynthetic bacterium Rhodobacter sphaeroides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes . Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II . Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes . Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant . However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R . sphaeroides . We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus . The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system . Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described . The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing . Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA . We also report the cloning, organization and structure of some of the hup genes from R . sphaeroides and construction of a Hup- strain.

Genetics, 1995 Aug, 140(4), 1307 - 17
Wolbachia infections and the expression of cytoplasmic incompatibility in Drosophila sechellia and D . mauritiana; Giordano R et al.; Various stocks of Drosophila mauritiana and D . sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods . Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D . sechellia but no expression of incompatibility in D . mauritiana . To determine whether absence of cytoplasmic incompatibility in D . mauritiana was due to either the bacterial or host genome, we transferred bacteria from D . mauritiana into an uninfected strain of D . simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia . We also performed the reciprocal transfer of the natural D . simulans Riverside infection into a tetracycline-treated stock of D . mauritiana . In each case, the ability to express incompatibility was unaltered by the different host genetic background . These experiments indicate that in D . simulans and D . mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D . mauritiana harbors a neutral strain of Wolbachia.

Int Immunol, 1995 Aug, 7(8), 1205 - 12
CD4-independent in vivo priming of murine CTL by optimal MHC class I-restricted peptides derived from intracellular pathogens; Dyall R et al.; CTL combat intracellular pathogens by killing infected cells . The molecular targets of their attack are foreign peptides bound to self MHC encoded class I molecules . Immunization of mice with peptides containing CTL determinants was shown to elicit CD4-dependent CTL . Here, we have achieved in vivo CTL priming with naturally processed 8-10 amino acid long class I-restricted peptides emulsified in an adjuvant . A potent, reproducible and physiologically relevant response was obtained using peptides from an intracellular bacterium and five viruses (including HIV) in two murine MHC haplotypes . This method is suitable for multiple vaccination, since a 'cocktail' of peptides derived from three pathogens elicited effector CTL against each pathogen . Most importantly, peptide-induced CD8+CD4- CTL were CD4(+)-independent . These results have implications for CTL induction in situations where CD4 T cells are depleted or compromised, as is the case in HIV infection.

Mol Cell Probes, 1995 Aug, 9(4), 259 - 64
Direct detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction; Begum D et al.; We recently reported the development and assessment of a technique for the detection of Shiga-like toxin-producing Escherichia coli (SLTEC) using the polymerase chain reaction (PCR) and a digoxigenin-11-dUTP-labelled DNA probe . This technique has now been adapted for the direct identification of SLTEC in ground beef . Ground beef homogenates were diluted 1000-fold to reduce the concentration of components which inhibit the thermostable polymerase . Assessment of four different ground beef samples using the PCR detection technique revealed that fat content was a major inhibitory component . As few as 30 SLTEC ml-1 of a ground beef homogenate were detected using the PCR technique, although it was necessary to enrich six of the samples for positive detection . These findings indicate that the PCR detection technique is suitable for the identification of SLTEC directly from contaminated ground beef without isolation of the bacterium or purification of its DNA.

J Biol Chem, 1995 Jul 28, 270(30), 17672 - 3
Proofreading in vivo . Editing of homocysteine by aminoacyl-tRNA synthetases in Escherichia coli; Jakubowski H; Editing reactions are an essential part of biological information transfer processes that require high accuracy, such as replication, transcription, and translation . The editing in amino acid selection for protein synthesis by an aminoacyl-tRNA synthetase, the first proofreading process discovered in the flow of genetic information, prevents attachment of incorrect amino acids to tRNA . Of numerous editing reactions studied in vitro, only one, editing of homocysteine by methionyl-tRNA synthetase, has also been demonstrated in vivo . It is therefore unclear to what extent editing of errors is physiologically relevant . Here we show that isoleucyl- and leucyl-tRNA synthetases also edit homocysteine by cyclizing it to homocysteine thiolactone in the bacterium Escherichia coli . These and other data also suggest that metabolite compartmentation or channeling governs which synthetase participates in editing in bacterial cells.

Biochem J, 1995 Jul 15, 309 ( Pt 2), 543 - 50
Complete nucleotide sequence of the gene encoding bacteriophage E endosialidase: implications for K1E endosialidase structure and function; Long GS et al.; Bacteriophage E specifically recognizes and infects strains of Escherichia coli which display the alpha-2,8-linked polysialic acid K1 capsule . Bacteriophage E endosialidase, which is thought to be responsible for initial absorption of the phage to the host bacterium, was purified, and the N-terminal amino acid sequences of the polypeptide monomer and cyanogen bromide fragments were determined . Synthetic oligonucleotide probes were designed from the N-terminal amino acid sequences and used to identify restriction fragments of bacteriophage E DNA encoding the endosialidase . The primary nucleotide sequence of the bacteriophage E endosialidase gene contains an open reading frame encoding a 90 kDa polypeptide which is processed to give a mature 74 kDa protein . The native enzyme is probably a trimer of identical 74 kDa subunits . In the bacteriophage E genome the K1E endosialidase open reading frame is preceded by a putative upstream promoter region with homology to a bacteriophage SP6 promoter . A central region of 500 amino acids of the deduced protein sequence of the K1E endosialidase was found to have 84% identity to K1F endosialidase . Both endosialidases contain two copies of a sialidase sequence motif common to many bacterial and viral sialidases . These sequences flank the region of greatest identity between the two endosialidase forms, which suggests that this central domain is involved in binding and hydrolysis of the polysialic acid substrate.

FEBS Lett, 1995 Jul 10, 368(1), 23 - 6
A novel 6Fe (2 x {3Fe-4S}) ferredoxin from Mycobacterium smegmatis; Imai T et al.; A novel ferredoxin was purified from Mycobacterium smegmatis by a series of hydrophobic chromatographies in the presence of high concentrations of ammonium sulfate and sodium chloride . The ferredoxin exhibited the same peptide map and N-terminal amino acid sequence as the known 7Fe ferredoxin from the same bacterium . On the other hand, this ferredoxin was found to contain approximately 6 Fe/mol ferredoxin and was also shown to contain only {3Fe-4S} clusters by resonance Raman spectroscopy, indicating that it is a novel 6Fe ferredoxin which contains two {3Fe-4S} clusters.

Gene, 1995 Jul 4, 160(1), 55 - 8
SgfI, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5'-GCGAT/CGC-3'; Kappelman JR et al.; A new restriction endonuclease (ENase), SgfI, has been isolated from the bacterium Streptomyces sp . SgfI recognizes the 8-bp palindrome 5'-GCGATCGC-3' and cleaves double-stranded DNA after the T in this sequence, producing a two-base 3' overhang compatible with PvuI termini . SgfI is a rare-cutting ENase and should be useful for megabase mapping experiments.

Biochemistry, 1995 Jul 4, 34(26), 8390 - 7
Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus; Sebban P et al.; Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy . These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB . In the wild-type Rb . sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site . These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant . As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant . The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds . In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively . The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1995 Jul 3, 1250(1), 49 - 59
Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus; Pieulle L et al.; We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus . The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg . D . africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters . EPR spectroscopic study of the enzyme indicates the presence of three {4Fe-4S}2+/1- centers/subunits . The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV . The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added . This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor . The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation . The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process . D . africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme . POR was shown to be located in the cytoplasm by immunogold labelling.

Emerg Infect Dis, 1995 Jul-Sep, 1(3), 79 - 85
Spiral bacteria in the human stomach: the gastric helicobacters; Dubois A; During the past decade, Helicobacter pylori has become recognized as one of the most common human pathogens, colonizing the gastric mucosa of almost all persons exposed to poor hygienic conditions from childhood . It also is often found, albeit with a lower frequency, in groups of high socioeconomic status . H . pylori causes chronic active gastritis and is a major factor in the pathogenesis of duodenal ulcers and, to a lesser extent, gastric ulcers . In addition, the presence of this bacterium is now recognized as a risk factor for gastric adenocarcinoma and lymphoma . Nevertheless, most infections appear without clinical consequences . In this second decade of intensive research, it is important to understand why H . pylori is sometimes a dangerous pathogen, and to determine how it can be eradicated in those at highest risk for severe disease.

Infect Immun, 1995 Jul, 63(7), 2674 - 81
Helicobacter pylori-induced gastritis in the domestic cat; Fox JG et al.; Helicobacter pylori has been cultured from the inflamed gastric mucosae of naturally infected cats; the lesions in H . pylori-infected cat stomachs mimic many of the features seen in H . pylori-infected human stomachs . To determine whether H . pylori-negative specific-pathogen-free cats with normal gastric mucosae were susceptible to colonization by this bacterium and whether gastritis developed after infections, four H . pylori-negative cats treated with cimetidine were orally dosed three times with 3 ml (1.5 x 10(8) CFU/ml) of H . pylori every 4 days . All four cats became persistently colonized as determined by gastric cultures and PCRs from serial gastric biopsy samples and necropsy samples at 7 months postinfection . H . pylori was not isolated from the two control cats, nor were their gastric tissues positive by PCR; one of the two cats had a few focal lymphocytic aggregates in the body submucosa, whereas the second cat had a normal gastric mucosa . All four H . pylori-infected cats had multifocal gastritis consisting of lymphoid aggregates plus multiple large lymphoid nodules, which were most noticeable in the antral mucosa . In addition, one H . pylori-infected cat had a moderate diffuse infiltration of polymorphonuclear leukocytes in the subglandular region of the antrum . H . pylori-like organisms were focally distributed in glandular crypts of the antrum . Two of the H . pylori-infected cats had significant (eightfold) increases over baseline in levels of immunoglobulin G H . pylori serum antibody . The H . pylori isolates from the four experimentally infected cats had restriction fragment length polymorphism patterns specific for the flaA gene that were identical to those of the inoculating strain . H . pylori readily colonizes the cat stomach and produces persistent gastritis.

Infect Immun, 1995 Jul, 63(7), 2625 - 31
Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin; Sandberg AL et al.; Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria . In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin . The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R . communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs . These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces . The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae . Therefore, the PMN glycoprotein receptor for A . naeslundii is clearly distinct from those recognized by E . coli . Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides . Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs.

Gut, 1995 Jul, 37(1), 30 - 4
Recrudescence of Helicobacter pylori after apparently successful eradication: novel application of randomly amplified polymorphic DNA fingerprinting; Xia HX et al.; The aim of this study was to find out if reinfection or recrudescence accounted for the recurrence of Helicobacter pylori infections after apparent eradication of the bacterium . Three hundred and twenty patients were treated with colloidal bismuth subcitrate (120 mg four times daily for four weeks), metronidazole and tetracycline (400 mg and 500 mg, respectively, thrice daily for the first week) . H pylori was eradicated four weeks after the end of treatment as assessed by the rapid urease test, histological examination, Gram staining, and culture . However, the infection recurred in 29 (9.1%) of the patients one year after apparent eradication . Pre and posteradication isolates from five patients were available . DNA was extracted and used for restriction endonuclease analysis with Hind III and Hae III, and for polymerase chain reaction (PCR) based randomly amplified polymorphic DNA fingerprinting with a combination of two 10 nucleotide primers . Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis was performed also . Randomly amplified polymorphic DNA fingerprinting was unique in that it yielded highly discriminatory fingerprints, which showed that the pretreatment and recurrent isolates obtained from each of the five patients were indistinguishable from one another . This shows that recurrence of H pylori infection is probably caused by recrudescence and that the discriminatory power of randomly amplified polymorphic DNA fingerprinting is a practicable and discriminatory typing scheme for H pylori.

Biophys J, 1995 Jul, 69(1), 280 - 6
Mechanical limits of bacterial flagellar motors probed by electrorotation; Berry RM et al.; We used the technique of electrorotation to apply steadily increasing external torque to tethered cells of the bacterium Escherichia coli while continuously recording the speed of cell rotation . We found that the bacterial flagellar motor generates constant torque when rotating forward at low speeds and constant but considerably higher torque when rotating backward . At intermediate torques, the motor stalls . The torque-speed relationship is the same in both directional modes of switching motors . Motors forced backward usually break, either suddenly and irreversibly or progressively . Motors broken progressively rotate predominantly at integral multiples of a unitary speed during the course of both breaking and subsequent recovery, as expected if progressive breaking affects individual torque-generating units . Torque is reduced by the same factor at all speeds in partially broken motors, implying that the torque-speed relationship is a property of the individual torque-generating units.

Trends Biotechnol, 1995 Jul, 13(7), 269 - 78
Unique peptide modifications involved in the biosynthesis of lantibiotics; Jack RW et al.; The lantibiotics are a unique class of bacterium-derived peptide antibiotics, all of which contain the rare amino acid lanthionine, as well as a number of other nonprotein amino acids . Unlike most other peptide antibiotics, lantibiotics are produced on the ribosome as a prepeptide, and a series of post-translational modifications converts this precursor into the biologically active peptide . A complex set of gene products involved in lantibiotic biosynthesis have been identified, including the genes for specific amino acid modifications, as well as other ancillary biosynthetic functions . In the future, it should be possible to use some of these enzymes to engineer novel, non-protein amino acids into other proteins of biotechnological interest and importance.

J Bacteriol, 1995 Jul, 177(14), 4089 - 96
Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development; Laue BE et al.; The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation . The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores . During the growth portion of the M . xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants . Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase . In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores . To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population . Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently . Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores . The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture . In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores . These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.

J Clin Pathol, 1995 Jul, 48(7), 662 - 6
High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay; Li C et al.; AIMS--To investigate the prevalence of Helicobacter pylori in the saliva of patients infected with this bacterium . METHODS--A novel polymerase chain reaction (PCR) assay was developed to detect H pylori in saliva and gastric biopsy specimens from patients undergoing endoscopy . RESULTS--Our PCR assay amplified a 417 base pair fragment of DNA from all 21 DNAs derived from H pylori clinical isolates but did not amplify DNA from 23 non-H pylori strains . Sixty three frozen gastric biopsy and 56 saliva specimens were tested . H pylori specific DNA was detected by PCR in all 39 culture positive biopsy specimens and was also identified from another seven biopsy specimens which were negative by culture but positive by histology . H pylori specific DNA was identified by PCR in saliva specimens from 30 (75%) of 40 patients with H pylori infection demonstrated by culture or histological examination, or both, and in three patients without H pylori infection in the stomach . CONCLUSION--The results indicate that the oral cavity harbours H pylori and may be the source of infection and transmission.

Appl Biochem Biotechnol, 1995 Jul-Sep, 54(1-3), 271 - 5
Use of a pentachlorophenol degrading bacterium to bioremediate highly contaminated soil; Colores GM et al.; A Sphingomonas species that mineralizes high concentrations of pentachlorophenol (PCP) was isolated from a PCP-contaminated EPA Superfund site . This bacterium, identified as Sphingomonas sp . strain RA2, is able to degrade PCP at concentrations of up to 300 micrograms/mL in liquid culture . This organism was tested for its ability to degrade high concentrations of PCP in a soil that did not contain organisms capable of degrading high concentrations of PCP . When inoculated into contaminated soil, Sphingomonas sp . RA2 mineralized PCP at concentrations of 300, 600, 900, and 1200 micrograms PCP/g of soil, but was unable to mineralize 1500 micrograms PCP/g of soil . Only very minimal loss of PCP was seen in uninoculated soils . The results of this study demonstrate that Sphingomonas sp . RA2 may be a useful organism for remediation of sites contaminated with high concentrations of PCP.

Proteins, 1995 Jul, 22(3), 226 - 44
Structural model of the photosynthetic reaction center of Rhodobacter capsulatus; Foloppe N et al.; The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis . However, no three-dimensional structure is available for this protein . We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations . In the Rb . capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps . viridis and Rb . sphaeroides . The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements . A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps . viridis, Rb . sphaeroides, and Rb . capsulatus.

Biochim Biophys Acta, 1995 Jun 30, 1230(3), 147 - 54
Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum; Visschers RW et al.; Spectroscopic properties, including low-temperature absorbance, linear and circular dichroism and site-selection fluorescence of the antenna complexes from Rhodospirillum molischianum have been determined . The unique 'LH1-like' character of the amino acid sequence from LH2 of this bacterium is reflected in the circular dichroism of the B850 band of this complex . The wavelength dependence of the polarization of the LH2 complex shows an unusual shape that is attributed to the octameric state of this complex . The complete amino acid sequence for the LH1 alpha-polypeptide and most of the beta-polypeptides are presented . These conform to the general features of other LH1 polypeptides . This result, in combination with spectroscopic data for LH1 imply that the organisation of the core in this bacterium is not much different from that in other purple non-sulphur bacteria.

Biochim Biophys Acta, 1995 Jun 30, 1230(3), 119 - 29
Cloning, sequencing, and mutagenesis of the cytochrome c4 gene from Azotobacter vinelandii: characterization of the mutant strain and a proposed new branch in the respiratory chain; Ng TC et al.; Azotobacter vinelandii is a free-living, nitrogen-fixing bacterium with a branched electron transport chain terminating with two terminal oxidases, cytochromes d and o . Cytochrome o is thought to receive its electrons from cytochromes c . The gene encoding cytochrome c4 has been cloned and sequenced (termed the cycA locus) . The deduced amino acid sequence contains a 20 residue signaling peptide sequence on the N-terminal end . Mutagenesis was performed by inserting a Kmr cassette into the structural gene . The subsequent mutant strains showed reduced amounts of cytochromes c (approximately 60% of wild-type levels) based on difference absorption spectra measurements . Heme staining confirmed the complete loss of cytochrome c4 protein in the mutant strains . These mutants could grow and respire normally, like the wild type, under both diazotrophic or non-diazotrophic conditions . Surprisingly, the cytochrome o terminal oxidase was still turning over in membranes from the cycA mutants as evidenced by substrate-reduced CO difference spectra and inhibition experiments with the use of the cytochrome o inhibitor, chlorpromazine . Still, the levels of oxidation by ascorbate-TMPD were greatly reduced in the cycA mutants . Therefore, it is proposed that cytochrome c4 does not exist in complex with cytochrome o as a multi-component terminal oxidase complex, yet still passes electrons to it in parallel like cytochrome c5, as opposed to in an obligate sequential manner with cytochrome c5 . In this pathway the proposed new branch is at the ubiquinone to cytochromes c level.

J Mol Biol, 1995 Jun 30, 250(1), 1 - 10
Two-dimensional crystallization and preliminary structure analysis of light harvesting II (B800-850) complex from the purple bacterium Rhodovulum sulfidophilum; Montoya G et al.; Light-harvesting complex II (B800/850) from the purple bacterium Rhodovulum (Rhv.) sulfidophilum has been isolated using a new protocol . It has been shown by analytical ultracentrifugation and native gels to be most likely an octamer . Two-dimensional crystals have been obtained by microdialysis . The plane group is p4212 with a = b = 157 A . The crystals diffract to 18 A in negative stain . Projection maps show clearly that LHII is organized in ring-like particles with an outer diameter of about 76 A.

Biochem Biophys Res Commun, 1995 Jun 26, 211(3), 892 - 900
Evidence for close in vivo association of protein with mitochondrial ribosomal RNA in a trypanosome; Tittawella I; Riboprotein particles containing the ribosomal RNA-like 9S and 12S RNAs in the trypanosome mitochondrion have never been isolated . Using the formaldehyde cross-linking procedure I show here that one or more of three proteins, q2 (16.5 kDa), r (15 kDa) and s (13 kDa), are closely associated in vivo with the 9S and 12S RNAs of the trypanosomatid parasite, Crithidia fasciculata . These proteins were also found to be associated with the parasite's cytosolic ribosomal RNA and to have strong immunological cross reactivity with riboprotein S11 of the bacterium Escherichia coli . These data provide the first evidence for the association of riboprotein-like proteins with the 9S and 12S mitochondrial RNA in a trypanosome, possibly as components of a mitochondrial ribosome.

Arch Biochem Biophys, 1995 Jun 20, 320(1), 149 - 54
Magnetic resonance of Fe-S clusters: isolation and characterization of a 7Fe ferredoxin from Rhodopseudomonas palustris; Battistuzzi G et al.; A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties . Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and 1H NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one {3Fe-4S} and one {4Fe-4S}, which places this protein in the class of 7Fe ferredoxins . The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0 . The molecular mass around 19 kDa makes this protein the heaviest known in this class . This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.

J Biol Chem, 1995 Jun 16, 270(24), 14439 - 44
Comparison of the enzymatic properties of the two Escherichia coli lysyl-tRNA synthetase species; Brevet A et al.; In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU . The two corresponding enzymes could be purified at homogeneity from a delta lysU and a delta lysS strain, respectively . Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PPi exchange as well as in the tRNALys aminoacylation reaction . Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex . In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase . This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium . Remarkably, these conditions also induce lysU expression . Homogeneous LysU and LysS were also compared in Ap4A synthesis . LysU is only 2-fold more active than LysS in the production of this dinucleotide . This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of Ap4A inside stressed Escherichia coli cells . This conclusion could be strengthened by determining the concentrations of Ap4N (N = A, C, G, or U) in a delta lysU as well as in a lysU+ strain, before and after a 1-h temperature shift at 48 degrees C . The measured concentration values were the same in both strains.

Biochim Biophys Acta, 1995 Jun 9, 1244(2-3), 269 - 76
Characterisation of glucose transport in Helicobacter pylori; Mendz GL et al.; The nature of the glucose transport system in the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis . Fast D-glucose uptake was demonstrated by using two methods of measuring glucose transport . The transport of 2-deoxy-D-glucose was inhibited competitively by D-glucose; and the efflux of 2-deoxy-D-glucose from cells also was affected by the presence of D-glucose . The transport of 2-deoxy-D-glucose was saturable with a Km of 4.8 mM and Vmax of 146.6 pmol (microliter cell water)-1 at 20 degrees C . The transport was temperature-dependent with energies of activation of 6.8 and 51.0 kJ mol-1 for 0.2 and 20 mM 2-deoxy-D-glucose, respectively . The temperature dependence and saturable nature of transport suggested the presence of one or more glucose carriers . The substrate specificity of the transport system was studied by measuring the effects of mono- and disaccharides on the rates of transport of the glucose analogue . The most significant inhibitory effects were obtained with D-galactose and L-arabinose . Lack of transport inhibition by L-glucose established the stereospecificity of the transporters for the D-isomer of glucose . Higher rates of 2-deoxy-D-glucose transport were measured in the presence of sodium ions than for other monovalent cations, and the presence of amiloride inhibited transport of the monosaccharide . No inhibition was observed with cytochalasin B, phloretin or phloridzin . The results suggested the existence of specific D-glucose transporters and that the glucose transport system of H . pylori is significantly different from other known bacterial transporters.

Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5572 - 6
Genomic DNA fingerprinting by restriction fragment end labeling; van Steenbergen TJ et al.; A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans . Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with {alpha-32P}dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel) . Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides . For A . actinomycetemcomitans, all strains had bands in common . Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types . Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated . The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods . When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found . Isolates from different locations of one patient showed indistinguishable patterns . Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients . It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

J Biol Chem, 1995 Jun 2, 270(22), 13186 - 91
The flavinylation reaction of trimethylamine dehydrogenase . Analysis by directed mutagenesis and electrospray mass spectrometry; Packman LC et al.; The flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from Methylophilus methylotrophus (bacterium W3A1) and Escherichia coli were studied by electrospray mass spectrometry (ESMS) . The ESMS analyses demonstrated for the first time that wild-type enzyme expressed in M . methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme . ESMS demonstrated that the deflavo forms of the recombinant wild-type and mutant enzymes are not post-translationally modified and therefore prevented from assembling with flavin mononucleotide (FMN) because of previously unrecognized modifications . The data suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expression levels in E . coli . Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative proportion of flavinylated product was substantially less than that seen for the recombinant wild-type enzyme . No flavinylation products were observed for a double mutant (His-29 to Cys-29; Cys-30 to His-30), in which the positions of the putative flavinylation base and cysteine nucleophile were exchanged . Taken together, the data indicate that the assembly of trimethylamine dehydrogenase with FMN occurs during the folding of the enzyme, and in the fully folded form, deflavo enzyme is unable to recognize FMN . Results of site-directed mutagenesis experiments in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced . Consequently, in the folded mutant enzymes, less flavin is trapped in the active site, and reduced levels of flavinylated product are obtained.

Zhonghua Wai Ke Za Zhi, 1995 Jun, 33(6), 339 - 41
{Local arterial infusion of 5-FU in treatment of acute necrotic pancreatitis}; Gu F et al.; In 19 patients with acute necrotic pancreatitis the cordis tube was inserted in the arterial suppling blood . If the necrotic site at the head of pancreata, the tube was placed in the arteria gastroduodenalis or arteria pancreaticoduodenalis . When the site at the trunk or rearby, the tube was placed in the arteria lienalis or coeliaca, and when the necrosis all over the pancreata, the tube in the arteria coeliaca . Through the tube, infusion drugs mainly 5-FU were given within 20 days from onset of the disease . Compared with the controlled group of 68 patients, the mortalities were 10.5% and 30.9%, and the rates of organ falls 5.3% and 42.7% . The second infection of bacterium was 5.3% and 44.1% respectively . We suggest that local arterial infusion of 5-FU is an effective treatment for acute necrotic pancreatitis and creats conditions for delayed operations, clears the remaining tissue of necrotic pancreatitis easily.

Mol Microbiol, 1995 Jun, 16(6), 1067 - 73
Treponema pallidum and the quest for outer membrane proteins; Radolf JD; Treponema pallidum, the syphilis spirochaete, has a remarkable ability to evade the humoral and cellular responses it elicits in infected hosts . Although formerly attributed to the presence of an outer coat comprised of serum proteins and/or mucopolysaccharides, current evidence indicates that the immuno-evasiveness of this bacterium is largely the result of its unusual molecular architecture . Based upon a combination of molecular, biochemical, and ultrastructural data, it is now believed that the T . pallidum outer membrane (OM) contains a paucity of poorly immunogenic transmembrane proteins ('rare outer membrane proteins') and that its highly immunogenic proteins are lipoproteins anchored predominantly to the periplasmic leaflet of the cytoplasmic membrane . The presence in the T . pallidum OM of a limited number of transmembrane proteins has profound implications for understanding syphilis pathogenesis as well as treponemal physiology . Two major strategies for molecular characterization of rare outer membrane proteins have evolved . The first involves the identification of candidate OM proteins as fusions with Escherichia coli alkaline phosphatase . The second involves the characterization of candidate OM proteins identified in outer membranes isolated from virulent T . pallidum . Criteria to define candidate OM proteins and for definitive identification of rare OM proteins are proposed as a guide for future studies.

Environ Health Perspect, 1995 Jun, 103 Suppl 5, 33 - 6
Bacterial growth with chlorinated methanes; Leisinger T et al.; Chlorinated methanes are important industrial chemicals and significant environmental pollutants . While the highly chlorinated methanes, trichloromethane and tetrachloromethane, are not productively metabolized by bacteria, chloromethane and dichloromethane are used by both aerobic and anaerobic methylotrophic bacteria as carbon and energy sources . Some of the dehalogenation reactions involved in the utilization of the latter two compounds have been elucidated . In a strictly anaerobic acetogenic bacterium growing with chloromethane, an inducible enzyme forming methyltetrahydrofolate and chloride from chloromethane and tetrahydrofolate catalyzes dehalogenation of the growth substrate . A different mechanism for the nucleophilic displacement of chloride is observed in aerobic methylotrophic bacteria utilizing dichloromethane as the sole carbon and energy source . These organisms possess the enzyme dichloromethane dehalogenase which, in a glutathione-dependent reaction, converts dichloromethane to inorganic chloride and formaldehyde, a central metabolite of methylotrophic growth . Sequence comparisons have shown that bacterial dichloromethane dehalogenases belong to the glutathione S-transferase enzyme family, and within this family to class Theta . The dehalogenation reactions underlying aerobic utilization of chloromethane by a pure culture and anaerobic growth with dichloromethane by an acetogenic mixed culture are not known . It appears that they are based on mechanisms other than nucleophilic attack by tetrahydrofolate or glutathione.

Crit Care Med, 1995 Jun, 23(6), 1040 - 7
Long-term survival after intensive care unit admission with sepsis; Sasse KC et al.; OBJECTIVE: To evaluate the long-term survival of critically ill patients with sepsis and to assess the factors predictive of long-term survival (> 1 month after admission date) . DESIGN: Prospective, cohort study . SETTING: Medical/surgical intensive care unit (ICU) in a multidisciplinary community hospital . PATIENTS: All patients admitted to the ICU from January 1, 1987 to March 31, 1991 who both demonstrated clinical evidence of the systemic inflammatory response syndrome and yielded blood cultures positive for a bacterium or fungus (n = 153) . INTERVENTIONS: Random set of procedures normally performed in an ICU setting . MEASUREMENTS AND MAIN RESULTS: Patient characteristics, including age, blood culture results, comorbid conditions, and severity of illness as estimated by the Acute Physiology Score of the Acute Physiology and Chronic Health Evaluation II prognostic system were recorded . Follow-up evaluation utilizing the National Death Index provided survival outcome for all patients 1 yr after hospital discharge . The mortality rate at hospital discharge was 51.0%, and mortality rates at 1 month, 6 months, and 1 yr after admission date were 40.5%, 64.7%, and 71.9%, respectively . A total of 33 patients survived beyond the period of observation . The analyses demonstrated the following findings: a) the survival rate was negatively correlated with the Acute Physiology Score up to 1 month after hospital admission date, but uncorrelated thereafter; b) fungal infections, such as Candida, had the shortest survival prospects of any blood-borne infection; and c) both malignancy and human immunodeficiency virus infection contributed to poorer outcomes, but differed in their patterns of long-term survival . CONCLUSIONS: The most critical period for surveillance of bacteremic patients was in months 2 through 6 after discharge, during which time, the percentage of patients surviving decreased dramatically . The degree of physiologic derangement, as measured by the Acute Physiology Score, was a useful measure of prognosis within the first month after the score was assessed at ICU admission . However, beyond this period, prognostic utility decreased significantly . Healthcare providers should use caution concerning the expected survival of hospitalized patients with human immunodeficiency virus, based on experience with distinct conditions, such as malignancies.

Biochem J, 1995 Jun 1, 308 ( Pt 2), 501 - 5
N-acetyl-D-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-D-mannosamine and pyruvate; Rodriguez-Aparicio LB et al.; Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate . When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity) . However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared . A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak . Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate . Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation . Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected . These findings suggest that NeuAc synthase is not present in E . coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.

J Bacteriol, 1995 Jun, 177(12), 3579 - 86
ccrA1: a mutation in Streptomyces coelicolor that affects the control of catabolite repression; Ingram C et al.; The regulation of carbon utilization is of central importance in the gene expression pathways for both morphological development and antibiotic production in Streptomyces species . We report the identification and characterization of a mutation in Streptomyces coelicolor, ccrA1, that affects the expression of several catabolite-controlled promoters . ccrA1 mutants are altered in expression of galP1, the glucose-sensitive, galactose-dependent promoter of the galactose utilization operon; in expression of the glycerol utilization operon, which is glucose sensitive and glycerol dependent; and in expression of chi63, the glucose-sensitive chitin-dependent promoter of a gene involved in chitin utilization . ccrA1 has no effect on the expression of galP2, a promoter that directs constitutive transcription of the galE and galK genes . ccrA1 maps to a region of the S . coelicolor genome which distinguishes it from other mutations known to be involved in catabolite control . We suggest that ccrA1 identifies a gene whose product may be involved in the general regulation of carbon catabolite repression in this complex bacterium.

Lab Anim Sci, 1995 Jun, 45(3), 239 - 43
Adaptation of the {13C}urea breath test as a noninvasive method for detection of Helicobacter pylori infection in squirrel monkeys (Saimiri spp.); Stadtlander CT et al.; The {13C}urea breath test was adapted for use in squirrel monkeys (Saimiri spp.) for identification of experimentally induced infection with Helicobacter pylori, the bacterium causing gastric ulcer in humans . A canine anesthesia inhalation mask was modified with a volume-reducing insert allowing sufficient breath collection from these small primates within 30 sec . Fourteen milligrams of {13C urea per kilogram of body weight was adequate for clear distinction between experimentally infected and noninfected animals . Initial infection of five squirrel monkeys resulted in increased 13CO2 in breath within 3 days after inoculation with H . pylori . Additional inoculation with H . pylori superimposed on an existing gastric population caused a transient increase in breath 13CO2 values, which gradually declined over the following 15 days . Breath test results indicating H . pylori infection were confirmed by high {13C} concentration in blood, by urease-positive culture, modified Steiner stain reaction, and Western blot analysis . This modified {13C}urea breath test provides a rapid, reproducible, noninvasive method for screening small primates used as nonhuman models for the study of gastric infection with H . pylori.

Biosci Biotechnol Biochem, 1995 Jun, 59(6), 1135 - 6
Purification and characterization of beta-N-acetylglucosaminidase from Alteromonas sp . strain O-7; Tsujibo H et al.; beta-N-Acetylglucosaminidase (EC 3.2.1.30) was purified from the outer membrane of a marine bacterium, Alteromonas sp . strain O-7 . The enzyme (GlcNAcase A) was purified by successive column chromatographies . The purified enzyme was found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis . The molecular mass and pI of GlcNAcase A were 92kDa and 4.9, respectively . The optimum pH and temperature were 6.0-7.0 and 45 degrees C, respectively . GlcNAcase A was stable up to 40 degrees C at pH 7.0, and hydrolyzed N-acetylchitooligosaccharides from dimer to hexamer . The amino-terminal 16 amino acid residues of GlcNAcase A were sequenced.

Eur J Biochem, 1995 Jun 1, 230(2), 666 - 75
The molybdenum nitrogenase from wild-type Xanthobacter autotrophicus exhibits properties reminiscent of alternative nitrogenases; Schneider K et al.; In the presence of molybdate (1 microM) 2-3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm) . The maximal specific activity obtained was 145 nmol ethylene reduced . min-1 . mg protein-1 (crude extract) . The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source . Vanadium compounds stimulated neither growth nor nitrogenase activity . Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level . The nitrogenase, responsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom . These results indicate that, in X . autotrophicus, a Mo-independent nitrogenase does not exist . However, the molybdate-containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases . The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane . Under certain conditions, i.e . in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured . Parallel to the pH-dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities {E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)} . The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH . They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.

Arch Microbiol, 1995 Jun, 163(6), 400 - 6
Purification, primary structure, and evolution of cytochrome c-550 from the magnetic bacterium, Magnetospirillum magnetotacticum; Yoshimatsu K et al.; Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined . The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form . Its midpoint redox potential at pH 7.0 was determined to be +289 mV . The primary structure of cytochrome c-550 was determined . Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c . Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c2, suggesting that M . magnetotacticum is phylogenetically related to photosynthetic bacteria.

J Hosp Infect, 1995 Jun, 30 Suppl, 372 - 82
Mycobactericidal testing of disinfectants: an update; Sattar SA et al.; Tuberculosis, a major killer in developing countries, is on the rise again in industrialized nations . AIDS, increased use of immunosuppression and the emergence of multiple drug-resistant Mycobacterium tuberculosis (MDR-TB) have further enhanced its significance . TB is projected to cause 3.5 million deaths per year by 2000 . Also, other types of mycobacteria are being incriminated in human infections with increasing frequency . Thus, the enhanced risk of nosocomial and iatrogenic spread of mycobacteria is forcing a review of infection control in general and claims of mycobactericidal activity of disinfectants in particular . Mycobacteria are more resistant to disinfection than enveloped viruses and other types of vegetative bacteria, but a proper comparison with non-enveloped viruses requires more data . Flaws in currently used protocols for mycobacterial activity are: (i) a lack of proper quantitation; (ii) unrealistically long contact times at higher than ambient temperatures; (iii) absence of a suitable organic load; (iv) ineffective neutralizers; (v) unsuitable surrogates for M . tuberculosis; (vi) improper recovery media; and (vii) inappropriate types of carriers . Furthermore, we have recently found a product meant for 14 day reuse to become non-mycobactericidal after only a week under actual use in an endoscopy unit . These considerations make the available data on product efficacy unreliable, especially in view of the increasing threat from MDR-TB . Recent findings suggest that the use of Mycobacterium terrae as a surrogate, better recovery media, flat surfaces as carriers, elimination of neutralizers, proper removal of cell clumps and a required > or = 4 log10 reduction in the number of colony forming units of the test bacterium after disinfectant treatment should make mycobacteridal tests more precise and reliable, thus making product registration and selection easier . There is also an urgent need to develop standardized protocols to determine the mycobactericidal activity of disinfectants under conditions of reuse.

FEBS Lett, 1995 May 29, 365(2-3), 172 - 8
1H and 13C NMR assignment and secondary structure of Chlorobium limicola f . thiosulfatophilum ferrocytochrome c555; Morelle N et al.; The 1H resonances of the ferrocytochrome c555 from the anaerobic green sulfur bacterium Chlorobium limicola f thio-sulfatophilum (strain Tassajara) have been assigned . Identification of spin systems and sequential assignment of 1H was accomplished by automated assignment computer programs followed by manual verification . In addition, 13C resonances have been extensively assigned by HSQC experiments at natural abundance . As determined by short-range NOE connectivities, 13C alpha chemical shifts, and HN exchange experiments, the secondary structure consists of 3 helices ranging from residues 3-13, 43-53 and 70-86 . Interestingly, the second helix is significantly longer than observed by X-ray crystallography {1977, Proc . Natl . Acad . Sci . USA 74, 5244-5247} . A topological model of the cytochrome c555 is presented based on a small number of long-range NOE contacts . The helices are shown to pack onto the heme according to the pattern common to all class I cytochromes c.

J Mol Biol, 1995 May 26, 249(1), 59 - 68
Pseudoknot in domain II of 23 S rRNA is essential for ribosome function; Rosendahl G et al.; The structure of domain II in all 23 S (and 23 S-like) rRNAs is constrained by a pseudoknot formed between nucleotides 1005 and 1138, and between 1006 and 1137 (Escherichia coli numbering) . These nucleotides are exclusively conserved as 1005C.1138G and 1006C.1137G pairs in all Bacteria, Archaea and chloroplasts, whereas 1005G.1138C and 1006U.1137A pairs occur in Eukarya . We have mutagenized nucleotides 1005C-->G, 1006C-->U, 1137G-->A and 1138G-->C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E . coli . The ability of 23 S rRNA to support cell growth is reduced when either of these base-pairs is disrupted, and it is completely abolished upon disruption of both base-pairs . Each mutant 23 S rRNA is assembled into 50 S subunits, but the mutant subunits do not stably interact with 30 S to engage in protein synthesis . Enzymatic and chemical probing of ribosomal particles reveals increased accessibility in the rRNA structure close to the sites of the mutations . The degree to which the mutations increase rRNA accessibility correlates with the severity of their phenotypic effects . Nucleotide 1131G is extremely reactive to dimethyl sulphate modification in wild-type subunits and ribosomes, but is rendered unreactive when either the pseudoknot is broken or when the r-proteins are removed . The structure of the pseudoknot region is possibly influenced by interaction of an r-protein at or close to the pseudoknot . Re-establishing the pseudoknot Watson-Crick interactions with one "eukaryal" (1005G.1138C or 1006U.1137A) pair and one "bacterial" C.G pair largely restores the structure and function of the rRNA . Bacterial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the structure of their pseudoknot region is partially open and accessible.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 5189 - 93
Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins; Gobin J et al.; Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection . To do so, it releases high-affinity iron-binding siderophores called exochelins . Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium . In this paper, we describe the purification of exochelins of M . tuberculosis and their characterization by mass spectrometry . Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state . The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain . These series further subdivide into serine- or threonine-containing species . The virulent M . tuberculosis Erdman strain and the avirulent M . tuberculosis H37Ra strain produce a similar set of exochelins . Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins . However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester . These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

FEBS Lett, 1995 May 22, 365(1), 10 - 2
4-Hydroxyphenethyl alcohol--a new cytokinin-like substance from the phototrophic purple bacterium Rhodospirillum rubrum 1R; Serdyuk OP et al.; Three compounds with cytokinin activity have been isolated from the medium of Rhodospirillum rubrum grown photosynthetically . Two N-6 aminopurine cytokinins revealed in the medium were identical with those obtained from R . rubrum cells previously . The third compound with cytokinin activity in Amaranthus caudatus bioassay proved to be a simple phenolic compound with elemental composition C8H10O2 . This cytokinin-like substance according to absorption spectra, mass spectrometry and 1H NMR spectra data was identified as 4-hydroxyphenethyl alcohol.

Gene, 1995 May 19, 157(1-2), 43 - 7
Organization and gene expression within restriction-modification systems of Herpetosiphon giganteus; Kroger M et al.; We have characterized a family of related restriction-modification (R-M) systems from the soil bacterium Herpetosiphon giganteus (Hgi) . A comparison of their genetic organization reveals two types of regulatory proteins, called controlling ORF C . While one of these small reading frames derived from RM.HgiCI seems to be an enhancer of its own promoter, evidence is provided for a silencer function of the other ORF C derived from the closely related AvaII-type systems RM.HgiBI/CII/EI . The respective silencer function is detected during our various attempts to clone three isoschizomers with unusually high differences in their specific activity . Sequencing and site-directed mutagenesis revealed just two amino acids as being responsible for a massive increase in specific activity of these endonucleases.

Biochemistry, 1995 May 2, 34(17), 5904 - 12
NMR assignment of Rhodobacter capsulatus ferricytochrome c', a 28 kDa paramagnetic heme protein; Caffrey M et al.; The cytochromes c' are paramagnetic heme proteins generally consisting of two identical 14 kDa subunits . The 1H and 15N resonances of the ferricytochrome c' from the purple phototrophic bacterium Rhodobacter capsulatus have been extensively assigned by the TOCSY-HSQC, NOESY-HSQC, HSQC-NOESY-HSQC, and HNHA 3D heteronuclear experiments performed on an 8 mM sample labeled with 15N . In addition, the 13C alpha and 13CO resonances were assigned by the HNCA and multiple-quantum HNCOCA 3D experiments performed on a 0.5 mM sample labeled with 13C and 15N . The assignment of the backbone 13C resonances was used to confirm the 1H and 15N assignments and to better define secondary structure . On the basis of medium-range NOEs, 3JHN alpha coupling constants, and backbone 13C chemical shifts, the secondary structure consists of four helices: helix-1 (3-29), helix-2 (33-49), helix-3 (78-97), and helix-4 (103-117) . On the basis of long-range NOE contacts, the Rb . capsulatus ferricytochrome c' is a four-helix bundle protein in which consecutive helices are antiparallel with respect to one another.

J Pept Sci, 1995 May-Jun, 1(3), 184 - 90
Synthetic S-(2,3-dihydroxypropyl)-cysteinyl peptides derived from the N-terminus of the cytochrome subunit of the photoreaction centre of Rhodopseudomonas viridis enhance murine splenocyte proliferation; Metzger JW et al.; Various lipopeptides representing the N-terminal part of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas virdis were prepared by solid-phase peptide synthesis . These lipopeptides consisted of a S-{2,3-dihydroxypropyl}-cysteinyl (Dhc) residue N-terminally coupled to the nonapeptide FEPPPATTT . Different numbers of palmitoyl (Pam) chains were attached to Dhc via ester and/or amide bonds . The lipopeptide Dhc(Pam)2-FEPPPATTT containing two ester-bonded palmitoyl residues and a free N-terminus was a potent polyclonal activator of murine (BALB/c) spleen cells at subnanomolar concentrations . The lipopeptide Pam-Dhc(Pam)2-FEPPPATTT containing three palmitoyl residues, the two-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residue, and the N-terminally elongated lipopeptide SLVAG-Dhc(Pam)2-FEPPPATTT were less active . The nonapeptide FEPPPATTT and the decapeptide Dhc-FEPPPATTT were only marginal splenocyte activators, even at concentrations as high as 1 microM . Thus, lipopeptide Dhc(Pam)2-FEPPPATTT constitutes the first potent splenocyte stimulation Dhc-lipopeptide described so far that contains only two fatty acid residues.

Fogorv Sz, 1995 May, 88(5), 163 - 6
{The role of dalacin C in the management of odontogenic inflammations}; Gyenes V et al.; The penicillin-metronidazole combination is well tried antibiotic therapy for the treatment of inflammations of dental origin . However, because of the rising number of penicillin-resistant bacterium strains and penicillin allergies, other antibiotics too are being applied ever more frequently . The literature data and the authors' own experience indicate that clindamycin (Dalacin C) may be used effectively against mixed infections of the oral cavity . A detailed account of the patients and the methods will be reported in the following article.

Arch Microbiol, 1995 May, 163(5), 313 - 21
The control of asymmetric gene expression during Caulobacter cell differentiation; Marczynski GT et al.; The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation . Each cell division produces two distinct cell types: a swarmer cell and a stalked cell . These cells possess distinct functional morphologies and differential programs of transcription and DNA replication . The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators . Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system . Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry . The molecular details of these universal cellular processes in C . crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Eur J Biochem, 1995 May 1, 229(3), 658 - 68
1H nuclear-magnetic-resonance investigation of oxidized Fe4S4 ferredoxin from Thermotoga maritima . Hyperfine-shifted resonances, sequence-specific assignments and secondary structure; Wildegger G et al.; The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range . The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins . A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented . Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given . They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T . maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein . This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas {Kissinger, C.R., Sieker, L.C., Adman E.T . & Jensen, L.H . (1991) J . Mol . Biol . 219, 693-715} and therefore suggesting different mechanisms of stabilization for T . maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR {Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S . C., Howard, J.B., Adams M.W.W . & La Mar, G.N . (1994) Biochemistry 33, 6316-6326}.

J Bacteriol, 1995 May, 177(10), 2946 - 9
Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene; Afseth G et al.; Characterization of the rRNA operon from the obligate intracellular bacterium Coxiella burnetii has determined the order of the rRNA genes to be 16S-23S-5S . A 444-bp intervening sequence (IVS) was identified to interrupt the 23S rRNA gene beginning at position 1176 . The IVS is predicted to form a stem-loop structure formed by flanking inverted repeats, and the absence of intact 23S rRNA molecules suggests that the loop is removed . An open reading frame in the IVS has been identified that shows 70% similarity at the amino acid level to IVS open reading frames characterized from four species of Leptospira.

J Bacteriol, 1995 May, 177(10), 2804 - 12
Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis; Zhao G et al.; One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH) . To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate . gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH) . The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene . We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study . The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence . Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa . E4PDH had an apparent Km value for E4P {Kmapp(E4P)} of 0.96 mM, an apparent kcat catalytic constant for E4P {kcatapp(E4P)} of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined . From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C . Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate . To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene . Implications of these findings in the evolution of GA3PDHs are also discussed . On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

J Bacteriol, 1995 May, 177(10), 2637 - 43
Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans; Klasen R et al.; Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp . oxydans DSM3503 was purified to homogeneity . This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate . GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000 . In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits . The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP . The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate . D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced . Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction . The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme . The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli . Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa . Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E . coli and with 6.80 U/mg of cell-free protein in G . oxydans, which corresponded to 85-fold overexpression of the G . oxydans wild-type GNO activity . Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.

Infect Immun, 1995 May, 63(5), 1999 - 2003
Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis; Imamura T et al.; Infection with Porphyromonas gingivalis is strongly associated with adult periodontitis, and proteinases are considered to be important virulent factors of the bacterium . In order to investigate the function of proteinases in disease development we examined vesicles, a biological carrier of these enzymes, for the generation of vascular permeability enhancement (VPE) activity, believed to correlate with the exudation of gingival crevicular fluid . The vesicles generated VPE activity from human plasma in a dose-dependent manner which could be inhibited 90% by antipain, a specific inhibitor of the Arg-specific cysteine proteinases (Arg-gingipains {RGPs} from P . gingivalis . Incubation of vesicles with high-molecular-weight-kininogen (HMWK)-deficient plasma did not result in VPE activity . On this basis, RGPs associated with vesicles were assumed to be responsible for most of the VPE activity generation via plasma prekallikrein activation and subsequent bradykinin production . The secondary pathway for VPE activity production was dependent on the direct release of bradykinin from HMWK by the concerted action of RGP and a Lys-specific cysteine proteinase (Lys-gingipain {KGP}), also associated with vesicles . These results indicate that RGP and KGP are biologically important VPE factors acting either via prekallikrein activation (RGP) and/or HMWK cleavage (RGP and KGP) to release BK and, thereby, contributing to the production of gingival crevicular fluid at periodontal sites infected with P . gingivalis.

Infect Immun, 1995 May, 63(5), 1893 - 8
Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures; Nishihara T et al.; The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system . When mouse bone marrow cells were cultured with A . actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed . The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine . In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A . actinomycetemcomitans Y4 capsular-like polysaccharide . Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A . actinomycetemcomitans Y4 capsular-like polysaccharide . However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation . IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A . actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures . The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A . actinomycetemcomitans Y4 capsular-like polysaccharide . These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A . actinomycetemcomitans Y4 capsular-like polysaccharide . We sought to determine whether osteoclast-like cell formation induced by A . actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004 . The formation of osteoclast-like cells was suppressed by H8 and HA1004 . These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A . actinomycetemcomitans Y4 capsular-like polysaccharide . Furthermore, a correlation between IL-1 alpha and prostaglandin E2 in the osteoclast recruitment induced by A . actinomycetemcomitans Y4 capsular-like polysaccharide is discussed.

Scand J Immunol, 1995 May, 41(5), 433 - 42
Characterization of the gene encoding the MPB51, one of the major secreted protein antigens of Mycobacterium bovis BCG, and identification of the secreted protein closely related to the fibronectin binding 85 complex; Ohara N et al.; The secreted protein MPB51 is one of the major proteins in the culture filtrate of Mycobacterium bovis BCG (BCG) and is a protein immunologically cross-reacting with the fibronectin binding 85 complex secreted by this bacterium . The gene encoding MPB51 (mpb51) was cloned, sequenced, and expressed in Escherichia coli . The mpb51 gene was mapped downstream of the gene for 85A component with 179 bp spaces . The mpb51 gene encoded 299 amino acids, including 33 amino acids for the signal peptide, followed by 266 amino acids for the mature protein with a molecular mass of 27807.37 Da . This is the first complete sequence of MPB51 . MPB51 showed 37-43% homology to the components of 85 complex . Two-dimensional electrophoresis of culture fluids of BCG and Western blotting indicated the existence of the other novel protein(s) which strongly cross-reacted with the alpha antigen (85B) and MPB51.

Appl Environ Microbiol, 1995 May, 61(5), 1917 - 22
Aromatic-degrading Sphingomonas isolates from the deep subsurface; Fredrickson JK et al.; An obligately aerobic chemoheterotrophic bacterium (strain F199) previously isolated from Southeast Coastal Plain subsurface sediments and shown to degrade toluene, naphthalene, and other aromatic compounds (J . K . Fredrickson, F . J . Brockman, D . J . Workman, S . W . Li, and T . O . Stevens, Appl . Environ . Microbiol . 57:796-803, 1991) was characterized by analysis of its 16S rRNA nucleotide base sequence and cellular lipid composition . Strain F199 contained 2-OH14:0 and 18:1 omega 7c as the predominant cellular fatty acids and sphingolipids that are characteristic of the genus Sphingomonas . Phylogenetic analysis of its 16S rRNA sequence indicated that F199 was most closely related to Sphingomonas capsulata among the bacteria currently in the Ribosomal Database . Five additional isolates from deep Southeast Coastal Plain sediments were determined by 16S rRNA sequence analysis to be closely related to F199 . These strains also contained characteristic sphingolipids . Four of these five strains could also grow on a broad range of aromatic compounds and could mineralize {14C}toluene and {14C}naphthalene . S . capsulata (ATCC 14666), Sphingomonas paucimobilis (ATCC 29837), and one of the subsurface isolates were unable to grow on any of the aromatic compounds or mineralize toluene or naphthalene . These results indicate that bacteria within the genus Sphingomonas are present in Southeast Coastal Plain subsurface sediments and that the capacity for degrading a broad range of substituted aromatic compounds appears to be common among Sphingomonas species from this environment.

J Clin Microbiol, 1995 May, 33(5), 1354 - 6
Detection of Tropheryma whippelii DNA in a patient with AIDS; Maiwald M et al.; A case of an AIDS patient infected with the Whipple's disease bacterium, Tropheryma whippelii, is reported . A DNA fragment with sequence specificity for the 16S rRNA gene of the bacterium was detected by PCR in a duodenal biopsy specimen from a 55-year-old male patient with AIDS and diarrhea . The biopsy specimen contained periodic acid-Schiff stain-positive macrophages which did not, however, resemble the sickleform-particle-containing cells characteristic of Whipple's disease . This observation raises two possibilities: either the patient had a coincidence of AIDS and Whipple's disease or Tropheryma whippelii acted as an opportunistic pathogen in this immunodeficient patient . The latter explanation is of interest in light of the ongoing discussion of immunologic abnormalities as predisposing factors for Whipple's disease.

Biochemistry, 1995 Apr 18, 34(15), 5294 - 302
Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier; Heller BA et al.; We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide . Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding . In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL . The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs . This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine . In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta) . This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle . Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type . The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.

Biochemistry, 1995 Apr 18, 34(15), 5288 - 93
Near-infrared resonance Raman spectra of Chloroflexus aurantiacus photosynthetic reaction centers; Cherepy NJ et al.; Resonance Raman spectra of the photosynthetic reaction center isolated from the green bacterium Chloroflexus aurantiacus have been obtained with excitation in the near-infrared absorption bands of the special pair (P) and the accessory bacteriochlorophyll (B) using shifted-excitation Raman difference spectroscopy (SERDS) . These spectra are compared with the previously reported Raman spectra of P and B in reaction centers from the purple bacterium Rhodobacter sphaeroides . The spectra of P and B from the two species are nearly identical . Common and distinctive attributes of these spectra include enhanced low-frequency (30-200 cm-1) modes in P and the absence of strong Raman activity in modes higher than 1200 cm-1 in both P and B . Also, the absolute scattering cross sections with excitation in the P band are unusually weak in both reaction centers, indicating that their excited states are rapidly vibronically dephased . The striking similarities between the P and B spectra in reaction centers from two very different bacterial species suggest that the common nuclear and electronic dynamics identified here are characteristic of photosynthetic reaction centers.

Presse Med, 1995 Apr 15-22, 24(15), 708 - 10
{Antral gastritis in chronic alcoholism . Role of cirrhosis and Helicobacter pylori}; Altman C et al.; OBJECTIVES: Antral gastritis is frequent in alcoholics . The role of H . pylori in the pathogenesis of gastritis in these patients is not well known . The aim of our study was to study the role of H . pylori and cirrhosis in the pathogenesis of antral gastritis in alcoholic patients . METHODS: Seventy-nine patients were included in the study . All underwent upper gastrointestinal tract endoscopy with antral biopsies, independently of the presence of abdominal pain, and had serological examination for H . pylori antibodies . RESULTS: Cirrhosis and gastritis were present in 50 and 40 patients respectively, H . pylori serological assay and histological identification of the bacterium were positive in 35 (44%) and 19 (24%) patients respectively . Discrepancy between the 2 tests were observed more frequently in cirrhotic patients . A positive serology with a negative histologic examination for H . pylori was present for 18 cirrhotic and 4 noncirrhotic patients (p < 0.05) . A gastritis without evidence of H . pylori was more frequent in cirrhotic than in noncirrhotic patients . H . pylori was histologically present in 11 of 29 cirrhotic patients and in 8 of the 11 noncirrhotic patients with a gastritis (p < 0.05) . CONCLUSIONS: Discrepancies between histological examination and H . pylori serology in patients with cirrhosis might be due to the inhospitable environment for H . pylori in case of portal hypertension; the positive serology could be in relation with a past infection.

Orv Hetil, 1995 Apr 2, 136(14), 709 - 12
{Helicobacter pylori in benign gastroduodenal diseases}; Joos A et al.; (A light- and electronmicroscopic study) . We have examined the occurrence of Helicobacter pylori (HP) in 2937 gastric antral biopsy specimens from 979 patients with upper gastrointestinal symptoms . The incidence of HP proved to be 50.5% . The endoscopic diagnoses were: gastritis (62 cases), gastric erosion (425), gastric ulcer (51), duodenitis (22), duodenal erosion (119), duodenal ulcer (122) and the HP incidence was 29, 46, 63,, 50, 66 and 73%, respectively . Microscopic findings were: chronic gastritis (442), chronic active gastritis (356) and normal mucosa (181) . The prevalence of HP in these groups was 43%, 78% and 11%, respectively . The activity of gastritis was in good correlation with HP infection . We did not see epithelial damage, and found only a few instances of mucus depletion . Electronmicroscopic examination was performed in order to investigate the morphology of bacterium and its relation to the mucosal cells . We observed mild loss of microvilli on the cell surface and did not see any cell invasion by bacteria.

J Wildl Dis, 1995 Apr, 31(2), 159 - 65
Analyses of mammalian sera in enzyme-linked immunosorbent assays with different strains of Borrelia burgdorferi sensu lato; Magnarelli LA et al.; Blood samples were collected from cottontail rabbits (Sylvilagus floridanus), raccoons (Procyon lotor), white-footed mice (Peromyscus leucopus), and white-tailed deer (Odocoileus virginianus) between 1977 and 1991 in southern Connecticut and New York State (USA) and were tested for antibodies against eight strains of Borrelia burgdorferi sensu lato in enzyme-linked immunosorbent assays . Among these spirochetes were six strains of B . burgdorferi sensu stricto, one strain of B . garinii (=IP90) and a strain (IPF) in group VS461 . Sera from each study group reacted positively to all strains having origins in North America and Eurasia . Assay sensitivities normally ranged between 85% and 100% for all study groups . The lowest sensitivity (66%) was noted when mouse sera were tested with B . garinii, an isolate from Ixodes persulcatus in the former Soviet Union . Differences in serum reactivity to various strains were noted for all study groups, but because of multiple shared antigens among the closely related spirochetes tested, the selection of a particular North American strain of B . burgdorferi sensu stricto did not appear to be a critical factor for optimal assay performance . Locally obtained strains of this bacterium are preferred as coating antigens for serologic testing because of their availability.

Infect Immun, 1995 Apr, 63(4), 1516 - 20
Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen; Sethi S et al.; Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children . Little is known about the human immune response to this bacterium . In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M . catarrhalis . Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M . catarrhalis sputum isolates . In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1 . Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1 . Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M . catarrhalis . OMP B1 is therefore an important OMP antigen on the surface of M . catarrhalis for the human immune response to infection by this bacterium.

Infect Immun, 1995 Apr, 63(4), 1478 - 83
Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth; Fortier AH et al.; Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h . LVS was internalized and remained in a vacuolar compartment throughout its growth cycle . The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain) . Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS . Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10(4) CFU) at 72 h . In contrast, over 10(8) CFU was consistently recovered from untreated cultures . Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria . Further, the effects of the inhibitors were completely reversible: inhibitor-pretreated LVS-infected macrophages washed free of the agent and cultured in medium fully supported LVS growth over 72 h . Endosome acidification is an important cellular event essential for release of iron from transferrin . The growth-inhibitory effects of both chloroquine and NH4Cl were completely reversed by addition of ferric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin . Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

Infect Immun, 1995 Apr, 63(4), 1176 - 82
The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain; Potempa J et al.; Porphyromonas gingivalis contains high concentrations of numerous cysteine proteinases with trypsin-like activity which have been implicated as important virulence factors in adult-onset periodontitis . We have analyzed the subfractions of six P . gingivalis strains for the presence of arginine-X- and lysine-X-specific proteinases (Arg-gingipain {RGP} and Lys-gingipain {KGP}) previously purified from P . gingivalis H66 . Western blot (immunoblot) analysis using antibodies produced against RGP and the N-terminal peptides of RGP or the catalytic subunit of KGP indicated that these enzymes are synthesized by the strains studied and exist as multiple molecular mass species . The major forms of RGP were identified as 110-, 95-, 70- to 90-, and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinins, with or without an added membrane anchorage peptide . The other forms are single-chain enzymes . While the 95- and 50-kDa RGP were found predominantly in culture medium, the 110- and 70- to 90-kDa forms associated with membranous fractions of the bacteria . The predominant form of KGP in all strains was a complex of the 60-kDa catalytic domain with hemagglutinins, and vesicle- and membrane-associated KGP was about 15 kDa larger than the 105-kDa enzyme present in culture media . These data explain the apparent complexity of P . gingivalis proteinases and indicate that in all strains tested there are two identical enzymes, one with arginine-X specificity and the other with lysine-X specificity, which, working in concert, are responsible for the trypsin-like activity associated with this bacterium.

Biophys J, 1995 Apr, 68(4 Suppl), 163S - 166S; discussion 166S-167S
Torque generation by the flagellar rotary motor; Berg HC; A review is given of the structure and dynamics of the flagellar rotary motor . Force-generating elements in a motor driving a tethered bacterium (a cell fixed to the substratum by a single flagellum) exert forces of order 20 pN while moving at speeds of order 1 micron/s . Force-generating elements in a motor driving a flagellar filament in a bundle exert forces some 10-fold lower but move at speeds more than 10-fold higher . The motor torque-speed relationship has been measured over a wide dynamic range . Motors strongly resist being driven backwards and are easily broken.

Int J Exp Pathol, 1995 Apr, 76(2), 111 - 23
Studies on early events of Borrelia burgdorferi-induced cytokine production in immunodeficient SCID mice by using a tissue chamber model for acute inflammation; Hurtenbach U et al.; Lyme arthritis, one of the common features of Borrelia burgdorferi infection in the human, is associated with the production of various monocyte derived cytokines . To investigate the expression and regulation of cytokines during the acute phase of spirochete induced inflammation, a perforated Teflon chamber was implanted under the dorsal skin of severe combined immunodeficiency (SCID) and immunocompetent co-isogenic C.B-17 mice . The histology of the surrounding chamber tissue exhibited sterile inflammation with several features reminiscent of an inflamed synovium, i.e . infiltration of polymorphonuclear and mononuclear cells, fibroblast-like cells and neovascularization . The experimental inoculation of Borrelia burgdorferi into the chamber resulted in the production of TNF-alpha, IL-1 and IL-6 into the chamber exudate, in both the immunodeficient, disease susceptible SCID and the immunocompetent, disease resistant C.B-17 mice . Peak levels of TNF-alpha were reached at 2 hours and of IL-1 and IL-6 at 6 hours after infection; by 24 hours, cytokine levels were only marginal (IL-1, IL-6) or non-detectable (TNF-alpha) . Experimental infection by s.c . injection distant from the tissue chamber led to colonization of the spirochetes into the chamber, suggesting a tropism of the bacteria for this tissue . Thus, this model provides a system for studying acute events of Borellia burgdorferi induced cytokine regulation in a complex cellular, synovium-like environment that the bacterium encounters in vivo.

Appl Environ Microbiol, 1995 Apr, 61(4), 1431 - 7
Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools; Balayeva NM et al.; The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains . Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB) . The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains . Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups . Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced . The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110 . Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia . Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.

Appl Environ Microbiol, 1995 Apr, 61(4), 1399 - 407
Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials; Bronnenmeier K et al.; A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed . Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography . Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, p-nitrophenyl-alpha-L-arabinofuranoside, and 4-O-methyl-glucuronosyl-xylotriose . The activities of two cellulases, one laminarinase, one xylanase, two putative beta-D-xylosidases, alpha-D-glucuronidase, and alpha-L-arabinosidase were identified . Because of their selective retardation on a Superdex gel filtration column, the two cellulases could be purified to homogeneity . According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular masses of 27 and 29 kDa, respectively, were determined for cellulase I and cellulase II . Maximal activities of both enzymes were observed at 95 degree C between pH 6.0 and 7.5 . In the presence of 2.5 M NaC1 the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80 degree C . On the basis of its activity towards CMC, cellulase I was classified as endo-beta-1,4-glucanase . Cellulase II was able to attack Avicel in addition to CMC, beta-glucan, and p-nitrophenyl-beta-D-cellobioside . It releases cellobiose and cellotriose from Avicel . The latter product is further cleaved into glucose and cellobiose . Cellulase II may therefore be classified as exo-beta-1,4-glucanase.

J Bacteriol, 1995 Apr, 177(8), 2157 - 63
Carbon monoxide-induced activation of gene expression in Rhodospirillum rubrum requires the product of cooA, a member of the cyclic AMP receptor protein family of transcriptional regulators; Shelver D et al.; Induction of the CO-oxidizing system of the photosynthetic bacterium Rhodospirillum rubrum is regulated at the level of gene expression by the presence of CO . In this paper, we describe the identification of a gene that is required for CO-induced gene expression . An 11-kb deletion of the region adjacent to the previously characterized cooFSCTJ region resulted in a mutant unable to synthesize CO dehydrogenase in response to CO and unable to grow utilizing CO as an energy source . A 2.5-kb region that corresponded to a portion of the deleted region complemented this mutant for its CO regulation defect, restoring its ability to grow utilizing CO as an energy source . When the 2.5-kb region was sequenced, one open reading frame, designated cooA, predicted a product showing similarity to members of the cyclic AMP receptor protein (CRP) family of transcriptional regulators . The product, CooA, is 28% identical (51% similar) to CRP and 18% identical (45% similar) to FNR from Escherichia coli . The insertion of a drug resistance cassette into cooA resulted in a mutant that could not grow utilizing CO as an energy source . CooA contains a number of cysteine residues substituted at, or adjacent to, positions that correspond to residues that contact cyclic AMP in the crystal structure of CRP . A model based on this observation is proposed for the recognition of CO by Cooa . Adjacent to cooA are two genes, nadB and nadC, with predicted products similar to proteins in other bacteria that catalyze reactions in the de novo synthesis of NAD.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1995 Apr, 177(8), 1959 - 66
Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers; Wiencek KM et al.; The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required . Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two . Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively . SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers . The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min . The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing . The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca . 2 min) decreased with increasing substratum hydrophobicity . With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.

Vet Immunol Immunopathol, 1995 Apr, 45(3-4), 321 - 32
Temporal effects of tumor necrosis factor-alpha on intracellular survival of Mycobacterium paratuberculosis; Stabel JR; The causative agent in Johne's disease is Mycobacterium paratuberculosis, an intracellular pathogen which causes enteritis in ruminants . Little is known about interactions between the host cell (macrophage) and M . paratuberculosis; however, this bacterium is able to evade normal host immune defenses and cause a chronic infective state . In the present study, we evaluated whether activation of a murine macrophage cell line (J774.16) by pretreatment with recombinant murine tumor necrosis factor alpha (TNF) prior to infection with M . paratuberculosis would affect their ability to restrict growth and kill the ingested bacteria . A murine cell line was utilized owing to difficulty in obtaining bovine reagents and lack of a continuous bovine macrophage cell line for repeated experimentation . After 4 h of infection, numbers of viable bacteria in cell lysates were significantly lower for macrophages pretreated with 1000 IU TNF ml-1 . The rate of bacterial growth as assessed by BACTEC radiometric culture system was also reduced at this time point . Upon further extension of the infection period to 72 h, we observed that moderate doses of TNF (10-1000 IU ml-1) significantly increased the number of viable M . paratuberculosis recovered whereas the highest dose of TNF (4000 IU ml-1) effectively reduced bacterial numbers . These data indicate that TNF can either enhance or reduce macrophage mycobactericidal and mycobacteriostatic activity depending upon both the level of TNF to which cells are exposed and the duration of infection.

Mol Microbiol, 1995 Apr, 16(1), 45 - 55
Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli; Ma D et al.; Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules . Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these beta-lactams across the outer membrane was not increased . This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi-drug efflux pump . The natural environment of an enteric bacterium such as E . coli is enriched in bile salts and fatty acids . An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate . In addition, acrAB expression was elevated by growth in 5 mM decanoate . These results suggest that one major physiological function of AcrAB is to protect E . coli against these and other hydrophobic inhibitors . Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria-Bertani medium . Finally, acrAB expression was shown to be increased in mar (multiple-antibiotic-resistant) mutants.

Mol Microbiol, 1995 Apr, 16(2), 171 - 5
Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development; Downard J et al.; The esg locus is required for the formation of multicellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus . Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes . Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase, a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms . During vegetative growth in M . xanthus, this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism . M . xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells . These effects include: (i) the lysis of M . xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants . These and other results suggest a model in which the branched-chain fatty acids, synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation . This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M . xanthus development.

Lab Anim Sci, 1995 Apr, 45(2), 131 - 9
Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology; Clifford CB et al.; The purpose of this study was to characterize a spontaneous disease condition causing hyperkeratosis in nude mice and to explore the etiologic role of a particular species of coryneform bacteria in this disease, colloquially known as "scaly skin disease." The study was divided into two parts . In the first phase, a series of inoculation experiments was conducted with a field isolate of the coryneform species used to study the clinical and histopathologic development of the disease syndrome . Athymic nude mice (4 to 5 weeks old) were inoculated on the skin of the back with a suspension of a pure culture of the coryneform bacterium that had been isolated from a field case . The culture was applied with a sterile cotton swab in concentrations varying from 6.1 x 10(4)/ml to 5.0 x 10(7)/ml . All inoculated mice became persistently infected throughout the 33 days of the experiment . Clinically evident hyperkeratosis in inoculated animals developed more frequently in mice housed in a microisolator cage than in a semi-rigid isolator and more frequently in mice inoculated with higher numbers of organisms . In all animals in which hyperkeratosis developed, it was first noted on day 7 after inoculation . The second series of experiments was designed to determine the success of various housing methods in excluding the infection, mechanisms of transmission, susceptibility of other stocks and strains of mice to the organism, and whether the other strains might serve as a source of the organism . Results of the study in various strains indicated that both immunocompetent and immunodeficient mice, whether glabrous or hirsute, could be infected with the organism, but only glabrous animals developed hyperkeratosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Syst Bacteriol, 1995 Apr, 45(2), 207 - 11
16S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus Ehrlichia; Pretzman C et al.; Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae . This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector . The nucleotide sequence of the N . helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria . The N . helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, > 95%) . All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%) . Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N . helminthoeca with other ehrlichial species . The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species . Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus . The close relationships among N . helminthoeca, E . risticii, and E . sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

J Mol Biol, 1995 Mar 24, 247(2), 224 - 35
Fine structure of the peptidyl transferase centre on 23 S-like rRNAs deduced from chemical probing of antibiotic-ribosome complexes; Rodriguez-Fonseca C et al.; Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1 all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms . The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyl transferase centre of the 23 S-like rRNAs . The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA . The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre . The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre . (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia coli numbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or modulate, the start of the peptide channel . No drug effects were found that could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins . However, two drugs T2 toxin and virginiamycin M1 protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site . Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.

Gene, 1995 Mar 21, 155(1), 19 - 25
Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system; Taron CH et al.; NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC . The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli {Van Cott and Wilson, Gene 74 (1988) 55-59} . However, none of these clones expressed detectable levels of the restriction endonuclease (ENase) . The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans . The complete NaeI system was finally cloned using E . coli AP1-200 {Piekarowicz et al., Nucleic Acids Res . 19 (1991) 1831-1835} and less stringent MTase-selection conditions . The naeIR gene was expressed first by cloning into S . lividans, and later by cloning under control of a regulated promoter in an E . coli strain preprotected by the heterologous MspI MTase (M.MspI) . The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.

Eur J Biochem, 1995 Mar 15, 228(3), 719 - 26
Properties of the soluble polypeptide of the proton-translocating transhydrogenase from Rhodospirillum rubrum obtained by expression in Escherichia coli; Diggle C et al.; Transhydrogenase, which catalyses the reduction of NADP+ by NADH coupled to proton translocation across a membrane, may be unique in the photosynthetic bacterium Rhodospirillum rubrum . Unlike the homologous enzyme from animal mitochondria and other bacterial sources, it has a water-soluble polypeptide, which exists as a dimer (Ths), that can be reversibly dissociated from the membrane component {Williams, R., Cotton, N . P . J., Thomas, C . M . & Jackson, J . B . (1994) Microbiology, 140, 1595-1604} . We have expressed the gene for Ths in cells of Escherichia coli under control of the tac promoter and a strong ribosome binding site . The protein, purified by column chromatography, fully reconstituted transhydrogenation activity to everted membrane vesicles of Rhs . rubrum that had been washed to remove Ths . The purified expressed protein was prepared in quantities over 100-fold greater than were obtained from wild-type Rhs . rubrum . The fluorescence spectrum of purified expressed Ths had an intense and unusually short wavelength emission maximum at 310 nm with shoulders at 298 and 322 nm . Time-resolved measurements indicated that the fluorescence decay was almost monoexponential with a lifetime of 5.2 ns . On denaturation with 4 M guanidine hydrochloride, the emission band shifted to 352 nm and decreased in intensity . In the native protein, the fluorophore was relatively inaccessible to quenching solutes, such as iodide ions and acrylamide . It is concluded that the fluorescence emission arises mainly from the single tryptophan residue of Ths (Trp72), which is locked into a rigid conformation and is located in highly non-polar environment . The 310-nm fluorescence of Ths was quenched by NADH, maximally to 46% . The apparent binding constant was 18 microM . The fluorescence of Ths-bound NADH was enhanced relative to the nucleotide in free solution and its emission maximum was shifted to a shorter wavelength (440 nm) . These data support previous indications that the NADH binding site is located in domain I of proton-translocating transhydrogenase . Excitation of Ths at 280 nm did not lead to sensitized emission at 440 nm from bound NADH . This indicates that the quenching of fluorescence of Ths by NADH does not result from resonance energy transfer from Trp72 to the bound nucleotide . NAD+, NADP+ and NADPH had little effect on the protein fluorescence . The kinetics of quenching of Ths fluorescence by NADH were examined after mixing in a stopped-flow device . The 'on' rate constant for nucleotide binding was approximately 8 x 10(6) M-1 s-1 and the 'off' constant approximately 150 s-1.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1941 - 4
Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli; Gibson LC et al.; Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways . In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction . The bchH and the bchI and -D genes from R . sphaeroides were expressed in Escherichia coli . When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner . This was possible only when all three genes were expressed . The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis . The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1826 - 30
Femtosecond infrared spectroscopy of reaction centers from Rhodobacter sphaeroides between 1000 and 1800 cm-1; Hamm P et al.; Time-resolved pump-and-probe experiments of reaction centers of the purple bacterium Rhodobacter sphaeroides (R26) in the mid-IR region between 1000 and 1800 cm-1 are recorded with a time resolution of 300-400 fs . The difference spectra of the states P*, P+HA-, and P+QA- with respect to the ground state P predominantly reflect changes of the special pair . They show positive and negative bands due to changes of distinct vibrational modes superimposed on a broad background of enhanced absorption . A number of certain bands can be assigned to the special pair P, to the bacteriopheophytin HA, and to the quinone QA . The temporal evolution of the IR absorbance changes is well described by the time constants known from femtosecond spectroscopy of the electronic states . Differences occur only at very early times, which are indicative of fast vibrational relaxation with a time constant of a few hundred femtoseconds.

J Theor Biol, 1995 Mar 7, 173(1), 1 - 13
A model of error propagation in the presence of an error-enhancing drug; Johansson J et al.; A mathematical model is considered that describes error propagation in protein synthesis, with emphasis on the role of an error-enhancing drug such as streptomycin . The subject of error propagation has been investigated in a number of works since an original proposal by Orgel in 1963 . From experiments, it is known that streptomycin given to a bacterium culture in small concentration increases the error level, but at a certain concentration threshold the bacteria die . This two-fold behavior has, in some papers, lead to the proposal that another effect of the drugs besides that of error propagation leads to the death of the bacteria . In the present work, we use a model related to kinetic models of selection in protein synthesis which include the combined effect of ribosome and synthetase action . The model shows very clear threshold effects: for small values of the parameter that represents the drug action, a stable situation is found that has an increased error level but still attains a rather high accuracy . At a certain threshold, this is no longer maintained . The main emphasis here is on the time evolution of relevant parameters, and it is also shown how this can be quite drastic: the accuracy may decrease rather smoothly to a critical point, where it is drastically lost and where the bacteria may die out very suddenly.

J Biol Chem, 1995 Mar 3, 270(9), 4712 - 20
Copper/topa quinone-containing histamine oxidase from Arthrobacter globiformis . Molecular cloning and sequencing, overproduction of precursor enzyme, and generation of topa quinone cofactor; Choi YH et al.; The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis . The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain . Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence . To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase . The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions . Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402) . Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K . (1994) FEBS Lett . 351, 360-364).

Hepatology, 1995 Mar, 21(3), 668 - 73
Escherichia coli capsular polysaccharide and spontaneous bacterial peritonitis in cirrhosis; Soriano G et al.; Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of cirrhosis . Escherichia coli is the most frequent bacterium isolated in this condition . The presence of capsular antigens, mainly the K1 capsular polysaccharide, has been associated with invasiveness in E coli infections . Capsular serotypes of E coli causing SBP were determined in 37 cirrhotic patients . Twenty-seven strains were encapsulated (72.9%), 9 of them (24.3%) with K1 capsular polysaccharide, and 10 were nonencapsulated . Patients with encapsulated E coli showed a significantly higher incidence (92.5% vs . 50%; P < .01) and number of complications per patient (1.9 +/- 1.1 vs . 0.8 +/- 1.0; P < .01) than patients with nonencapsulated strains . Although mortality was higher in patients with encapsulated strains (44.4% vs . 20%), the difference did not reach statistical significance . Considering patients infected by encapsulated strains, the incidence of complications and mortality were similar in patients with or without K1 strains . These data suggest that the presence of encapsulated strains could have a prognostic significance in SBP caused by E coli in cirrhotic patients.

J Bacteriol, 1995 Mar, 177(5), 1348 - 56
Adaptation of Xanthobacter autotrophicus GJ10 to bromoacetate due to activation and mobilization of the haloacetate dehalogenase gene by insertion element IS1247; van der Ploeg J et al.; Monobromoacetate (MBA) is toxic for the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 at concentrations higher than 5 mM . Mutants which are able to grow on higher concentrations of MBA were isolated and found to overexpress haloacid dehalogenase, which is encoded by the dhlB gene . In mutant GJ10M50, a DNA fragment (designated IS1247) had copied itself from a position on the chromosome that was not linked to the dhlB region to a site immediately upstream of dhlB, resulting in a 1,672-bp insertion . IS1247 was found to encode an open reading frame corresponding to 464 amino acids which showed similarity to putative transposases from two other insertion elements . In most of the other MBA-resistant mutants of GJ10, IS1247 was also present in one more copy than in the wild type, which had two copies located within 20 kb . After insertion to a site proximal to dhlB, IS1247 was able to transpose itself together with the dhlB gene to a plasmid, without the requirement of a second insertion element being present downstream of dhlB . The results show that IS1247 causes bromoacetate resistance by overexpression and mobilization of the haloacid dehalogenase gene, which mimics steps during the evolution of a catabolic transposon and plasmid during adaptation to a toxic growth substrate.

Infect Immun, 1995 Mar, 63(3), 1102 - 6
Helicobacter pylori-specific CD4+ T-cell clones from peripheral blood and gastric biopsies; Di Tommaso A et al.; Colonization of human gastric mucosa with cytotoxic strains of the bacterium Helicobacter pylori is associated with peptic ulcer and with chronic gastritis . Since little is known about the T-cell response to H . pylori, we investigated the CD4+ T-cell response both in peripheral blood mononuclear cells (PBMCs) and at the site of infection . First, we compared the bulk PBMC proliferative response to the bacterium in individuals with and without symptoms of gastroduodenal disease . We found that the PBMCs from virtually all individuals proliferate in response to heat-inactivated bacteria . Second, we cloned H . pylori-specific CD4+ T lymphocytes from the PBMCs of three patients and from both the gastric mucosa and PBMCs of a fourth patient . We have found that CD4+ T-cell clones specific for H . pylori from peripheral blood samples and gastric mucosae of infected patients are major histocompatibility complex class II restricted and discriminate between several cytotoxic and noncytotoxic bacterial strains . Moreover, they are polyclonal in terms of T-cell receptor usage and major histocompatibility complex restriction . Our results demonstrate that the T-cell response to the whole bacterium in PBMCs does not correlate with antibody response, infection, or disease . However, H . pylori-specific CD4+ T cells are detectable, at the clonal level, in both the periphery and gastric mucosa of infected patients . Localization of these cells at the site of disease suggests they are effectors of the immune response to the bacteria.

Curr Microbiol, 1995 Mar, 30(3), 137 - 41
Detection and characterization of the African citrus greening liberobacter by amplification, cloning, and sequencing of the rplKAJL-rpoBC operon; Planet P et al.; Greening disease of citrus is caused by a phloem-restricted, uncultured bacterium, recently characterized and named Liberobacter . As shown previously, a probe encoding ribosomal protein genes (rplKAJL-rpoBC operon) from an Asian liberobacter could detect all Asian liberobacter strains tested, but not African strains . Using the sequence of the rplKAJL-rpoBC operon of the Asian liberobacter strain from Poona (India), we have defined primers for PCR amplification of the equivalent genes of an African liberobacter strain . The amplified fragment was cloned in pUC18 and successfully used as a probe to detect African liberobacter strains by Southern and dot hybridizations . Sequence comparisons of the African and Asian liberobacter operons indicate that they represent two different species in the proposed genus Liberobacter.

J Clin Microbiol, 1995 Mar, 33(3), 541 - 4
Borrelia burgdorferi in an urban environment: white-tailed deer with infected ticks and antibodies; Magnarelli LA et al.; Ticks and blood samples were collected from white-tailed deer (Odocoileus virginianus) in forests located in an insular, urban area of Bridgeport, Conn., and in rural south central Connecticut during 1992 and 1993 . Immature and adult Ixodes scapularis ticks were tested for Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, by indirect fluorescent-antibody staining methods . Deer sera were analyzed for antibodies to this bacterium by an enzyme-linked immunosorbent assay . Infected ticks parasitized deer in Bridgeport from May through December; the prevalence of infection varied from 1.1% of 93 larvae to 28.1% of 114 adult females . The percentages of infected males (10.5% of 380 ticks) and females (13.7% of 328 ticks) were relatively lower in south central Connecticut . In antibody tests, the prevalence of seropositive specimens collected in Bridgeport (61% of 146 serum specimens) was more than twofold greater than that of specimens obtained in south central Connecticut (26.7% of 116 serum specimens) . Foci for Lyme borreliosis can occur in forested, urban settings as well as in rural areas if there are ticks, rodents, birds, and large mammals present . Human exposure to ticks in such sites should be considered as a possible source of B . burgdorferi infection.

Ann Acad Med Singapore, 1995 Mar, 24(2), 218 - 23
Peptic ulcer--a new look; Kang JY; This review covers major advances in peptic ulcer disease over the last 25 years . Flexible endoscopy enables accurate diagnosis of peptic ulcer to be made and its introduction made possible the large number of controlled clinical trials on the use of various agents in peptic ulcer treatment . The histamine H-2 receptor antagonists, which reduce gastric acid output, were the first major group of potent ulcer healing drugs introduced . Subsequently, other ulcer healing agents with different modes of action, e.g . colloidal bismuth, sucralfate, prostaglandin analogues, omeprazole were also shown to be effective . The identification of Helicobacter pylori ten years ago was another major advance in peptic ulcer disease . This bacterium is now thought to be the most important cause of peptic ulcer disease and its eradication cures the disease . There are still unanswered questions regarding varying sequelae of Helicobacter pylori infection, the effect of the infection on gastric acid and gastrin secretion while a convenient and effective treatment regime remains to be developed . Non-steroidal anti-inflammatory drugs are now thought to be the second most important cause of peptic ulcer disease and a major risk factor for ulcer complications and mortality . While misoprostol and histamine H-2 antagonists can prevent peptic ulcer associated with these substances, indications for prophylaxis have not yet been defined . If Helicobacter pylori infection could be controlled through improved socio-economic standards and possible development of a vaccine, and if anti-inflammatory drugs devoid of gastric side effects can be developed, peptic ulcer may become a less important health problem in the future.

Rev Sci Tech, 1995 Mar, 14(1), 75 - 9
Testing disinfectants for efficacy; Tamasi G; Testing under laboratory conditions is undoubtedly useful in assessing the activity of disinfectants . However, such testing must be regarded as no more than a preliminary to field trials . This not only indicates the unreliability of laboratory tests, but also poses the wider problem of how any laboratory evaluation can be correlated with the field requirements . Many attempts have therefore been made to devise tests which are suitable to evaluate the in-use requirements of disinfection (e.g . on farms) . The aim of these techniques is to record the end-point of a disinfection procedure on the surfaces in animal houses . In view of the need to standardise testing conditions in the field, the author provides details of the optimum time and place for taking samples under field conditions . Samples for culture media should be taken from the floor when dry . The various techniques (i.e . swabbing, agar cylinder, agar carrier, 'ready-to-use' set) should provide information on the end-point of the disinfection . The desired end-points is approximately one viable bacterium per cm2 of surface area . The technique used should demonstrate whether the disinfection has been effective.

Mol Microbiol, 1995 Mar, 15(6), 1115 - 25
Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1; Mamat U et al.; Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsular polysaccharide (CPS) and the O-specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium . A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli . Independently of the O-type, transformation of various E . coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains . The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp . However, attempts to express phage-encoded peptides were not successful . Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (LPS biosynthesis-interfering) RNA . This RNA contains sequence complementarity to E . coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA . The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1.

Biochem Mol Biol Int, 1995 Mar, 35(3), 549 - 57
Purification and characterization of a raw-starch digesting amylase from a soil bacterium--Cytophaga sp; Jeang CL et al.; A newly isolated bacterium from soil, identified as Cytophaga sp . was found to produce raw-starch digesting amylase . The enzyme was purified from 24-hr cultured medium through ammonium sulfate fractionation, DEAE-Sepharose CL 6B ion exchange chromatography and Sephacryl S-200 gel filtration . The preparation was proved to be homogeneous by SDS-PAGE . The subunit molecular weight determined by SDS-PAGE was 59 KD . The optimum temperature was 50 degrees C on soluble starch and 60 degrees C on raw starch . The optimum pH was in the range of 4.5 to 6.5 on soluble starch and 6.5 to 9.5 on raw starch . In the presence of Mn+2, Cu+2 or Zn+2, the enzyme activity on either substrate was inhibited . Dinitrofluorobenzene, N-bromosuccinimide and trinitrobenzene sulfonic acid all showed inhibitory effect on the enzyme acting on both substrates.

FEMS Microbiol Lett, 1995 Mar 1, 126(3), 277 - 82
Phylogenetic analysis of a novel sulfate-reducing magnetic bacterium, RS-1, demonstrates its membership of the delta-Proteobacteria; Kawaguchi R et al.; Most of the 16S ribosomal RNA gene of a sulfate-reducing magnetic bacterium, RS-1, was sequenced, and phylogenetic analysis was carried out . The results suggest that RS-1 is a member of the delta-Proteobacteria, and it appears to represent a new genus . RS-1 is the first bacterium reported outside the alpha-Proteobacteria that contains magnetite inclusions . RS-1 therefore disrupts the correlation between the alpha-Proteobacteria and possession of magnetite inclusions, and that between the delta-Proteobacteria and possession of greigite inclusions . The existence of RS-1 also suggests that intracellular magnetite biomineralization is of multiple evolutionary origins.

Infect Immun, 1995 Mar, 63(3), 874 - 83
Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi; Leong JM et al.; The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues . We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans . Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious . The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells . Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans . Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells . The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis . Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding . Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway . These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.

J Mol Biol, 1995 Feb 24, 246(3), 429 - 57
Crystallographic refinement at 2.3 A resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis; Deisenhofer J et al.; The atomic model of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis has been refined to an R-value of 0.193 at 2.3 A resolution . The refined model contains 10,288 non-hydrogen atoms; 10,045 of these have well defined electron density . A Luzzati-plot indicates an average co-ordinate error of 0.26 A . During refinement, the positions of a partially ordered carotenoid, a unibiquinone in the partially occupied QB site, a detergent molecule, seven putative sulphate ions, and 201 water molecules were found . More than half of these waters are bound at interfaces between protein subunits and therefore contribute significantly to subunit interactions . Water molecules also play important structural and probably functional roles in the environment of some of the cofactors . Two water molecules form hydrogen bonds to the accessory bacteriochlorophylls and to the protein in the vicinity of the special pair of bacteriophylls, the primary electron donor . A group of about 10 water molecules is bound near the binding site of the secondary quinone QB . These waters are likely to participate in the transfer of protons to the doubly reduced QB.

Arch Biochem Biophys, 1995 Feb 20, 317(1), 103 - 11
Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation; Benning C et al.; Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids . Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes . Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth . Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-{alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl}glycerol . The second lipid was identified as the betaine lipid 1,2-di-O-acyl-{4'-(N,N,N-trimethyl)-homoserine}glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy . Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae . Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R . sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

EMBO J, 1995 Feb 15, 14(4), 631 - 8
The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits; Karrasch S et al.; Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes . Frozen-hydrated samples have been analysed by electron microscopy . The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A . The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre . These dimensions are sufficient to incorporate a reaction centre in vivo . Within each subunit, density for the alpha- and the beta-polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned . The experimentally determined structure contradicts models of the LHC I presented so far.

FEMS Microbiol Lett, 1995 Feb 15, 126(2), 105 - 11
Overproduction and properties of the mannuronate alginate lyase AlxMB; Malissard M et al.; In previous studies (Malissard et al., FEMS Microbiol . Lett . (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 micrograms per litre of culture . The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge . AlxM has now been overproduced in E . coli BL21(DE3)/pAL-Sur/pLysS . Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture . It has been purified to protein homogeneity using a one-step procedure . The nicked AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.

FEMS Microbiol Lett, 1995 Feb 1, 126(1), 7 - 11
The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: characterization and formation under different growth conditions; Hagemann GE et al.; The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities . In contrast to other facultative phototrophs, R . sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark . The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux) . After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4 . Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

EMBO J, 1995 Feb 1, 14(3), 442 - 51
Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex; Schurig H et al.; Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity . A second larger enzyme with PGK activity and identical N-terminal sequence was also found . Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity . No other TIM is detectable in T . maritima crude extracts . As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa . SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein . Both enzymes show high thermostability . Measurements of the catalytic properties revealed no extraordinary results . pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far . The corresponding pgk and tpi genes are part of the apparent gap operon of T . maritima . This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene . On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene . A programmed frameshift may be responsible for this fusion . A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.

J Bacteriol, 1995 Feb, 177(3), 608 - 13
Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration; Hochkoeppler A et al.; We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F.E . Jenney and F . Daldal, EMBO J . 12:1283-1292, 1993; F.E . Jenney, R.C . Prince, and F . Daldal, Biochemistry 33:2496-2502, 1994) . One pathway is via the well-characterized, water-soluble cytochrome c2 (cyt c2), and the other is via a novel membrane-associated c-type cytochrome named cyt cy . In this work, we probed the role of cyt cy in respiratory electron transport by isolating a set of R . capsulatus mutants lacking either cyt c2 or cyt cy, in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway . The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt cy, like cyt c2, connects the bc1 complex and the cyt c oxidase during respiratory electron transport . Whether cyt c2 and cyt cy are the only electron carriers between these two energy-transducing membrane complexes of R . capsulatus is unknown.

Mol Microbiol, 1995 Feb, 15(3), 431 - 44
Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima; Winterhalter C et al.; A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo-1,4-beta-D-xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined . With a half-life of about 40 min at 90 degrees C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known . XynA is a 1059-amino-acid (approximately 120 kDa) modular enzyme composed of an N-terminal signal peptide and five domains, in the order A1-A2-B-C1-C2 . By comparison with other xylanases of family 10 of glycosyl hydrolases, the central approximately 340-amino-acid part (domain B) of XynA represents the catalytic domain . The N-terminal approximately 150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal approximately 170-amino-acid repeated domains (C1-C2) . Cellulose-binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding . C1 and C2 did not share amino acid sequence similarity with any other known cellulose-binding domain (CBD) and thus are CBDs of a novel type . Structurally related protein segments which are probably also CBDs were found in other multidomain xylanolytic enzymes . Deletion of the N-terminal repeated domains or of all the non-catalytic domains resulted in substantially reduced thermostability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Immunol Med Microbiol, 1995 Feb, 10(3-4), 271 - 80
Cell surface characteristics of Helicobacter pylori; Moran AP; Helicobacter pylori is an important gastroduodenal pathogen of humans . Immunological and structural studies have been performed on the phospholipids, lipopolysaccharides (LPS) and some surface proteins of H . pylori strains . H . pylori LPS has, in general, low immunological activity and this property may aid the survival of this chronic infection . Nevertheless, H . pylori LPS has been found to influence the quality of gastric mucin and to stimulate pepsinogen secretion, thereby contributing to gastric disease . A number of putative adhesins of the bacterium have been described . This multiplicity of adhesins may reflect that H . pylori adherence is a multi-step process involving different interactions, and that different adhesins may mediate adherence to various sites in gastric tissue.

J Ind Microbiol, 1995 Feb, 14(2), 154 - 8
Tributyltin-resistant marine bacteria: a summary of recent work; Suzuki S et al.; A tributyltin chloride (TBTCl)-resistant bacterium, Alteromonas sp . M-1, was isolated from coastal seawater . This bacterium grew in medium containing 125 microM TBTCl . TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system . A genetic library was constructed using plasmid vector pUC 19 . Three positive clones were obtained, by which E . coli was transformed to TBTCl resistance . Of the three clones, the shortest fragment from HindIII-library was analyzed . This fragment was 1.8 kb long and contained one complete open reading frame . The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage and E . coli . TBTCl-tolerant marine bacteria other than Alteromonas sp . M-1 were obtained from natural seawater to which TBTCl was added . DNA-DNA hybridization was performed between the three cloned fragments from Alteromonas sp . M-1 and chromosomal DNA of the TBTCl-tolerant bacteria . Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.

J Ind Microbiol, 1995 Feb, 14(2), 132 - 41
Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance; Rouch DA et al.; Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases . Metal resistance may be chromosomally-, plasmid- or transposon-encoded, and one or more genes may be involved: at the biochemical level at least six different mechanisms are responsible for resistance . Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species . To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria . How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion . Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms . The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations . It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations . The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals . Potentially, models for selection of resistance to any substance can be derived using this approach.

Protein Sci, 1995 Feb, 4(2), 228 - 36
Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: purification, characterization, and image processing; Schurig H et al.; Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity . The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources . As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa . Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry . Averaging of the aligned particles proves the enzyme to be a tetramer of dimers . The enzyme requires divalent cations in the activity assay, Mg2+ being most effective . The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C . The pH optimum of the enzyme lies between 7 and 8 . The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively . Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM . The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

Wei Sheng Wu Xue Bao, 1995 Feb, 35(1), 28 - 32
{A new zoonosis--investigation of Gardnerella vaginalis disease of fox . I . Causative agent isolation and artificial infection}; Yan Z et al.; It is the first time that this paper reports on using improved rabbit blood agar culture medium to isolate 145 strains Gardnerella vaginalis from the foxes and their abortion fortus organs of main fox farms . Among the strains, 26 strains were isolated from the abortion foetus organs, isolation rate was 92.86%; 118 strains were isolated from the vaginal exeretion of the abortion and empty foxes, isolation rate was 34.01%; 1 strain was isolated from inguinal lymph node of the pelted positive fox, isolation rate was 2% . None was isolated from blood . By causative agent isolation, we revealed the bacterium survival position in vivo, and the best isolation route isolation opportunity and isolation methods were selected . By artificial infection test, we have proved pathogenicity of the bacterium.

Oral Microbiol Immunol, 1995 Feb, 10(1), 35 - 41
Lactoferrin interaction with Actinobacillus actinomycetemcomitans; Alugupalli KR et al.; The interaction of lactoferrin with Actinobacillus actinomycetemcomitans was examined in a 125I-labeled protein binding assay . The binding of human and bovine lactoferrins reached maximum within 1 h . Lactoferrin binding to the bacterium was pH-dependent and reversible . Scatchard analysis indicated the existence of two different types of binding sites on the bacterium, one with a high affinity constant k alpha approximately 8.8 x 10(-7) M) and the other with a low one (k alpha approximately 1.8 x 10(-6) M) . Bacteria in the exponential phase of growth showed higher binding than cells in the stationary phase . Bacteria grown in medium containing serum and/or lysed erythrocytes bound lactoferrin to a lesser extent . Heat-inactivated serum, lysed erythrocytes and other proteins such as mucin and laminin inhibited lactoferrin binding to A . actinomycetemcomitans in a competitive binding assay . Sodium dodecyl sulfate polyacrylamide-gel electrophoresis and Western blot analysis of the cell envelope as well as the outer membrane of A . actinomycetemcomitans revealed lactoferrin-reactive protein bands at 29 kDa and 16.5 kDa . The 29-kDa band displayed a heat-modifiable lactoferrin-reactive form with a molecular weight of 34 kDa . Neither proteinase K-treated cell envelope nor lipopolysaccharide of this bacterium showed reactivity with lactoferrin . These data suggests a specific interaction of lactoferrin with outer membrane proteins of A . actinomycetemcomitans.

J Clin Microbiol, 1995 Feb, 33(2), 445 - 54
Helicobacter bilis sp . nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice; Fox JG et al.; A fusiform bacterium with 3 to 14 multiple bipolar sheathed flagella and periplasmic fibers wrapped around the cell was isolated from the liver, bile, and lower intestine of aged, inbred mice . The bacteria grew at 37 and 42 degrees C under microaerophilic conditions, rapidly hydrolyzed urea, were catalase and oxidase positive, reduced nitrate to nitrite, did not hydrolyze indoxyl acetate or hippurate, and were resistant to both cephalothin and nalidixic acid but sensitive to metronidazole . On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel helicobacter, Helicobacter bilis . This new helicobacter, like Helicobacter hepaticus, colonizes the bile, liver, and intestine of mice . Although the organism is associated with multifocal chronic hepatitis, further studies are required to ascertain whether H . bilis is responsible for causing chronic hepatitis and/or hepatocellular tumors in mice.

Curr Microbiol, 1995 Feb, 30(2), 117 - 20
Induced mutagenesis by bleomycin in the purple sulfur bacterium Thiocapsa roseopersicina; Pavon V et al.; Cell death and mutagenesis in bleomycin-treated cells of Thiocapsa roseopersicina (a purple sulfur bacterium) was studied by cultivation in a semisolid medium (agar-shake technique) . This technique has also proven useful in assessing the frequency of antibiotic mutations by detecting and counting individual colonies of Thiocapsa roseopersicina . The frequencies of spontaneous mutants resistant to ampicillin, rifampicin, cloramphenicol, tetracycline, kanamycin, streptomycin, and neomycin were also studied: they ranged between 2 x 10(-9) and 9 x 10(-8) . Bleomycin (4 micrograms/ml) sharply increased the frequency of ampicillin-resistant mutants, from 10(-8) (spontaneous) to 4 x 10(-4) (induced), in 17 h . An inducible, error-prone mechanism of DNA synthesis seems to be responsible for this enhancement of the mutagenic effect . this is the first report on the sensitivity to several antibiotics, and capacity of lethality and mutagenesis by bleomycin has been studied in a purple sulfur bacterium.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 724 - 8
A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: identification and mutagenesis of two conserved residues that are essential for enzyme activity; Wilks HM et al.; Protochlorophyllide reductase (NADPH:protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway . We have developed an expression system in which the protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme . By protein sequence comparisons, we have identified the plant protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases . Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity . A model of the enzyme reaction mechanism for light-dependent protochlorophyllide reduction is proposed.

Presse Med, 1995 Jan 21, 24(3), 183 - 8
{Cat-scratch disease and disease caused by Bartonella (Rochalimaea)}; Drancourt M et al.; The aetiology of cat scratch disease remains controversial since both Afipia felis and Bartonella (Rochalimaea) henselae have been isolated from diseased lymph nodes . Bartonella henselae, Bartonella (Rochalimaea) quintana and Bartonella (Rochalimaea) elizabethae cause endocarditis and Bartonella bacilliformis cause septicemia (Oroya's fever) in non-immunocompromized patients, and Bartonella henselae and Bartonella quintana cause fever, bacillary angiomatosis, and visceral peliosis in human immunodeficiency virus-infected patients . Bartonella quintana is the historical agent of trench fever and we recently isolated it from chronic adenopathy . The diagnosis of Afipia felis and Bartonella infections relies upon the isolation of the bacterium from blood, node tissue after inoculation of cell cultures systems and molecular identification, and upon the serology . In vitro both species are sensitive to aminoglycosides, and we recommend aminoglycosides be included in antibiotic regimens for treating cat scratch disease and Bartonella infections.

Proc Natl Acad Sci U S A, 1995 Jan 17, 92(2), 477 - 9
Cells of Escherichia coli swim either end forward; Berg HC et al.; Chemotactic cells of the bacterium Escherichia coli were marked asymmetrically by growth on a rich medium containing tetrazolium red . When this dye is reduced, it tends to form a refractile granule near one end of the cell, readily visualized by dark-field microscopy . In smooth-swimming cells, the marker was found with equal probability in front or behind . In wild-type cells, tumbles changed the cell orientation nearly as often as not . Some cells formed flagellar bundles at one end more frequently than at the other, but the run-interval distributions were the same either way . We conclude that the sensory system does not favor one end of the cell over the other . Thus, chemoreceptors that appear in patches at only one pole do not serve as a nose.

FEMS Microbiol Lett, 1995 Jan 15, 125(2-3), 317 - 21
Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries; Duenas M et al.; Phagemid vectors for display of protein/peptides on the surface of filamentous phage utilize a plasmid genome carrying the phage origin of replication, along with the gene fused to a fragment of gene III . Generation of phage particles displaying the fusion protein also requires superinfection of the host bacterium with a helper virus . We describe here the construction of a new gene III mutant of M13 KO7 bacteriophage and compare its ability to act as helper phage with two mutants derived from Fd tet (fKN 16 and fCA 55) . Furthermore, we investigate their capability to act as helper phages in SAP selection, where non-infectious helper phage, expressing antibody fragments but not protein 3, can still infect by first reacting with a soluble antigen-protein 3 fusion protein . Gene III mutants were found to be non-infectious, and high titers of infective particles were obtained only when the helper phage was grown in cells harbouring a gene III-containing plasmid . An amplification of the phage titer of 10(6) x was achieved in M13-derived phages, when used for the selection of specific antibody fragments.

FEMS Microbiol Lett, 1995 Jan 15, 125(2-3), 305 - 10
The effect of carbohydrates on the expression of the Prevotella ruminicola 1,4-beta-D-endoglucanase; Gardner RG et al.; The beta-1,4-endoglucanase of the ruminal bacterium, Prevotella ruminicola B14, hydrolysed carboxymethylcellulose and barley glucan but not xylan or mannan . Endoglucanase activity was present in 88- and 82-kDa proteins, and there was at least a 20-fold variation in endoglucanase activity when P . ruminicola B14 was grown on different sugars . The highest activities were observed with mannose, cellobiose or xylose and little activity was observed with sucrose, arabinose or rhamnose, P . ruminicola B14 also had significant xylanase and mannanase activities, but these activities were present in proteins that had lower molecular masses than the endoglucanase and these proteins did not cross-react with antibody made against the endoglucanase . Mannanase activity has a similar pattern of expression to the endoglucanase, while the xylanase was not induced or repressed by the same sugars or combinations of sugars . The xylanase activity was greatest when xylan was the energy source for growth, but xylose was a very poor inducer of xylanase activity.

Presse Med, 1995 Jan 14, 24(2), 67 - 73, 75-6, 78-9
{Helicobacter pylori, a rediscovered bacterium . Implication in gastroduodenal diseases}; Sobhani I et al.; Helicobacter pylori is a microaerophilic bacterium initially found in the gastric antrum of patients with peptic ulcer disease . As a result, H . pylori is now believed to have a pathophysiologic role in gastritis as well as in peptic ulcer disease . Several recent studies showed that it may be associated with duodenal ulcer relapse and that eradication therapy using antibiotics may significantly decrease the ulcer recurrence rate in duodenal ulcer patients . Moreover, epidemiological studies suggest that it may increase the relative risk of carcinoma in the stomach and preliminary studies seem to indicate that some low-grade lymphoma in the stomach may regress after H . pylori eradication . Although the mechanisms by which H . pylori induces mucosal injury and/or neoplasm is not clearly understood, several modifications in gastric functions have been reported . The most specific way of detecting H . pylori in tissue is a combination of culture and histologic staining of mucosal biopsy specimens obtained by endoscopy . Rapid urease test, cytology and PCR procedures performed on biopsies may give rapid, sensitive and specific results . Breath test using 13C- or 14C-radiolabelled urea and serology tests are of particular importance when H . pylori diagnosis is needed via no invasive procedures . Helicobacter pylori is supposed to interact with G and D cells . Gastrin and somatostatin are synthetized and released from antral G and gastric D cells respectively . The gastric D cells are in close contact with either G and parietal cells . Gastrin stimulates gastric acid secretion and epithelial gastric cell proliferation (parietal and EC-L cells) while somatostatin inhibits these effects . Chronic gastritis is associated with fundic duodenal ulcer disease . In this situation, basal gastrin and meal- or bombesin-stimulated gastrin in the serum (especially gastrin G17) have been found to be higher in H . pylori positive than in negative patients . Moreover, gastrin decreases up to normal levels after eradication of H . pylori . The long term effect of a such hypergastrinemia is not so far established . The mechanism underlaying hormonal modification is poorly understood . Since no G/D cell ratio modification could be found after H . pylori eradication while the amount of somatostatin increases, one would suggest functional alteration of either G or D cells in the H . pylori-related chronic gastritis . The role of inflammatory mediators on the gastrin release and the processing of progastrin induced by the bacterium need further investigations.

Mol Gen Genet, 1995 Jan 6, 246(1), 91 - 9
A gene proposed to encode a transmembrane domain of an ABC transporter is expressed in wheat mitochondria; Bonnard G et al.; In a study of transcribed regions of the wheat mitochondrial genome, we identified an open reading frame of 720 bp, which was consequently designated orf240 . The amino acid sequence deduced from orf240 shows a high level of similarity with HelC, a protein essential for c-type cytochrome biogenesis in the photosynthetic purple bacterium Rhodobacter capsulatus . HelC is part of a putative heme ABC transporter . An open reading frame homologous to orf240 is present in the mitochondrial genome of Marchantia polymorpha . The wheat gene is expressed as an mRNA of 2.8 kb, which is further processed to smaller transcripts . The transcript is highly edited, with 36 C to U modifications found in the coding region of all cDNAs sequenced . RNA editing is responsible for changes in 14% of the amino acids specified by the transcript.

Scand J Gastroenterol Suppl, 1995, 212, 34 - 7
Triple therapy with ranitidine, clarithromycin, and metronidazole in the treatment of Helicobacter pylori; Gotz JM et al.; BACKGROUND: To determine whether a triple therapy regimen for the treatment of Helicobacter pylori infection, consisting of ranitidine 300 mg q.i.d., clarithromycin 500 mg t.i.d., and metronidazole 500 mg t.i.d . would provide a safe and effective treatment regimen, we performed an open prospective study in 20 consecutive patients with proven H . pylori-associated non-ulcer dyspepsia or peptic ulcer disease . METHODS: The percentage of patients in whom eradication of H . pylori succeeded was determined . A semiquantitative assessment of histology was performed, and the results were analysed using Wilcoxon's matched-pairs ranks tests; side effects were noted and graded . RESULTS: Eradication was achieved in 19 of 20 patients, i.e . in 95% (confidence interval 85-100%) . Eradication of the bacterium led to a significant improvement in semiquantitative histology scores; active antral inflammation decreased from (mean +/- SEM) 1.84 +/- 0.19 to 0.21 +/- 0.16 (p = 0.0004) and chronic antral inflammation from 2.47 +/- 0.14 to 1.16 +/- 0.14 (p = 0.0002); active gastric body inflammation decreased from 0.95 +/- 0.19 to 0.00 +/- 0.00 (p = 0.0015) and chronic inflammation from 1.68 +/- 0.17 to 0.32 +/- 0.11 (p = 0.0007) . Side effects occurred in 45% of patients, but in over half of these patients only mild side effects occurred . Severe side effects did not occur, none of the patients discontinued the triple therapy . CONCLUSIONS: Triple therapy with ranitidine, clarithromycin, and metronidazole provides a safe and effective treatment of H . pylori infection, resulting in a high eradication rate, and in significant decrease in semiquantitative histology scores . Further prospective studies are warranted.

Scand J Gastroenterol Suppl, 1995, 210, 77 - 81
Sucralfate counteracts the inhibition of gastric mucosal mucin receptor by Helicobacter pylori lipopolysaccharide; Slomiany A et al.; BACKGROUND: Among the disturbances in gastric mucosal defense associated with Helicobacter pylori infection is the loss of mucus coat continuity, which results in a severe disturbance in the ability of mucus coat to maintain its functions as the pre-epithelial element of gastric mucosal defense . Here, we show that H . pylori, through its cell-wall lipopolysaccharide, disrupts the interaction between gastric mucin and its mucosal receptor, and that sucralfate is capable of counteracting this untoward effect of the bacterium . METHODS: The receptor was isolated from octylglucoside-solubilized gastric mucosal epithelial cell membranes by affinity chromatography on Sepharose-bound wheat germ agglutinin and following iodination with 125I, used in the binding assays for mucin in the presence of H . pylori lipopolysaccharide and sucralfate . RESULTS: Preincubation of the receptor protein with H . pylori lipopolysaccharide led to a decrease in mucin binding . The inhibitory effect was proportional to the concentration of lipopolysaccharide and reached a maximum of 91% at 30 micrograms/ml . The effect of H . pylori lipopolysaccharide was countered by sucralfate, which caused a dose-dependent relief of the inhibitory effect . The maximum (75%) restoration in mucin-receptor binding occurred at 60 micrograms/ml sucralfate . CONCLUSIONS: The results provide strong evidence for the effectiveness of sucralfate in preventing the loss of gastric mucus coat continuity caused by H . pylori.

Microbiol Immunol, 1995, 39(10), 745 - 51
Construction of a combined NotI/SmaI physical and genetic map of Moraxella (Branhamella) catarrhalis strain ATCC25238; Furihata K et al.; Using pulsed field gel electrophoresis (PFGE) and Southern hybridization techniques, a physical map of Moraxella catarrhalis strain ATCC25238 was constructed to provide basic genetic knowledge of this bacterium that has attracted attention in recent years as a human pathogen . Restriction endonuclease NotI cut the genome into 10 fragments and SmaI into 9, and the molecular size of the genome was estimated to be 1,940 kilobases . Location of the 12 genes participating in the biosynthesis of purine, pyrimidine and nine kinds of amino acids were determined on the circular physical map of the strain.

Aliment Pharmacol Ther, 1995, 9 Suppl 2, 71 - 6
The prevalence of Helicobacter pylori infection in gastric cancer; Forman D; The prevalence of Helicobacter pylori infection in gastric cancer patients is difficult to estimate because the infection may be lost from individuals with cancer or its precursor conditions . The bacterium does not colonize cancerous tissue and therefore studies using histopathology as a means of assessing infection require gastric biopsy material sampled separately from the tumour itself . Results from five such studies, with more than 50 patients, have demonstrated an H . pylori prevalence of 43%-78% . Studies using serological assessment of antibody presence have the advantage of enabling adequate case-control comparisons . Ten reported studies that were retrospective in design showed a range in prevalence from 52% to 89% among the cases and from 38% to 78% among the controls . Five studies showed a significant increase in infection prevalence among the cases, with odds ratios ranging from 1.6 to 4.2 . The odds ratio increased in subjects who were younger . Retrospective studies may be biased in that the prevalence of H . pylori among cases may be systematically under-estimated . Three prospective serological studies showed a prevalence of 69%-95% among cases and 47%-76% among controls . All three studies had a significantly elevated odds ratio and a pooled estimate of this was 3.8 (95% CI: 2.3-6.2) . In the pooled analysis there was a significant trend towards an increased odds ratio with increased time between the assessment of infection and cancer diagnosis . After 14 years of follow-up the overall infection prevalence among cases was 90% and the odds ratio was 8.7 (95% CI: 2.7-45.0) . This might represent the best estimate of infection prevalence and associated risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Aliment Pharmacol Ther, 1995, 9 Suppl 2, 33 - 9
The prevalence of Helicobacter pylori infection in different countries; Pounder RE et al.; The prevalence of Helicobacter pylori infection in a community is related to three factors: firstly, the rate of acquisition of infection with H . pylori--that is, incidence; secondly, the rate of loss of the infection; thirdly, the prolonged persistence of the bacterium in the gastroduodenal mucosa between infection and eradication . Variation in the prevalence of H . pylori is dominated by the great differences between communities in the incidence of H . pylori infection during childhood . The countries of the world form two groups: Group One is made up of those where the majority of children become infected with H . pylori during childhood and chronic infection continues during adult life; in Group Two only a minority of children are infected during childhood, but the prevalence of infection rises in proportion to age during adult life . Understanding the ages at which people acquire infection with H . pylori is crucial to the interpretation of H . pylori prevalence data.

Appl Environ Microbiol, 1995 Jan, 61(1), 297 - 302
Degradation and mineralization of atrazine by a soil bacterial isolate; Radosevich M et al.; An atrazine-degrading bacterial culture was isolated from an agricultural soil previously impacted by herbicide spills . The organism was capable of using atrazine under aerobic conditions as the sole source of C and N . Cyanuric acid could replace atrazine as the sole source of N, indicating that the organism was capable of ring cleavage . Ring cleavage was confirmed in 14CO2 evolution experiments with {U-14C-ring}atrazine . Between 40 and 50% of ring-14C was mineralized to 14CO2 . {14C}biuret and {14C}urea were detected in spent culture media . Cellular assimilation of 14C was negligible, in keeping with the fully oxidized valence of the ring carbon . Chloride release was stoichiometric . The formation of ammonium during atrazine degradation was below the stoichiometric amount, suggesting a deficit due to cellular assimilation and metabolite-N accumulation . With excess glucose and with atrazine as the sole N source, free ammonium was not detected, suggesting assimilation into biomass . The organism degraded atrazine anaerobically in media which contained (i) atrazine only, (ii) atrazine and glucose, and (iii) atrazine, glucose, and nitrate . To date, this is the first report of a pure bacterial isolate with the ability to cleave the s-triazine ring structure of atrazine . It was also concluded that this bacterium was capable of dealkylation, dechlorination, and deamination in addition to ring cleavage.

J Appl Bacteriol, 1995 Jan, 78(1), 61 - 5
Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization; Hay J et al.; The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar . DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg . pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture . A combination of co-cultivation of samples with Leg . pneumophila-naive A . polyphaga or Hartmannella vermiformis, incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg . pneumophila which were viable although not culturable.

J Leukoc Biol, 1995 Jan, 57(1), 80 - 7
LPS inhibits the intracellular growth of Legionella pneumophila in thioglycolate elicited murine peritoneal macrophages by iron-dependent, tryptophan-independent, oxygen-independent, and arginine-independent mechanisms; Gebran SJ et al.; Thioglycolate-elicited murine macrophages from genetically susceptible A/J mice activated with lipopolysaccharide (LPS) and infected with Legionella pneumophila in vitro evince marked inhibition of intracellular growth of this bacterium . The mechanism of inhibition by LPS-activated macrophages in terms of replication of this intracellular pathogen is unclear . LPS activation of murine macrophages induced a downshift in transferrin receptor (TfR) expression and reduction in cellular iron content, and this was correlated with augmented intracellular growth of Legionella in the cells . When LPS-stimulated macrophages were first saturated with iron, partial reversion of L . pneumophila growth restriction was observed . However, an excess of exogenous L-tryptophan (Trp) did not reverse this growth inhibition, nor did supplementation of the macrophage culture medium with both iron and Trp . The antilegionella activity of the macrophages induced by LPS activation was independent of reactive oxygen intermediates (ROI), since the scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on growth restriction . Likewise, notwithstanding the ability of LPS-activated macrophages to synthesize reactive nitrogen intermediates (RNI), which was inhibited by L-arginine analogs NG-monomethyl-L-arginine and L-aminoguanidine), as well as by incubation in arginine-free medium, their ability to inhibit the intracellular replication of L . pneumophila was not affected . Thus, we conclude that LPS-activated macrophages inhibit the intracellular growth of L . pneumophila partially by iron-dependent, Trp-independent, and ROI- and RNI-independent mechanisms . We also suggest that additional unknown mechanisms are involved, since complete reversion was not obtained.

J Bacteriol, 1995 Jan, 177(2), 277 - 82
Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus; Janssen PH et al.; Acetone degradation by cell suspensions of Desulfococcus biacutus was CO2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO2 dependent . Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded . In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate . Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA . In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thioyltically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway . Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone . This is postulated to be the carboxylation reaction (delta G(o)' for the carboxylation of acetone to acetoacetate, +17.1 kJ.mol-1) . At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ.mol of acetone-1, if one mol of ATP was invested . In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10(13) times lower than that measured . Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of teh energetic equivalent of (at lease) one ATP molecule . Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate . Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme . Carboxylation of pyruvate of phosphoenolpyruvate could not be detected.

Clin Exp Immunol, 1995 Jan, 99(1), 76 - 81
Interferon-gamma (IFN-gamma) production by human T lymphocytes upon Legionella pneumophila stimulation in vitro; Kitsukawa K et al.; In vitro immune responses to Legionella pneumophila were investigated . When human peripheral blood lymphocytes (PBL) from healthy volunteers were stimulated with formalin-killed L . pneumophila for 7 days in vitro, strong proliferative responses were observed . The responding cells were shown to be a CD4 T cell subset . It was also found that the CD4 T cells secreted significant amounts of IFN-gamma into the PBL culture supernatant . The production of IFN-gamma and IL-4 by PBL was measured semiquantitatively by reverse transcriptase-assisted polymerase chain reaction (RT-PCR) methods . Formalin-killed or live L . pneumophila-stimulated PBL expressed the mRNA for IFN-gamma but not the mRNA for IL-4 . The results suggest that the whole bacterium, as opposed to the supernatant, predominantly stimulates Th1 type helper T cells . The cloned T cells specific for L . pneumophila expressed the mRNA for IFN-gamma but not for IL-4 . In contrast to formalin-killed or live L . pneumophila stimulation, when PBL were stimulated with the bacterial culture supernatant, the proliferating T cells produced the mRNA for IL-4 as well as for IFN-gamma . A significant correlation between the proliferative response to formalin-killed L . pneumophila and IFN-gamma release in culture was observed (r = 0.6932, P < 0.001) in PBL from 30 healthy volunteers . From these in vitro studies, it is suggested that the whole L . pneumophila bacterium and their soluble antigens stimulate T cells in a manner which results in a different pattern of cytokine production.

Infect Immun, 1995 Jan, 63(1), 366 - 74
Rickettsia conorii entry into Vero cells; Teysseire N et al.; The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole . In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact . The entry occurred within 3 min after bacterium-cell contact, and R . conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol . Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed . By 12 min, 90% of bacteria were internalized and half were free in the cytosol . This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.

Infect Immun, 1995 Jan, 63(1), 27 - 37
Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils; Jansen R et al.; The Actinobacillus pleuropneumoniae RTX-toxins ApxI, ApxII, and ApxIII are important virulence factors of this swine pathogen . It is hypothesized that the Apx toxins are deleterious to defense cells of the host, enabling the bacterium to infect the host . To confirm this, we studied the effect on porcine polymorphonuclear neutrophils of mutant strains of A . pleuropneumoniae that were devoid of Apx toxins . For this purpose, we developed a system for targeted mutagenesis of A . pleuropneumoniae based on the conditionally replicating plasmid pVE6063 and insertional mutagenesis by homologous recombination . Employing this system on the reference strain of serotype 1, a strain that secretes ApxI and ApxII, we generated mutant strains that were devoid of ApxI and/or ApxII . We compared the ability of the parent strain and the mutant strains to provoke an oxidative burst in porcine neutrophils and to kill these cells . The parent strain and mutants that secreted either ApxI or ApxII provoked an oxidative burst and killed the neutrophils, whereas mutant strains that were devoid of ApxI and ApxII did not . These experiments indicate the importance of ApxI and ApxII to these profound effects on neutrophils and emphasize the importance of ApxI and ApxII in pathogenesis.

Arch Pathol Lab Med, 1995 Jan, 119(1), 30 - 2
Serology as a valid screening test for Helicobacter pylori infection in asymptomatic subjects; Blecker U et al.; Serologic testing is generally accepted as a valid noninvasive screening method for the detection of a Helicobacter pylori infection . To validate serology as an appropriate screening test for H pylori infection in symptom-free subjects, a recent-generation enzyme-linked immunosorbent assay for the detection of H pylori-specific IgG was performed in a large series of asymptomatic women . Blood samples for H pylori serology were taken from 542 apparently healthy women (aged 20 to 40 years) during prenatal screening . In this group, 120 (22.1%) had a positive titer for H pylori . We observed a significantly higher overall prevalence of H pylori seropositivity in nonwhites (62.3%) when compared with Belgian-born whites (17.8%) . In both groups there was a significant increase in seropositivity with increasing age . To investigate the correlation between a positive enzyme-linked immunosorbent assay and the actual presence of an active H pylori infection, carbon 13-labeled urea breath tests were performed in 85 seropositive and in 65 randomly selected seronegative subjects . These breath tests were positive in 82 (96.5%) of 85 seropositive and in none of the seronegative subjects, reflecting an actual presence of H pylori in the gastric mucosa of the seropositive women . We conclude that in our population of H pylori-seropositive subjects positive serologic findings correlates extremely well with an active infection with this bacterium . However, because all subjects who were investigated were actually symptom-free, it still should be determined whether these patients should undergo upper gastrointestinal endoscopy and/or be treated with an eradication therapy against H pylori . Further long-term follow-up studies will be required to answer this question.

J Bacteriol, 1995 Jan, 177(1), 235 - 7
Growth and buoyant density of Escherichia coli at very low osmolarities; Baldwin WW et al.; The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored . At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml . As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml . From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities . The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml . Both strains of E . coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9 . (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM . To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly . From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM . With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9 . Strain ML3 would only haltingly grow at 15 mosM . Four attempts were made to grow both ML3 and ML308 at 5 mosM . In three of the experiments, ML308 grew, while strain ML3 grew in one experiment . While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently . Therefore, from this study, we believe that E . coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.

Br Vet J, 1995 Jan-Feb, 151(1), 89 - 100
Attachment of Mycoplasma mobile 163 K to piscine gill arches and rakers--light, scanning and transmission electron microscopic findings; Stadtlander CT et al.; Explant cultures of gill arches and rakers were established to evaluate the attachment and colonization characteristics as well as the cytotoxic effects of the piscine bacterium, Mycoplasma mobile 163 K on piscine gill epithelium . Light, scanning and transmission electron microscopy were applied in this study and revealed heavy colonization of mycoplasmas on gill rakers, resulting in severe tissue damage of the gill epithelium . The complications for the function of the gills during breathing, the mechanisms of cytotoxicity, and the validity of this newly-established in vitro model are discussed in detail . In addition, anatomical specialities designated as spikes were identified on the inner surface of the gill rakers from trout; these could be used in the differentiation of fish.

Res Vet Sci, 1995 Jan, 58(1), 26 - 34
Grading the lesions of ovine footrot; Whittington RJ et al.; Sixteen methods of grading the lesions of ovine footrot were assessed on the basis of the effect of the lesions on the humoral immune response of the host to a causative bacterium, Dichelobacter nodosus . Methods that allowed for qualitative and quantitative differences in lesion scores between sheep were the best predictors of host response, and methods that assessed the lesions in each of the eight digits were more efficient than methods that did not grade the digits within feet . Weighting the scores for lesions that involved underrunning of the keratin of the hoof provided the most powerful means of predicting host response . The correlations between host response and the more elaborate weighted scores were close to the highest possible among additive linear estimators . Total weighted footscore, which is the sum of the footscores of the four feet weighted for underrun lesions, is proposed as a simple and effective grading system for sheep with lesions of footrot . There was a significant association within sheep between the number of underrun feet and the severity of lesions in individual feet.

Biofizika, 1995 Jan-Feb, 40(1), 60 - 73
{Does taxis exist in bacterial viruses?}; Ivanitskii GR et al.; By way of example of interaction of T4 bacteriophage with E . coli bacterium the scenario of enhancement of sorption of phages on bacteria through the remote mechanism of their cyclic interaction has been considered . An enlargement of the typical phage size, when its fibrillae are opened, underlies this mechanism . Modification of the structure of phages also occurs through the medium by release of products of bacterial metabolism into it . Allied questions related to investigation of physicochemical and biological processes, which require to take into account "fluffing" of microparticles . The medium-induced change of volume of microparticles leads to spatial cooperative effects with non-linear dynamics . Such phenomena are possible in all diffusional processes in biology, chemistry and physics.

Appl Biochem Biotechnol, 1995 Jan, 50(1), 35 - 43
Chemical synthesis and cloning of human beta-endorphin gene in Escherichia coli; Kim MH et al.; Total synthesis of human beta-endorphin gene has been designed for the expression in bacterial system . Eight individual oligonucleotides corresponding to the beta-endorphin gene were chemically synthesized and joined through the enzyme-catalyzed reaction . The final yield of the 111-nucleotide-long synthetic beta-endorphin gene construct was about 10% of the total oligonucleotide used . The synthetic human beta-endorphin gene was cloned into the bacterium Escherichia coli, using pUC8 vector and shown to have the correct nucleotide sequences as designed.

Wien Med Wochenschr, 1995, 145(7-8), 165 - 70
{The dermatologic spectrum of Lyme borreliosis}; Aberer E; The classical symptoms of Lyme borreliosis (LB) on the skin, erythema migrans, borrelia lymphocytoma and acrodermatitis chronica atrophicans (ACA) can be diagnosed clinically . In atypical cases, however, the diagnosis of a Borrelia burgdorferi (Bb) infection is based on the isolation of this bacterium from affected tissues . In early Lyme disease problems are arising in complicated erythema migrans which may represent reinfection, or with a failure of antibiotic treatment in some patients . The pathological changes in ACA presumably result from a chronic T-cell-mediated tissue injury with atrophy or sclerosis of connective tissue . On the other hand the presence of numerous plasma cells indicates activation of the humoral immune system where a progression into a malignant B-cell lymphoma is possible . In the recent past Bb could be cultivated from skin biopsies of a series of dermatoses of unknown origin, such as circumscribed scleroderma, dermatomyositis-like syndrome, relapsing nodular panniculitis, granuloma anulare and roseolar erythemas . New therapeutic strategies by administering antibiotics have been found to be effective in the described Bb induced dermatoses . Future studies should be focused on new standardized diagnostic procedures which make it possible to define the spectrum of dermatoses associated with a Bb infection.

Antonie Van Leeuwenhoek, 1995, 67(4), 345 - 50
Localization of the enzymes involved in H2 and formate metabolism in Syntrophospora bryantii; Dong X et al.; Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacterium Syntrophospora bryantii contained high hydrogenase activities (8.5-75.8 mumol.min-1mg-1 protein) and relatively low formate dehydrogenase activities (0.04-0.07 mumol.min-1 mg-1 protein) . The KM value and threshold value of the hydrogenase for H2 were 0.21 mM and 18 microM, respectively, whereas the KM value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 microM, respectively . Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction . Formate dehydrogenase and CO2 reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane . Results suggest that during syntrophic butyrate oxidation H2 is formed intracellularly while formate is formed at the outside of the cell.

J Enzyme Inhib, 1995, 8(4), 233 - 41
Regulation of arylsulfate sulfotransferase from a human intestinal bacterium by nucleotides and magnesium ion; Konishi-Imamura L et al.; Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group from phenylsulfate esters to phenolic compounds . Pentachlorophenol, one of the selective inhibitors of phenol sulfoconjugation in mammalian tissues, inhibited both phenol and tyramine sulfation by ASST . Nucleotide triphosphates such as ATP, GTP, UTP and CTP, and pyrophosphate inhibited the ASST activity, whereas Mg2+ and Mn2+ activated the enzyme and prevented its inhibition by ATP and pyrophosphate . Equimolar binding of {alpha-} and {gamma-32P}ATP to the enzyme showed that the enzyme protein was not phosphorylated, but bound ATP . These results suggest that nucleotide triphosphates and divalent cations are important modulators in the control of ASST activity.

Microbiol Immunol, 1995, 39(8), 591 - 8
Humoral immunity to commensal oral bacteria: quantitation, specificity and avidity of serum IgG and IgM antibodies reactive with Actinobacillus actinomycetemcomitans in children; Cole MF et al.; The levels, specificity and avidities of serum IgM and IgG antibodies reactive with Actinobacillus actinomycetemcomitans (Aa) serotypes a, b and c were determined in periodontally healthy (PH) children and compared with subjects with localized juvenile periodontitis (LJP) . All PH children exhibited IgM and IgG Aa-reactive antibodies whether or not Aa was detected subgingivally but the antibodies were not specific for Aa . In contrast, LJP sera contained high concentrations of IgM and IgG antibodies reactive with Aa that were largely specific for this bacterium . IgM and IgG antibodies in both PH and LJP subjects were of low avidity . With one exception, the avidities of IgG anti-Aa antibodies were significantly greater than those of IgM antibodies in both PH and LJP subjects . However, although the LJP subjects had as much as 115-fold more Aa-reactive IgG antibody than did the PH subjects the avidities of their IgG antibodies were no greater than those of the PH group . The induction by the host of low-avidity antibodies, that are ineffective in immune elimination, may be a reason why commensal bacteria persist at mucosal surfaces and why persons with LJP fail to eliminate Aa from their periodontal pockets.

Gene, 1994 Dec 30, 151(1-2), 191 - 6
Sequence analysis of the Rickettsia prowazekii gyrA gene; Wood DO et al.; The Rickettsia prowazekii (Rp) gyrA gene, which codes for a subunit of DNA gyrase in this obligate intracellular bacterium, has been isolated and characterized . Nucleotide sequence analysis revealed an open reading frame (ORF), initiating with a GTG start codon, of 2718 bp that could encode a protein of 905 amino acids (aa) with a calculated M(r) of 101,048 . The Rp gyrase subunit A (GyrA), when compared to GyrA analogs of other bacterial species, exhibited 43 to 50% identity . Alignment of the Rp GyrA aa sequence with the other analogs revealed the presence of a span of additional aa within the putative DNA-binding domain . The lack of an ORF within 865 bp upstream from the Rp gyrA demonstrates a Rp gene organization different from that of characterized gyrA from other species . Despite the similarity to Escherichia coli GyrA, Rp GyrA did not complement an E . coli gyrA temperature-sensitive mutant . However, Rp gyrA was dominant to an E . coli gyrA96 nalidixic-acid-resistant (NalR) mutant, conferring Nal sensitivity when introduced into the NalR E . coli strain.

Biochim Biophys Acta, 1994 Dec 15, 1201(3), 424 - 36
The growth of Escherichia coli in glucose-limited chemostat cultures: a re-examination of the kinetics; Senn H et al.; The relationship between specific growth rate (mu) and steady-state glucose concentration was investigated for Escherichia coli ML30 in carbon-limited chemostat culture . This was made possible by the development of a method for measuring reducing sugars in culture media in the microgram.1-1-range . Cells initially cultivated in batch culture at high glucose concentrations required long-term adaptation to nutrient-limited growth conditions in the chemostat (between 100-200 volume changes at D = 0.6 h-1) until steady-state with respect to residual glucose concentration was reached; for adapted cells, however, new steady-state glucose concentrations were usually obtained within less than 10 volume changes . A statistical evaluation of different kinetic models showed that between 0.2 h-1 < D < 0.8 h-1 the three models proposed by Monod (1942), Shehata and Marr (1971), and Westerhoff et al . (1982) described the data equally well and the applicability of the different models is discussed . Depending on the model used, calculated glucose concentrations supporting half maximum growth rate (Ks) were in the range of 40-88 micrograms.1-1 . The data strongly suggest that the large differences in Ks constants reported in the literature (ranging from 40 micrograms.1-1 up to 99 mg.1-1) are due to the use of E . coli cells adapted to different degrees to nutrient-limited growth conditions . This indicates that it is probably not possible to describe the kinetic properties of a bacterium with a single set of kinetic 'constants'.

J Biol Chem, 1994 Dec 9, 269(49), 30988 - 93
Production, purification, and characterization of recombinant maspin proteins; Sheng S et al.; Maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (Zou, Z., Anisowicz, A., Hendrix, M . J . C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R . (1994) Science 263, 526-529) . In this paper, we report the production of recombinant glutathione S-transferase-maspin fusion protein, expressed in the bacterium Escherichia coli, and recombinant maspin, expressed in the insect Spodoptera frugiperda cells . The fusion protein was purified by glutathione affinity chromatography . Maspin expressed in insect cells was purified by a combination of Bio-Rad AG1-2X anion exchange chromatography and heparin affinity chromatography . The recombinant maspin from insect cells was cleaved at the putative reactive center, as confirmed by protein sequencing . Both recombinant proteins demonstrated strong inhibitory effects on the invasion by two breast tumor cell lines across reconstituted basement membranes and such inhibitory effect was abolished in the presence of the polyclonal antibody made against the reactive center region of maspin . The trypsin-cleaved recombinant maspin did not inhibit invasion, indicating that the inhibitory activity requires the intact putative reactive center . This paper provides evidence that recombinant maspin protein itself inhibits invasion, and supports the role of maspin as a tumor suppressor.

Gene, 1994 Dec 2, 150(1), 97 - 100
An improved procedure and new vectors for transposon Tn5 mutagenesis of the phototrophic bacterium Rhodospirillum rubrum; Ghosh R et al.; A detailed examination of vectors and procedures used for Tn5 mutagenesis of the phototrophic purple non-sulfur bacterium Rhodospirillum rubrum has been performed . The mobilizable Tn5 suicide vectors currently available show a frequency of Tn5 mutagenesis for R . rubrum of approx . 10(-7)-10(-8), approx . 100-1000-fold lower than observed for the related bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides . Using the blue-to-red reversion of a blue-green mutant, R . rubrum ST6, containing a single Tn5 lesion in one of the early genes for carotenoid biosynthesis, we have shown that the frequency of precise excision of a chromosomally inserted Tn5 element, to restore the wild-type phenotype in the absence of selection, is 10(-6) . We have constructed three new suicide vectors for Tn5 mutagenesis, where the transposase encoded by the IS50R element was placed in the same (pSUPEG11, pSUPEG21) or in the opposing (pSUPEG22) orientation from the weak promoter of the RK2-derived tetR gene . With the vector pSUPEG11, the frequency of Tn5 mutagenesis was increased to 10(-5), approx . 100-fold higher than observed previously.

J Bacteriol, 1994 Dec, 176(24), 7694 - 702
Multiple chromosomes in bacteria: structure and function of chromosome II of Rhodobacter sphaeroides 2.4.1T; Choudhary M et al.; Although multiple chromosomes occur in bacteria, much remains to be learned about their structural and functional interrelationships . To study the structure-function relationships of chromosomes I and II of the facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T, auxotrophic mutants were isolated . Five strains having transposon insertions in chromosome II showed requirements for p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dHBA), serine, thymine, uracil, or histidine . The His, Thy, and pABA-dHBA mutants reverted to prototrophy at low frequency and concordantly lost their transposon insertions from the genome . The Ser, Ura, and pABA-dHBA mutants were complemented by cosmids that carried the region of chromosome II where the transposon insertions were located . The cosmids used for complementation analysis were selected, on the basis of map position, from a set of overlapping clones that had been ordered by a combination of hybridization and restriction endonuclease mapping . These experiments provide the basis for detailed studies of the structure, function, and interaction between each chromosome, and they demonstrate at this early stage of investigation that no fundamental differences exist between each chromosome.

J Bacteriol, 1994 Dec, 176(24), 7484 - 90
Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism; Chattopadhyay S et al.; A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis . The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S . Chattopadhyay, A . Mukherjee, and S . Ghosh, J . Bacteriol . 175:3240-3243, 1993) has now been completely sequenced . The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively . The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus . A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene . Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source . The reason for this is not clear . A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A . brasilense.

Eur J Biochem, 1994 Dec 1, 226(2), 613 - 8
A blue non-heme iron protein from Desulfovibrio gigas; Chen L et al.; A novel iron-containing blue protein, named neelaredoxin, was isolated from the sulfate-reducing bacterium Desulfovibrio gigas . It is a monomeric protein with a molecular mass of 15 kDa containing two iron atoms/molecule . The N-terminal sequence of neelaredoxin has similarity to the second domain of desulfoferrodoxin, a protein purified from Desulfovibrio vulgaris Hildenborough . This finding supports the hypothesis that the gene coding for desulfoferrodoxin (rbo) might have arisen from a gene fusion {Brumlik, M . J., Leroy, G., Bruschi, M . & Voordouw, G . (1990) J . Bacteriol . 172, 7289-7292} . The visible spectrum exhibits a single band at 666 nm, responsible for the blue color of the protein, which is completely bleached upon reduction with sodium ascorbate . In the oxidized state the EPR spectrum is complex, exhibiting well-resolved features at g = 7.6, 7.0, 5.9, and 5.8 which are assigned to two high-spin (S = 5/2) mononuclear-iron (III) centers with different rhombic distortions (E/D approximately 0.05 and approximately 0.08) . The two iron atoms contribute identically to the visible spectrum as judged from visible redox titrations, from which a reduction potential of +190 mV was determined for both iron sites at pH 7.5 . At high pH the visible and the EPR spectra become pH-dependent with a pKa above 9: the 666-nm band shifts to 590 nm and the EPR signals are converted into a signal with gmax approximately 4.7 . Neelaredoxin is readily reduced both by H2/hydrogenase/cytochrome c3 and by NADH/NADH-rubredoxin oxidoreductase.

Biochem J, 1994 Dec 1, 304 ( Pt 2), 441 - 7
Electronic properties of the dissimilatory sulphite reductase from Desulfovibrio vulgaris (Hildenborough): comparative studies of optical spectra and relative reduction potentials for the {Fe4S4}-sirohaem prosthetic centres; Lui SM et al.; The dissimilatory sulphite reductase (desulfoviridin) from the sulphate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) displays distinct optical and redox characteristics relative to the haem subunit of Escherichia coli assimilatory sulphite reductase . For high-spin pentaco-ordinate desulfoviridin there is minimal change in the absorbance of the oxidized chromophores both after reduction or after addition of exogenous ligands . A ligand-metal charge-transfer band approximately 702 nm is observed in both the oxidized and one-electron-reduced enzyme . E.p.r . spectroscopy has been used to define the relative reduction potentials for sirohaem and {Fe4S4} centres (delta E0 = Es0-Ec0) as a function of sirohaem axial co-ordination . Typically delta E0 lies in a range from -10 to -50 mV . These results show a correlation with the sigma-donor or pi-acceptor properties of the ligand and stand in sharp contrast with estimates for the E . coli enzyme . The electronic properties of the coupled {Fe4S4}-sirohaem redox centre common to both nitrite- and sulphite-reducing enzymes are apparently strongly dependent on the environment generated by protein side chains.

J Bacteriol, 1994 Dec, 176(23), 7299 - 308
Positive and negative regulation of sequences upstream of the form II cbb CO2 fixation operon of Rhodobacter sphaeroides; Xu HH et al.; The unlinked form I and form II Calvin cycle CO2 fixation (cbb) operons of the photosynthetic bacterium Rhodobacter sphaeroides are located on different genetic elements, yet both operons are positively regulated by the transcription activator protein CbbR, the product of the cbbR gene located immediately upstream of the form I operon . By employing deletion mutagenesis, and a newly constructed promoter probe vector, the form II operon promoter (cbbFIIp) and three other promoters (Up, Vp, and Wp) were localized within 2.1 kb upstream of the form II operon . Mutations in both cbbR and the first gene of the form I operon (cbbFI) elicited both positive and negative responses when transcriptional fusions controlled by these four promoters were examined . With the exception of Wp, all these upstream promoters were repressed by oxygen . In addition, these promoters were associated with open reading frames of unknown function whose deduced amino acid sequences showed no significant relationship to proteins in current databases . The results of these experiments suggest that the promoter sequences and genes upstream of the form II cbb operon may be intimately involved with control of the cbb regulon of this photosynthetic organism.

J Bacteriol, 1994 Dec, 176(23), 7260 - 6
Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation; Stibitz S et al.; We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis . A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B . pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B . pertussis bacterium has been constructed . This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B . pertussis genome . In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored . In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined . We have used this genetic system to map a newly described locus in B . pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).

Infect Immun, 1994 Dec, 62(12), 5603 - 7
CD4+ and CD8+ T-cell-dependent and -independent host defense mechanisms can operate to control and resolve primary and secondary Francisella tularensis LVS infection in mice; Conlan JW et al.; Immunity to experimental infection with the facultative intracellular bacterium Francisella tularensis is generally considered an example of T-cell-mediated, macrophage-expressed immunity . However, the results of the present study indicate that T-cell-independent mechanisms are also important in anti-Francisella defense . They show that mice selectively depleted of CD4+, CD8+, or both T-cell populations by treatment with T-cell subset-specific monoclonal antibodies remained capable of controlling and partly resolving a primary sublethal Francisella infection . Similarly, it was found that Francisella-immune mice depleted of either or both subsets of T cells retain a high degree of acquired immunity to reinfection . Together, these findings imply that resistance to primary and secondary tularemia can be mediated by cells other than CD4+ and CD8+ T cells.

J Appl Bacteriol, 1994 Dec, 77(6), 674 - 81
Salvage synthesis of purine nucleotides by Helicobacter pylori; Mendz GL et al.; The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy . The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine . Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed . The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established . The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected . Phosphorylase activities were not observed with any of the nucleosides . Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine . No nucleoside kinase activities were observed with any of the nucleosides . The presence of adenylate kinase was established, but no guanylate kinase activity was observed . The study provided evidence for the presence in H . pylori of salvage pathways for the biosynthesis of purine nucleotides.

Appl Environ Microbiol, 1994 Dec, 60(12), 4617 - 9
Demethylation of dimethylsulfoniopropionate to 3-mercaptopropionate by an aerobic marine bacterium; Visscher PT et al.; A bacterium, strain BIS-6, that grew aerobically on dimethylsulfoniopropionate (DMSP) was isolated from an intertidal mud sample . Strain BIS-6 quantitatively demethylated DMSP and 3-methiolpropionate to 3-mercaptopropionate . Strain BIS-6 was a versatile methylotroph growing on the osmolytes DMSP and glycine betaine and their methylated degradation products (dimethyl glycine, sarcosine, methylamines, and dimethyl sulfide.

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