Mol Gen Mikrobiol Virusol, 1990 Oct, (10), 28 - 30 {Regulation of phenylalanine biosynthesis in the obligate methylotroph Methylobacillus M75}; Maksimova NP et al.; Regulation of phenylalanine biosynthesis has been studied in the bacterium Methylobacillus M75 on the level of enzymes 3-deoxy-D-arabinoheptulose-7-phosphate-synthase, chorismatmutase, prephenatdehyrataze . The DAHP-synthase is shown to be synthesized constitutively and its activity is inhibited by all aromatic aminoacids and antranilate . The synthesis of chorismatmutase and prephenatdehydratase is repressed by tyrosine, the activity of the latter enzyme, besides that, is inhibited by phenylalanine, the effect of which is decreased in the presence of tyrosine. J Hosp Infect, 1990 Oct, 16(3), 257 - 61 Mycobacterium chelonei isolation from broncho-alveolar lavage fluid and its practical implications; Nye K et al.; Mycobacterium chelonei was isolated from the broncho-alveolar lavage fluid of seven patients on eight occasions over a 6-month period . The same bacterium was identified in the hospital water supply . Despite the use of a recommended disinfection procedure, it proved impossible to eradicate the organism until the bronchoscopes were treated with ethylene oxide and the use of tap water in rinsing was abandoned. Gene, 1990 Sep 28, 94(1), 69 - 75 Cloning, sequence and expression in Escherichia coli of the Methylobacillus flagellatum recA gene; Gomelsky M et al.; By means of interspecific complementation of an Escherichia coli recA- mutation with phasmids containing a gene bank from an obligate methylotroph, Methylobacillus flagellatum (Mf), the recA+ gene from this bacterium was identified . When expressed in an E . coli recA- host, it can function in recombination, DNA repair, and prophage induction . The nucleotide sequence of the gene has been determined . The coding region consists of 1032 bp specifying 344 amino acids . The deduced RecA protein structure shows a striking homology with RecA from other bacteria, except for the C-terminal region and some residues which were proposed to be responsible for the coprotease ability of RecA proteins . The region preceding the recA-Mf gene start codon has no SOS box--the LexA repressor binding site . Expression of the recA-Mf gene in E . coli proved to be DNA-damage independent. Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 777 - 86 Mode of action and substrate specificity of a purified exo-1,4-beta-D-glucosidase cloned from the cellulolytic bacterium Ruminococcus albus AR67; Ware CE et al.; A gene encoding exo-1,4-beta-D-glucosidase, from Ruminococcus albus AR67, was cloned in Escherichia coli, restriction mapped, and shown to be expressed from sequences within the insert that function as a promoter in E . coli . The cloned enzyme was located predominantly in the cytoplasm (40%) and attached to insoluble cell components (48%) . After purification to homogeneity, the enzyme (Mr = 64,000, monomeric) was specific for substrates with beta-D-glucopyranosyl configuration and was inactive against alpha-glucosides, lactosides and xylosides . Km values of the enzyme decreased with increasing chain length (G2-G5) . Glucose was the major product of hydrolysis from cellodextrins . Preference for longer chain cellodextrins is consistent with exo-1,4-beta-D-glucan glucohydrolase mode of action {E.C . 3.2.1.74}. Biochemistry, 1990 Sep 4, 29(35), 8085 - 93 The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis; Gibson JL et al.; Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation . Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants {for a review, see Tabita (1988)} . The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988) . The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity . In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster . In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources . In the case of FBPase, there are several regions that are conserved in the R . sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS) J Cell Physiol, 1990 Sep, 144(3), 408 - 15 Caffeine, an inhibitor of endocytosis in Dictyostelium discoideum amoebae; Gonzalez C et al.; The effect of the trimethylxanthine, caffeine, was examined on the growth and endocytosis pathways of the vegetative amoebae of the cellular slime mold Dictyostelium discoideum . Caffeine at concentrations of 1.5-3 mM was found to inhibit axenic growth, fluid-phase pinocytosis, and secretion of lysosomal enzymes . Cell viability was unaffected by incubation for 16 hours with 5 mM caffeine but decreased markedly thereafter . Phagocytosis of the bacterium Escherichia coli by Dictyostelium amoebae was also inhibited by caffeine, although at concentrations twofold to threefold higher . Caffeine rapidly entered into amoebae to reach an equilibrium between extracellular and intracellular concentrations, and it was not appreciably metabolized by Dictyostelium . Inhibition of growth and endocytosis was reversible upon removal of the drug and was partially counteracted by 10 mM adenosine . As caffeine discharged intracellular calcium stores in Dictyostelium (Abe et al., 1988), its inhibitory effect on endocytosis could result from the perturbation of calcium homeostasis . In agreement with this hypothesis, the cation La3+ (10 microM), a Ca2(+)-transport inhibitor, also strongly reduced fluid-phase pinocytosis. FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 95 - 9 Sizing of the Leptospira genome by pulsed-field agarose gel electrophoresis; Baril C et al.; Pulsed-field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family . The three restriction enzymes, NotI (5'GC/GGCCGC), NheI (5'G/CTAGC), ApaI (5'-GGGCC/C) generated DNA fragments of suitable size . The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira. FEMS Microbiol Immunol, 1990 Sep, 2(2), 83 - 8 Induction of tumoricidal activated macrophages by a liposome-encapsulated glycolipid, trehalose 2,3,6'-trimycolate from Gordona aurantiaca; Furukawa M et al.; A mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate, prepared from a non-pathogenic acid-fast bacterium Gordona aurantiaca, was shown to induce strong tumoricidal activity in peritoneal exudate cells by intravenous or intraperitoneal injection of liposome-encapsulated preparations . The mycolic acid derivative containing a high proportion of unsaturated fatty acids rendered macrophages cytotoxic against syngeneic mastocytoma cells in the absence of endotoxin, for over 14 days after the injection . The macrophages were ascertained to be at low intracellular levels of a lysosomal enzyme beta-galactosidase and an ectoenzyme alkaline phosphodiesterase, a specific pattern as previously described for "primed macrophages" . However the culture supernatants of the peritoneal exudate cells were not cytotoxic. Zentralbl Bakteriol, 1990 Sep, 273(4), 431 - 54 The technique of polymerase chain reaction--a new diagnostic tool in microbiology and other scientific fields (review); Thiele D; The polymerase chain reaction, a method of so far unknown sensitivity and specificity, is about to become an important diagnostic tool in microbiology . Practically even a single bacterium, virus particle, or parasite can be detected by it . Furthermore, this technique has been used with highly promising results in other scientific fields like genetics, forensic medicine and archeology . This article reviews technical aspects and variations of this new technique. J Med Microbiol, 1990 Sep, 33(1), 61 - 6 Ultrastructure of a spiral micro-organism from pig gastric mucosa ("Gastrospirillum suis"); Mendes EN et al.; The ultrastructural features of a helical-shaped bacterium occurring in the stomach of pigs, within the mucus on the mucosal surface of antral pits, were examined . The bacterial cell had three to eight spiral turns, flattened and truncated ends and was approximately 4.0 microns long and 0.6 microns wide . In some sections, up to six flagella, about 22 nm in diameter, were seen arising from each pole . The cytoplasm contained sparse, irregular granules, numerous ribosomes and large single-layered membrane-bound granules . In the flagella insertion area, there was a highly electron-dense component, the "polar membrane" . This micro-organism differed from similar bacteria described in cats, dogs and monkeys, and may cause inflammation in the antral mucosa of pigs similar to Helicobacter pylori infection in man . Furthermore, it was morphologically similar to the spiral micro-organism distinct from H . pylori which has been described recently in human antral mucosa from patients with gastritis and may be of potential significance as a pathogen in man . The name "Gastrospirillum suis" is proposed for this bacterium. Respir Med, 1990 Sep, 84(5), 377 - 85 Acute bronchitis in the community: clinical features, infective factors, changes in pulmonary function and bronchial reactivity to histamine; Boldy DA et al.; A descriptive study of acute bronchitis in patients without pre-existing pulmonary disease was undertaken in the community during the winter months of 1986-87 . Forty-two episodes were investigated in 40 individuals . The cardinal symptom was the acute onset of cough (100%), usually productive (90%) . Wheezing was noted by 62% of patients, but heard on auscultation in only 31% . A potential pathogen was isolated in 29% of cases with a virus (eight cases) being identified more frequently than either Mycoplasma pneumoniae (three cases) or a bacterium (three cases) . The acute illness was associated with significant reductions in forced expired volume in 1 second (P less than 0.02) and peak expiratory flow (P less than 0.001) but not forced vital capacity compared to 6 weeks later . Ten of the 27 (37%) patients who had a histamine challenge test performed at 6 weeks had a PD20 of less than 7.8 mumol histamine . Thirty-nine episodes (93%) were treated with antibiotics by the general practitioner, the clinical course being unremarkable apart from one patient who developed a lingular pneumonia despite antibiotic therapy . Further studies are required to assess whether acute bronchitis causes an acute increase in bronchial hyperresponsiveness and whether either antibiotics or inhaled bronchodilators or anti-inflammatory therapy has a useful role in the management of this predominantly viral illness. J Bacteriol, 1990 Sep, 172(9), 5425 - 31 Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2; Mitchell D et al.; Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2 . A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis . Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome . Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness . Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell . With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants . This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures . Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production. J Bacteriol, 1990 Sep, 172(9), 4877 - 87 Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD; McCleary WR et al.; Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation) . The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies . Individual frz cells cannot control the frequency at which they reverse direction while gliding . Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins . In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase . We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli . Thus, although M . xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria. Mol Gen Genet, 1990 Sep, 223(2), 205 - 10 Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus; Martinez-Laborda A et al.; Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light . We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants . A carR mutant produces the same carotenoids in the dark as the wild type grown in the light . This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system . A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA . In the dark, the carA mutant produces high levels of phytoene, the first C40 colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type . Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene . This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others . Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids . The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect. J Bacteriol, 1990 Sep, 172(9), 5299 - 306 CsgA, an extracellular protein essential for Myxococcus xanthus development; Shimkets LJ et al.; CsgA mutants of Myxococcus xanthus appear to be defective in producing an extracellular molecule essential for the developmental behaviors of this bacterium . The csgA gene encodes a 17.7-kilodalton polypeptide whose function and cellular location were investigated with immunological probes . Large quantities of the CsgA gene product were obtained from a lacZ-csgA translational gene fusion expressed in Escherichia coli . The chimeric 21-kilodalton protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Affinity-purified polyclonal antibodies raised against the fusion protein were used to determine the cellular location of the native CsgA protein by colloidal gold labeling and transmission electron microscopy . Between 1,100 and 2,200 extracellular molecules of CsgA per developing M . xanthus cell were detected, most of which were associated with the extracellular matrix . The anti-CsgA antibodies inhibited wild-type development unless they were first neutralized with the fusion protein . Together these results suggest that the CsgA gene product has an essential, extracellular function during development, possibly as a pheromone. Plasmid, 1990 Sep, 24(2), 90 - 9 Chloramphenicol acetyltransferase expression in marine Rhodobacter sp . NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid; Matsunaga T et al.; A vector, pUK318, was constructed to allow the expression of foreign genes in the marine photosynthetic bacterium Rhodobacter sp . NKPB 0021 . This strain has been cured of its two endogenous plasmids . pUK318 consists of a 2.3-kb PstI-BamHI restriction fragment, containing a marine Rhodobacter plasmid replication region, cloned into pUC18 . This fragment was derived from plasmid pRD31, a 3.1-kb endogenous plasmid purified from the marine strain Rhodobacter sp . NKPB 043402 . A kanamycin resistance gene from Tn903 was cloned into the PstI restriction site to provide antibiotic selection . pUK318 was transferred to Rhodobacter sp . NKPB 0021 by transformation, and efficiencies of 7.2 x 10(-5) were obtained . Furthermore, pUK318 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics . pUK318 was used to express the Escherichia coli chloramphenicol acetyl transferase (CAT) gene in Rb . NKPB 0021 . Transformants expressed a maximum CAT activity of 1.12 mmol/min/g dry cells . In addition, the DNA region essential for pUK318 replication in Rb . NKPB 0021 was localized to a 1.36-kb HincII-PstI fragment . This is the first report of a plasmid vector containing a marine Rhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection. Hua Xi Yi Ke Da Xue Xue Bao, 1990 Sep, 21(3), 252 - 5 {Construction of the human cytomegalovirus B gene clone and its restriction site analysis by computer}; Wu J et al.; We constructed two clones of the human cytomegalovirus strain AD169, using the plasmid pBR322 and the recipient bacterium Escherichia coli strain HB101 . The human cytomegalovirus B gene was isolated from the recombinant plasmid pAT153 that contained the 20.7-kb Hind III F fragment of the human cytomegalovirus genome . The recombinant plasmid pAT153 was digested with BamHI and a 8.5-kb fragment was isolated and ligated with BamHI-digested pBR322 to form the recombinant plasmid pB1 . pB1 was further digested with BamHI-Hind III and a 5.1-kb fragment was isolated, and ligated with BamHI-Hind III digested pBR322 to form the recombinant plasmid pBH1 . The human cytomegalovirus B gene sequence was analysed by Beckman Microgenie Systems . The results indicated that the human cytomegalovirus B gene could be further digested by 13 kinds of restrict endonucleases . The size of the restrict fragments was between 22 base pairs to 2.9 kilobase pairs . The construction of the expression plasmid that contained human cytomegalovirus B gene is now in progress. Mol Microbiol, 1990 Sep, 4(9), 1567 - 74 Mutagenesis, cloning and complementation analysis of C4-dicarboxylate transport genes from Rhodobacter capsulatus; Hamblin MJ et al.; Transposon mutagenesis was used to isolate insertion mutants of the photosynthetic bacterium Rhodobacter capsulatus which were unable to grow under aerobic conditions in the dark on malate, succinate or fumarate as sole carbon sources . Of five mutants isolated, all were deficient in C4-dicarboxylate transport . However, these mutants were still capable of photoheterotrophic growth, although at a slower rate than the wild type, on malate and succinate (but not fumarate) . The mutated locus (designated dct) was complemented in trans using a cosmid gene bank . Subcloning and complementation analysis indicated that at least three closely linked genes essential for aerobic dicarboxylate transport were contained within an 8.3 kb region of the Rhodobacter capsulatus chromosome. J Bacteriol, 1990 Sep, 172(9), 5071 - 8 Sequence analysis and transcriptional organization of the Rhodopseudomonas viridis cytochrome c2 gene; Grisshammer R et al.; The cytochrome c2 gene (cycA) of the purple nonsulfur bacterium Rhodopseudomonas viridis was isolated from a genomic library by using two degenerate oligonucleotides containing all possible DNA sequences predicted from the published amino acid sequence of this protein (Ambler et al., Proc . Natl . Acad . Sci . USA 73:472-475, 1976) . Cloning and sequence analysis of the cytochrome c2 gene indicated the presence of a typical procaryotic 20-residue signal peptide, suggesting that this periplasmic protein in synthesized in vivo as a precursor . In addition, four amino acids were found to be different by comparing the published sequence of the mature protein with that deduced from the isolated cycA gene (Lys-14----Leu, Ser-46----Ala, Ile-84----Val, Leu-97----Ile) . Northern (RNA) blot analysis and fine mapping of the 5' and 3' ends of the cycA gene transcript from photoheterotrophically grown R . viridis cells revealed one abundant transcript of 523 to 530 nucleotides in length, with the transcription start site at position -39 relative to the coding region of cytochrome c2 . A low-abundance transcript with an extended 3' end (about 600 bases in length) is thought to be processed by exonucleases, resulting in the slightly shorter main transcript. J Theor Biol, 1990 Aug 23, 145(4), 535 - 45 Evolutionary relationships between "Q-type" photosynthetic reaction centres: hypothesis-testing using parsimony; Beanland TJ; Hypotheses concerning the evolutionary relationships between "Q-type" photosynthetic reaction centres are tested using amino acid parsimony analysis of subunit sequences and an alignment based on dot matrix comparisons . Strong evidence is found for independent gene duplications having produced the L and M subunits of the photosynthetic purple bacterial reaction centre and D1 and D2 of Photosystem-II . Much support is also found for the L and M subunits of the green filamentous bacterium Chloroflexus aurantiacus arising from the same gene duplication as the purple bacterial subunits, suggesting there was an ancestral bacterial heterodimeric reaction centre . These conclusions caution against over-extrapolation from the purple bacterial reaction centre to Photosystem-II, and suggest that the latter is more ancient than previously supposed. Nature, 1990 Aug 16, 346(6285), 674 - 7 Isolation and sequence of an FK506-binding protein from N . crassa which catalyses protein folding; Tropschug M et al.; Slow protein-folding reactions are accelerated by a prolyl cis/trans isomerase isolated from porcine kidney which is identical to cyclophilin, a protein that is probably the cellular receptor for the immunosuppressant cyclosporin A . Catalysis probably involves the isomerization of prolyl peptide bonds in the folding protein chains . Cyclosporin A inhibits folding catalysis by cyclophilin . Here we report the isolation, cloning, sequencing and expression of another protein with prolyl isomerase activity from Neurospora crassa which is unrelated to cyclophilin and which also catalyses slow steps in protein folding . This protein does, however, show sequence similarity to a human protein that binds to another, recently discovered immunosuppressive drug, FK506 . Moreover, it shares 39% identity with the carboxy-terminal 114 residues of a cell-surface protein from the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease . Catalysis of folding by the FK506-binding protein from N . crassa is inhibited by FK506, but not by cyclosporin A . Thus, at least two different classes of conformationally active enzymes (conformases) exist that catalyse slow steps in protein folding . Both occur in a wide variety of cells and are inhibited by immunosuppressive drugs. J Biol Chem, 1990 Aug 15, 265(23), 13741 - 9 ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro; Woehle DL et al.; Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification . Incubation of R . rubrum cell supernatant with {alpha-32P}NAD results in the labeling of glutamine synthetase and two other unidentified proteins . Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins . The {alpha-32P}ATP- and {alpha-32P} NAD-dependent modifications of R . rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels . Loss of enzymatic activity by glutamine synthetase did not correlate with {alpha-32P}NAD labeling . This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R . (1984) J . Biol . Chem . 259, 5100-5104) . A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels . The adenylylation site of R . rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification. Appl Environ Microbiol, 1990 Aug, 56(8), 2494 - 8 Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction; Welch D et al.; A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid . Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h . By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C . trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light . DNA from intact cells from each of the 15 serovars of C . trachomatis could also be amplified for visualization . With this procedure, the presence or absence of C . trachomatis DNA in a sample could be established in less than 1.5 h . The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C . trachomatis, and similar techniques should be possible for any type of bacteria. Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6077 - 81 Juxtaposition of expressed variable antigen genes with a conserved telomere in the bacterium Borrelia hermsii; Kitten T et al.; Borrelia hermsii, an agent of relapsing fever, survives in mammals through antigenic variation . Change in serotype-specific variable outer membrane proteins (Vmps) occurs when a Vmp gene at an expression site is replaced with a previously silent gene for another Vmp . Silent and active genes are on separate linear plasmids . The upstream site for a nonreciprocal recombination between two linear plasmids is near the 5' ends of the expressed and silent genes . In the present study we sought the downstream recombination sites in two serotypes, 7 and 21 . Restriction fragments containing plasmid telomeres were identified by susceptibility to digestion with BAL-31 and rapid reannealment following denaturation . Whereas both silent genes and a minority population of both expression-linked genes were several kilobases from the telomeres, the predominant population of both expressed genes had 3' ends near plasmid telomeres . Sequence analysis of the predominant expression plasmids revealed that the telomeric sequences were the same in serotypes 7 and 21 . Identical sequence was also downstream of silent Vmp genes . Switching of Vmp genes appears to occur by recombination that involves both upstream and downstream sites . The expression plasmid's telomere is preserved in the recombination event. J Bacteriol, 1990 Aug, 172(8), 4497 - 504 Gene encoding the 5.7-kilodalton chlorosome protein of Chloroflexus aurantiacus: regulated message levels and a predicted carboxy-terminal protein extension; Theroux SJ et al.; The major light-harvesting pigment of the green filamentous bacterium Chloroflexus aurantiacus is bacteriochlorophyll (Bchl) c, localized in chlorosomes attached to the inner surface of the cytoplasmic membrane . Chlorosomes consist of four polypeptides and associated pigments and lipids . Previous studies of the inducible assembly of the photosynthetic apparatus had indicated that the major chlorosomal polypeptides are present as high-molecular-weight aggregates before the appearance of mature chlorosomes, and a mechanism for posttranslational processing of a polyprotein had been proposed . We have isolated the gene (csmA) encoding the 5.7-kilodalton chlorosomal polypeptide from C . aurantiacus in order to determine whether this protein is synthesized as part of a polyprotein . Analysis of the nucleotide sequence of csmA indicates that the gene is not large enough to encode more than one known chlorosome polypeptide . Transcriptional analysis indicates that csmA is transcribed as a small message whose abundance is regulated in response to oxygen, so that no csmA message is detectable in cells grown aerobically in the dark . Comparison of the sequence predicted by csmA with the peptide sequence of the Bchl c binding protein purified from chlorosomes indicates that this protein is synthesized with a carboxy-terminal extension of 27 amino acids . We discuss possible roles for this carboxy-terminal extension in the assembly of chlorosomes. FEMS Microbiol Lett, 1990 Aug, 58(3), 263 - 8 Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella; Zygmunt MS et al.; Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats . CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6 . Immunoblotting revealed most of the stained bands around pH 4.5-5.4 . CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested . Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection. J Bacteriol, 1990 Aug, 172(8), 4370 - 7 Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata; Marrs CF et al.; Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis . We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed . Using an M . bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M . lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M . lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli . Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations . Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids . These deletions and insertions thus localize a probable invertase necessary for the inversion event . The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M . bovis and called ORF2 appeared to be a strong candidate for the invertase . This conclusion was confirmed when a plasmid containing the M . bovis ORF2 supplied, in trans, the inversion function missing from one of the M . lacunata phase-locked inversion mutants . We have named these putative invertase genes piv(ml) (pilin inversion of M . lacunata) and piv(mb) (pilin inversion of M . bovis) . Despite previously noted sequence similarities between the M . bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases. Biotechnology (N Y), 1990 Aug, 8(8), 746 - 9 Applications of imaging spectroscopy in molecular biology . II . Colony screening based on absorption spectra; Arkin AP et al.; Digital imaging spectroscopy has been used to obtain the grayscale spectrum of colored bacterial colonies directly from petri dishes . Up to 500 individual colony spectra can be simultaneously recorded and processed from a single plate . Spectra can be obtained in the visible to near infrared region (400nm-900nm) with 10nm resolution . Instrument response is normalized through run-time radiometric calibration such that each grayscale spectrum can be converted to the ground-state absorption spectrum of the colony . In this study, mutants of the photosynthetic bacterium Rhodobacter capsulatus have been differentiated by the absorption spectra of their pigment-protein complexes . This imaging technique is applicable to chromogenic systems in which colony and/or media color (e.g . indicator plates) provides a quantitative indicator of gene expression. Biochemistry, 1990 Jul 24, 29(29), 6911 - 8 Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli; Taylor MF et al.; The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 {Bennett, L . T., Jacobsen, M . R., & Dean, D . R . (1988) J . Biol . Chem . 263 1364-1369} and the resulting plasmid transformed and expressed in Escherichia coli strain DH5 . Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E . coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions . Even higher levels were observed with flavodoxin expressed in E . coli under control of a tac promoter . Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form . The flavodoxin purified from E . coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein . The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate . Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions . The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV . This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin . Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 Jul 2, 267(1), 33 - 7 Electron microscopy of native and artificial methylreductase high-molecular-weight complexes in strain Gö 1 and Methanococcus voltae; Hoppert M et al.; The preparation of inside-out vesicles from methanogenic bacteria with protein cell walls was improved with regard to the preservation of structure and localization of membrane-bound proteins . Complexes similar to the methanoreductosome in the methanogenic bacterium Go 1 were also found attached to the inner aspect of the cytoplasmic membrane of Methanococcus voltae . Methanoreductosomes were purified from crude extracts of Go 1-cells by affinity chromatography . Under specific conditions at high protein concentrations methyl-CoM-methylreductase molecules isolated from Go 1-cells could be reassociated to spherical complexes of various sizes, with an appearance similar to that of methanoreductosomes isolated from strain Go 1. Appl Environ Microbiol, 1990 Jul, 56(7), 2193 - 9 Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium; Brooker JD et al.; Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures . Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S . ruminantium . A monoclonal antibody-based solid-phase immunoassay was established to quantify S . ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells . For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth . Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S . ruminantium reacted with the same antigen on each strain . For one strain, an additional antigen reacted with both monoclonal antibodies . In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies. Appl Environ Microbiol, 1990 Jul, 56(7), 2025 - 8 Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history; Nilsson M et al.; Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats . We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T . sacchari is probably one of the oldest known living organisms . More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T . vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes . In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes . In varved sediment cores from three lakes, we found a temporal relationship between records of T . vulgaris endospores and the pollen of plants indicating agriculture . Endospores were very rare in sediments deposited before agriculture, ca . 1100 A.D . From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded . High endospore accumulation rates were found with the subsequent agricultural expansion . This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses . Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies. J Cell Biol, 1990 Jul, 111(1), 87 - 94 Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus; Troschel D et al.; A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described . Synthesis of two proteins of the bacterium's photosynthetic apparatus (light-harvesting complex B870 alpha and beta) was performed by SP6 polymerase transcription of the subcloned genes, isolation of the mRNA and translation in vitro using a cell-free extract of R . capsulatus cells . The integration of these proteins in vitro into added intracytoplasmic membrane vesicles (ICM) is demonstrated . Without addition of ICM approximately 70% of the synthesized B870 proteins were soluble . If, however, ICM were present during synthesis, the majority of the soluble protein was found to associate with the membranes . The membrane-associated polypeptides could be solubilized only by detergent treatment but could not be extracted by treatment at alkaline pH (Na2CO3), suggesting that the proteins had been firmly inserted into the lipid bilayer . Moreover, the B870 alpha and beta proteins that integrated in vitro into ICM were also found to associate with pigment ligands and to assemble into a native reaction center/B870 complex . The native conformation of this complex isolated from ICM by Triton fractionation was demonstrated by microspectral analysis of the bound pigments. Mol Microbiol, 1990 Jul, 4(7), 1199 - 205 Protein transfer into the recipient cell during bacterial conjugation: studies with F and RP4; Rees CE et al.; Transfer of donor cell proteins to the recipient bacterium was examined in F- and RP4-mediated conjugation . Transfer of a 120 kD polypeptide, identified as the larger product of the plasmid DNA primase gene, was readily detected during RP4-promoted conjugation . The protein was transmitted to the cytoplasm of the recipient, presumably complexed to the transferred ssDNA . F DNA was transferred without detectable association with any cytoplasmic tra protein or with the ssDNA-binding protein encoded by the plasmid . However, a 92 kD protein, possibly F TraD product, was transmitted to the membrane fraction of the recipient cell. Infect Immun, 1990 Jul, 58(7), 2076 - 84 The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope; Stover CK et al.; Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific . Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes . The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R . tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium . In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli . DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide . A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with {35S}methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide . Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A) . Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene. Biochemistry, 1990 Jun 26, 29(25), 5968 - 74 Direct observation of the phase behavior of the lipid bilayers of phage PM2 and the intact host cells by 1H-31P cross-polarization NMR; Odahara T et al.; A method for obtaining the 31P NMR spectrum of a particular supramolecular structure in an intact biological system was developed by applying the 1H-31P cross-polarization technique to a lipid-containing bacteriophage, PM2, and its host bacterium, Alteromonas espejiana . It was shown that 31P NMR spectra of nucleic acids and lipid bilayers can be obtained separately with short and long thermal contact times, respectively . The temperature dependence of the chemical shift anisotropy (delta sigma = sigma parallel - sigma perpendicular) was examined for the separately obtained membrane spectra . Referring to the results of thermal analysis and 31P NMR spectra of bilayers of the extracted phospholipids, the phase transition of the biomembrane was identified for the PM2 phage and the host cell . The dynamic state of the biomembrane of the intact bacterium was directly monitored in detail . The phase behavior of the PM2 lipid bilayer showed good agreement with the earlier report (Akutsu et al., 1980) . It turned out that the phase behavior of the intact biomembrane is different from that of the bilayer of the extracted lipids for both PM2 and the host cell . Namely, the terminal temperatures of the phase transition of the host cell and PM2 membranes were lower and higher than those of the extracted phospholipids, respectively. J Mol Biol, 1990 Jun 5, 213(3), 411 - 4 Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis; Raghavan M et al.; Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis . One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000 . Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer) . The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A . The crystals are suitable for high-resolution X-ray diffraction analysis. J Exp Med, 1990 Jun 1, 171(6), 1931 - 42 Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo; Singh RP et al.; The Eta-1 gene specifies a secreted product of activated T cells and is associated with genetic resistance to infection by an obligate intracellular bacterium . Previous studies have suggested that eta-1 might affect the ability of macrophages to migrate to the site of bacterial infection and/or to inhibit intracellular bacterial growth . We therefore examined the interaction of eta-1 with macrophages in vitro and in vivo . We find that macrophages express approximately 10(4) eta-1 receptors/cell and each receptor has a Kd of approximately 5 x 10(-10) M . The subsequence of eta-1 containing an RGD motif is required for binding because a synthetic peptide containing the eta-1 RGD domain inhibited protein attachment to macrophages . We also found that subcutaneous inoculation of mice with eta-1 resulted in a cellular infiltrate comprised primarily of macrophages . We propose that the interaction between eta-1 and its receptor on macrophages results in a change in macrophage physiology resulting in accumulation of these cells at extravascular sites. J Bacteriol, 1990 Jun, 172(6), 3379 - 87 Surface proteins of the gliding bacterium Cytophaga sp . strain U67 and its mutants defective in adhesion and motility; Burchard RP et al.; Surface proteins of the gliding bacterium Cytophaga sp . strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen) . At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion . The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight . An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern . Two other Adh2 revertants resembled their adhesion-defective parent . The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type. J Bacteriol, 1990 Jun, 172(6), 3117 - 24 Use of nonmotile mutants to identify a set of membrane proteins related to gliding motility in Cytophaga johnsonae; Pate JL et al.; Nonmotile mutants of the gliding bacterium Cytophaga johnsonae were examined to identify proteins that might be involved in gliding motility . Wild-type and mutant cell proteins were solubilized and fractionated by using Triton X-114, and the proteins that partitioned into the aqueous phase or the detergent phase were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for proteins that differed between wild-type and mutant cells . Seventeen proteins, ranging in size from 16 to 150 kilodaltons, were implicated by this technique as having some relationship to gliding and were designated Gld-1 through Gld-17 . All Gld proteins behaved as integral membrane proteins, partitioning into the detergent phase . All 56 mutants examined exhibited changes in 1 or more of the Gld proteins, with the number of proteins altered in any mutant varying from 1 to 11 . Several lines of evidence suggested that proteins Gld-12 through Gld-15 are glycoproteins . Analysis of banding patterns of detergent-fraction proteins of motile revertants supported the idea that the Gld proteins have a role in gliding motility. J Bacteriol, 1990 Jun, 172(6), 3051 - 9 Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures; Gomes SL et al.; Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes . Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family . A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E . coli . Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment . Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message . S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence . A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E . coli . At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E . coli sigma 70 promoter consensus sequence . S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication . Thus, in both human cells (I . K . L . Milarski and R . I . Morimoto, Proc . Natl . Acad . Sci . USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions. Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4504 - 8 Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli; Jakubowski H; Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases . The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone . Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo . In E . coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase . One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo . These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E . coli but, in general, establish the importance of error-editing mechanisms in living cells. Mol Microbiol, 1990 Jun, 4(6), 977 - 89 Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides; Coomber SA et al.; Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction-centre and light-harvesting LH1 complexes . The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202 . We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction-centre H subunit . This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium. J Gen Microbiol, 1990 Jun, 136 ( Pt 6), 1161 - 6 DNA restriction fingerprint analysis of the soil bacterium Azospirillum; Giovannetti L et al.; Total DNAs of 18 strains of Azospirillum from different sources and geographical areas were compared by restriction endonuclease pattern analysis . Fragments obtained with HindIII or BglII were separated by PAGE and stained with silver nitrate . Each strain possessed a unique and reproducible fingerprint with each enzyme, thereby facilitating strain recognition . UPGMA analysis recovered clusters of band patterns that were compared to the distribution of species within the genus Azospirillum. Biochemistry, 1990 May 22, 29(20), 4886 - 92 Coordination and redox properties of a novel triheme cytochrome from Desulfovibrio vulgaris (Hildenborough); Tan JA et al.; A high molecular weight multiheme c-type cytochrome from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been spectroscopically characterized and compared with the tetraheme cytochrome c3 . The protein contains a pentacoordinate high-spin heme (gz 6.0) and two hexacoordinate low-spin hemes (gz 2.95, gy 2.27, gx 1.48) . From analysis of the g values for the low-spin hemes by the procedure of Blumberg and Peisach (Palmer, 1983) and comparison with with the optical spectra from a variety of c-type cytochromes, it is likely that these low-spin hemes are bound by two histidine residues . The NO derivative displayed typical rhombic EPR features (gx 2.07, gz 2.02, gy 1.99) . Addition of azide does not lead to coupling between heme chromophores, but the ligand is accessible to the high-spin heme . The use of a glassy-carbon electrode to perform direct (no promoter) electrochemistry on the cytochrome is illustrated . Differential pulse polarography of the native protein gave two waves with reduction potentials of -59 (5) and -400 (8) mV (versus NHE) . The cyanide adduct gave two waves with reduction potentials of -263 (8) and -401 (8) mV . The cytochrome was found to catalyze the reduction of nitrite and hydroxylamine. J Biol Chem, 1990 May 15, 265(14), 8329 - 38 Genetic and biochemical characterization of carotenoid biosynthesis mutants of Rhodobacter capsulatus; Armstrong GA et al.; We have used genetic and biochemical techniques to study carotenoid biosynthesis (crt) mutants of Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium . All nine identified crt genes are located within the 46-kilobase pair photosynthesis gene cluster, and eight of the crt genes form a subcluster . We have studied the operon structure of the crt gene cluster using transposon Tn5.7 mutants . The Tn5.7 insertion sites in 10 mutants have been mapped to high resolution (25-267 base pairs) by Southern hybridization . Two insertions each map within the coding regions of the crtA, crtC, crtE, and crtF genes, and one insertion lies within the crtI gene . The insertion in crtI is not polar on the downstream crtB gene, suggesting that crtI and crtB may form two separate operons . Another insertion located in the 5' noncoding region between the divergent crtA and crtI genes has no effect on wild-type pigmentation and apparently lies between the promoters for these operons . A Tn5.7 mutation in the 3' region of crtA yields a bacteriochlorophyll-minus phenotype, while a 5' insertion affects only carotenoid biosynthesis . Regulatory signals for transcription of a downstream operon required for bacteriochlorophyll biosynthesis may thus overlap the coding region of crtA . We also present the first evidence for the functions of the crtB, crtE, and crtJ gene products using a new in vitro assay for the incorporation of {14C}isopentenyl pyrophosphate into carotenoid precursors and phytoene in cell-free extracts . Extracts from a crtE mutant accumulate {14C}prephytoene pyrophosphate, while those from crtB and crtJ mutants accumulate {14C}geranylgeranyl pyrophosphate . We therefore propose that CrtE is the phytoene synthetase and that CrtB, and possibly CrtJ, are components of the prephytoene pyrophosphate synthetase. Am J Med, 1990 May 14, 88(5A), 41S - 45S Studies of the outer membrane proteins of Branhamella catarrhalis; Murphy TF; PURPOSE: Branhamella catarrhalis has emerged as an important human pathogen in the past several years . Therefore, studies of the outer membrane have been undertaken in order to identify virulence factors and begin to understand the immune response to infection . MATERIALS AND METHODS: The outer membrane of B . catarrhalis has been purified by sucrose density centrifugation . The outer membrane proteins (OMPs) of 50 strains from diverse sources were isolated by simpler methods and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Experiments were designed to identify OMPs that express determinants on the surface of the intact bacterium . RESULTS: Eight major OMPs have been identified (OMPs A through H) . The OMP patterns from diverse strains were strikingly similar . OMP E (approximately 56,000 daltons) and OMP G (approximately 28,000 daltons) have determinants that are surface-exposed and these determinants are shared among a majority of strains of B . catarrhalis . CONCLUSION: These observations have important implications with regard to the immune response to infection and future vaccine development. Mol Microbiol, 1990 May, 4(5), 811 - 20 Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi; Hinnebusch J et al.; Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends . DNA at the ends, or telomeres, of two linear plasmids of B . burgdorferi strain B31 was examined . Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced . An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid . The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats . The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid . The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region . These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons. Biophys J, 1990 May, 57(5), 1009 - 23 Mathematical analysis of cell-target encounter rates in three dimensions . Effect of chemotaxis; Charnick SB et al.; Efficient and rapid immune response upon challenge by an infectious agent is vital to host defense . The encounter of leukocytes (white blood cells of the immune system) with their targets is the first step in this response . Analysis of the kinetics of this process is essential not only to understanding dynamic behavior of the immune response, but also to elucidating the consequences of many leukocyte functional abnormalities . The motion of leukocytes in the presence of targets typically involves a directed, or chemotactic component . These immune cells orient the direction of their motion in the presence of gradients in chemical attractants generated by pathogens . Fisher and Lauffenburger (1987 . Biophys . J . 51:705-716) developed a model for macrophage/bacterium encounter in two dimensions which includes chemotaxis, and applied it to the particular system of alveolar macrophages (phagocytic leukocytes on the lung surface) . Their model showed that macrophage/target encounter is likely the rate-limiting step in clearance of bacteria from the lung surface (Fisher, E . S., D . A . Lauffenburger, and R . P . Daniele . 1988 . Am . Rev . Resp . Dis . 137:1129-1134) . We have extended this model to analyze the effects of cell motility properties and geometric parameters on cell-target encounter in three dimensions . The differential equation governing encounter time in three dimensions is essentially the same as that in two dimensions, except for changed probability values . Our results show that more highly directed motion is necessary in three dimensions to achieve substantially decreased encounter times than in two dimensions, because of the increased search dimensionality . These general results were applied to the particular system of neutrophils operating in three dimensions in response to a bacterial challenge in connective tissue . Our results provide a plausible rationalization for both the chemotactic and chemokinetic behavior observed in neutrophils . That is, these cells exhibit in vitro a greater chemotactic bias and a more dramatic variation of speed with attractant concentration than alveolar macrophages, and our results indicate that these behaviors can have a greater influence in three-dimensional connective tissue infection situations than in two-dimensional lung surface infection cases . In addition, we show that encounter apparently is not generally the rate-limiting step in this neutrophil response . These findings have important implications for correlating in vitro measured defects in cell motility and chemotaxis properties with in vivo functions of host defense against infection. Infect Immun, 1990 May, 58(5), 1195 - 200 Synthesis and secretion of Bordetella pertussis adenylate cyclase as a 200-kilodalton protein; Bellalou J et al.; Bordetella pertussis, the etiological agent of whooping cough, synthesizes a calmodulin-sensitive adenylate cyclase that is suspected to play a major role in the virulence of this bacterium . We show that adenylate cyclase synthesized as a 200-kilodalton protein is the product of the cyaA gene and that various virulent Bordetella species secrete this high-molecular-weight polypeptide without apparent proteolytic processing . When submitted to trypsin digestion, the 200-kilodalton protein was converted to a stable 45- to 50-kilodalton species . This corresponds to the size of the enzyme previously purified from a culture supernatant . The molecular heterogeneity reported for the various identified forms of adenylate cyclase could therefore result in part from proteolytic degradation or molecular aggregation of the major 200-kilodalton form of the enzyme. Izv Akad Nauk SSSR Biol, 1990 May-Jun, (3), 415 - 9 {The marine bacterium Alteromonas piscicida--a producer of enzymes with thrombolytic action}; Demina NS et al.; The ability of marine bacteria A . piscicida to produce exoproteases that were able to lyse human blood clots has been studied . Optimal conditions for biosynthesis of these enzymes have been found . The enzyme has been partially purified . In concentration of 1 mg/ml it has activity corresponding to that of 500 micrograms/l plasmine and 100 micrograms/ml trypsine . The enzyme activity was completely inhibited after incubation in human blood plasma. Z Naturforsch {C}, 1990 May, 45(5), 459 - 62 Sequence analysis of four atrazine-resistant mutants from Rhodopseudomonas viridis; Ewald G et al.; Four atrazine-resistant mutants from the purple bacterium Rhodopseudomonas viridis were isolated . Sequence analysis revealed three different mutant strains carrying mutations in the herbicide-binding pocket: i) MAV 2: L212-Glu----Lys, ii) MAV 3: L216-Phe----Ser and iii) MAV 4 = MAV 5: L217-Arg----His, L220-Val----Leu . Except MAV 3 all Rps . viridis mutants are different from those selected by their resistance towards the closely related triazine terbutryn. Postgrad Med, 1990 May 1, 87(6), 159 - 61, 164 Treatment of Lyme disease . Best use of antibiotics; Rahn DW; Lyme disease is a tick-borne illness caused by the spirochetal bacterium Borrelia burgdorferi . Recognition of the clinical manifestations and geographic range of this illness has expanded rapidly in recent years . Although much remains to be explained about this infection, most patients can be treated effectively on the basis of the clinical experience gathered to date . Antibiotics are the mainstay of therapy for all stages of illness . Development of a vaccine and improvement in laboratory diagnostic techniques glimmer on the horizon but will not replace careful clinical observation and follow-up as the foundations of management. Plasmid, 1990 May, 23(3), 226 - 36 Plasmid from photosynthetic bacterium Ectothiorhodospira Sp . carries a transposable streptomycin resistance gene; Deragon JM et al.; Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp . resolved a single extrachromosomal element, plasmid pDG1 . Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping . Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3 . The resistance to streptomycin and mercury found in Ectothiorhodospira sp . was transferred to E . coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3 . The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E . coli HB101, using a kanamycin resistance reporter gene . High stringency molecular hybridization with 32P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences . In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK100 used as a target . For this test, we regrouped in the same cells of E . coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tetr,kmr) . We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E . coli DH1 . The transfer frequency of streptomycin resistance into pVK100 was 10(-5), compatible with a transposition event . In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another. Pathol Biol (Paris), 1990 May, 38(5), 441 - 5 {Mathematical study of the sensitivity curves of Escherichia coli exposed to polymyxins}; Peyret M et al.; The time killing curves of five strains of Escherichia coli (ATCC 25922, ATCC 29194, CIP 54125, CIP 54127, CIP 54117 (K 12)) exposed to five concentrations of polymyxin B are similar: latency phasis, two decreasing phasis and for the low polymyxin B concentrations growth phasis . In our experimental conditions, the Mg(+)+ and Ca(+)+ concentrations of the medium (Mueller-Hinton; medium A: Ca(+)+ = 9 mg/l, Mg(+)+ = 0.5 mg/l; medium B: Ca(+)+ = 35 mg/l, Mg(+)+ = 15.5 mg/l; medium C: Ca(+)+ = 60 mg/l, Mg(+)+ = 20 mg/l) have no time effect upon the killing curves . A decreasing biexponential model can be fitted to the data . Such a model is compatible with interaction between antibiotic and bacterium and can be formalized accorrding to the equation: T + ATB K1 in equilibrium of K2 T* - ATB K3----ATB + dead with ATB: Polymyxine B in excess, T: target bacterium and T*: modification target bacterium. J Bacteriol, 1990 May, 172(5), 2765 - 8 FtsZ regulates frequency of cell division in Escherichia coli; Bi E et al.; Cell division is regulated so that it occurs only once per cell cycle . In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole . Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length . We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place . This result suggests that the level of FtsZ controls the frequency of cell division in E . coli. Gene, 1990 Apr 30, 89(1), 129 - 32 Expression of cloned restriction and modification genes, hjaIRM from Hyphomonas jannaschiana in Escherichia coli; Danaher RJ et al.; A type-II RM system, HjaI, was identified in the marine bacterium, Hyphomonas jannaschiana . The ENase recognizes GATATC, and DNA fragments generated after cleavage with this enzyme contain blunt ends . A DNA fragment encoding these enzymes was cloned and expressed in Escherichia coli, although the level of expression of the cloned genes was low . DNA methylated by M.HjaI was not restricted by the Mcr or Mrr restriction systems of E . coli . Although H . jannaschiana is a marine bacterium isolated near the thermal vents on the floor of the Pacific Ocean, the biochemical properties of the ENase were similar to those of EcoRV, an isoschizomer isolated from E . coli. J Biol Chem, 1990 Apr 25, 265(12), 6817 - 26 The role of methylation in chemotaxis . An explanation of outstanding anomalies; Weis RM et al.; The role of methylation in chemotaxis is understood generally, but several anomalies exist which bring into question the timing of methylation relative to sensing . A double mutant bacterium, deficient in both methyltransferase and methylesterase (Tr-Es-) is capable of chemotaxis even though the respective single mutants (Tr- and Es-) are not . This Tr-Es- mutant will accumulate in capillaries containing aspartic acid but not in capillaries containing serine despite the fact that both the aspartate and serine receptors are part of the methylation-dependent pathway . To understand these anomalies, a combination of theoretical analyses and experimental studies was performed . A mathematical analysis of the gradients of aspartate and serine in the capillary assay shows that outside the capillary the gradients are shallow, but just inside the mouth of the capillary they are very steep . Also, when the number of bacteria accumulated in the capillary is at a maximum, the range of attractant concentrations in the steep gradient just inside the mouth of the capillary is optimal for response and partial adaptation by the Tr-Es- mutant . We postulate that random motion brings the Tr-Es- mutant into the capillary, where it is able to move up the steep gradient . The difference in timing of the responses to serine and aspartate explains why the Tr-Es- mutant accumulates in aspartate- but not in serine-containing capillaries . A simple diffusion-capture model incorporating these concepts can account for experimental values of the number of Tr-Es- bacteria accumulating in the capillary . These studies provide a rational explanation for all of the apparent anomalies and lead to the conclusion that methylation/demethylation plays a crucial role in sensing as well as setting the zero point of the receptor. FEBS Lett, 1990 Apr 9, 263(1), 10 - 4 Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99; Teneberg S et al.; Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99 . There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium . However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction . The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGc alpha-3Gal beta 4Glc beta Cer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide . When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive . This ganglioside was dominating in adult pig . The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well as thus explain the resistance of adult pigs to infection with E . coli K99. Am J Gastroenterol, 1990 Apr, 85(4), 399 - 403 14C-urea breath test for the detection of Helicobacter pylori; Veldhuyzen van Zanten SJ et al.; The high urease activity of Helicobacter pylori can be used to detect this bacterium by noninvasive breath tests . We have developed a 14C-urea breath test which uses 5 microCi 14C with 50 mg nonradioactive urea . Breath samples are collected at baseline and every 30 min for 2 h . Our study compared the outcome of the breath test to the results of histology and culture of endoscopically obtained gastric biopsies in 84 patients . The breath test discriminated well between the 50 positive patients and the 34 patients negative for Helicobacter pylori: the calculated sensitivity was 100%, specificity 88%, positive predictive value 93%, and negative predictive value 100% . Treatment with bismuth subsalicylate and/or ampicillin resulted in lower counts of exhaled 14CO2 which correlated with histological improvement in gastritis . The 14C-urea breath test is a better "gold standard" for the detection of Helicobacter pylori than histology and/or culture. J Bacteriol, 1990 Apr, 172(4), 2113 - 23 A Caulobacter gene involved in polar morphogenesis; Driks A et al.; At specific times in the cell cycle, the bacterium Caulobacter crescentus assembles two major polar organelles, the flagellum and the stalk . Previous studies have shown that flbT mutants overproduce flagellins and are unable to form chemotaxis swarm rings . In this paper, we report alterations in both the stalk and the flagellar structure that result from a mutation in the flagellar gene flbT . Mutant strains produce some stalks that have a flagellum, produce some stalks that have an extra lobe protruding from their sides, have filaments lacking the 29-kilodalton flagellin, and produce several unusual cell types, including filamentous cells as well as predivisional cells with two stalks and predivisional cells with no stalk at all . We propose that flagellated stalks arise as a consequence of a failure to eject the flagellum at the correct time in the cell cycle and that the extra stalk lobe is due to a second site for the initiation of stalk biogenesis . Thus, a step in the pathway that establishes the characteristic asymmetry of the C . crescentus cell appears to be disrupted in flbT mutants . We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle . The polar particles appear as a cluster of approximately 1 to 10 stain-excluding rings, visible in electron micrographs of negatively stained wild-type cells . This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells. J Clin Microbiol, 1990 Apr, 28(4), 814 - 6 Isolation of a previously undescribed rickettsia from an aborted bovine fetus; Dilbeck PM et al.; A previously undescribed obligate intracellular bacterium was isolated from an aborted bovine fetus . The organism was resistant to penicillin, replicated within cytoplasmic vacuoles, exhibited structural characteristics compatible with the rickettsias, and shared antigenic determinants with Cowdria ruminantium. Proc Natl Acad Sci U S A, 1990 Apr, 87(8), 3235 - 9 Atomic-resolution structure of the cellulose synthase regulator cyclic diguanylic acid; Egli M et al.; Cyclic diguanylic acid acts as a regulator for cellulose synthase activity in the bacterium Acetobacter xylinum . We report the x-ray crystal structure of the regulator at atomic resolution . The structure contains two independent molecules that adopt almost identical conformations . The two molecules form self-intercalated units that are stacked on each other . Two different G.G base-pairing modes occur between the stacks . The more stable one has two or possibly three hydrogen bonds between two guanines and is related to the type of hydrogen bonding that is believed to exist between G-rich strands at the ends of chromosomes. J Immunol, 1990 Apr 1, 144(7), 2738 - 44 The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium; Marra A et al.; The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages . We show that L . pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells . The characteristics of the interaction between L . pneumophila and differentiated HL-60 cells closely resemble those between L . pneumophila and human peripheral blood monocytes . With both cell types, C receptors and serum C mediate attachment of L . pneumophila, which are taken up by coiling phagocytosis . The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell . As in human monocytes, an avirulent mutant derivative of L . pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells . Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L . pneumophila-mononuclear phagocyte interaction. J Immunol, 1990 Apr 1, 144(7), 2771 - 80 Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3; Schlesinger LS et al.; We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes . mAb against complement receptors (CR) inhibit adherence and phagocytosis of M . tuberculosis in fresh nonimmune serum . A mAb against the type 1 CR (CR1) inhibits adherence of M . tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4% . A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7% . Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2% . mAb against other monocyte surface Ag do not significantly influence adherence . In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M . tuberculosis in the presence of heat-inactivated serum . By electron microscopy, monocytes ingest all M . tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion . In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M . tuberculosis adherence or ingestion . Adherence of M . tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence . Heat inactivation of serum markedly reduces adherence of M . tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold . Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively . Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M . tuberculosis (71 +/- 1%) . C component C3 is fixed to M . tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA . Human monocytes ingest M . tuberculosis by conventional phagocytosis as viewed by electron microscopy . This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M . tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand. Oral Microbiol Immunol, 1990 Apr, 5(2), 49 - 56 Probe-specific DNA fingerprinting applied to the epidemiology of localized juvenile periodontitis; DiRienzo JM et al.; Although Actinobacillus actinomycetemcomitans has been recognized as a primary etiological agent in localized juvenile periodontitis, questions remain concerning the source of infection, mode of transmission, and relative virulence of strains . DNA fingerprinting analysis, using a randomly cloned chromosomal DNA fragment as a probe, revealed that previously characterized strains of A . actinomycetemcomitans displayed significant restriction site heterogeneity which could be applied to the typing of clinical isolates of this bacterium such that individual strains or variants could be traced within subjects from localized juvenile periodontitis families . Hybridization data derived from an analysis of bacterial isolates obtained from families participating in an ongoing longitudinal study of the disease showed that a single individual could be infected with more than one strain or variant of A . actinomycetemcomitans and that various members of the same family could harbor different strains or variants of the bacterium . In several cases the clinical isolates were matched to characterized laboratory strains by comparing hybridization patterns generated by digestion of the DNA with several restriction enzymes in independent reactions . Thus, probe-specific DNA fingerprinting of A . actinomycetemcomitans will permit us to determine if particular strains or variants are frequently associated with sites of periodontal destruction . Attention could then be focused on determining the virulence properties of those strains or variants that have in vivo significance. Infect Immun, 1990 Apr, 58(4), 868 - 73 Role of the putative "link" glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H7 strain CL-49; Sajjan SU et al.; Purified rat intestinal mucin was used to identify mucin-binding sites for type 1-piliated Escherichia coli O157:H7 strain CL-49 isolated from a patient with hemorrhagic colitis and hemolytic uremic syndrome . Optimum binding of bacteria in a microtiter binding assay occurred with a mucin coating concentration of 15 micrograms (protein)/150 microliters . In hapten inhibition studies, several nonmucin glycoproteins bearing exposed mannosyl residues in N-linked oligosaccharides were effective inhibitors, as was rat mucin . The same glycoproteins caused bacterial aggregation . High-molecular-mass glycoproteins of the mucin were separated from its 118-kilodalton "link" glycopeptide fraction, and the latter was shown to be the mucin-binding component for E . coli CL-49 and its purified type 1 pili . This was confirmed in hemagglutination inhibition studies . Treatment of the link glycopeptide with jack bean alpha-mannosidase or endo-beta-N-acetylglucosaminidase H destroyed bacterial binding activity . Chemical or enzymatic modifications of intact rat mucin were undertaken to evaluate the normal accessibility of the link glycopeptide receptors to E . coli CL-49 . Deglycosylation with trifluoromethane-sulfonic acid abolished binding, whereas pronase digestion had no effect . Reduction and alkylation as well as lipid extraction enhanced bacterial binding by the mucin, presumably by causing greater exposure of receptor sites . In summary, our binding studies revealed, for the first time, that intestinal mucin bears oligomannosyl receptors for type 1 pili and that these receptors are located on N-linked oligosaccharides of the 118-kilodalton link glycopeptide region of the mucin . Our experiments suggest the receptors are normally partly "covered" by noncovalently bound lipid . In addition, release of the link component from the rest of the mucin by disulfide bond reduction causes greater exposure of specific bacterium-binding sites. Appl Environ Microbiol, 1990 Apr, 56(4), 949 - 55 Identification of Francisella species and discrimination of type A and type B strains of F . tularensis by 16S rRNA analysis; Forsman M et al.; Tularemia is a zoonotic disease, occurring throughout the Northern Hemisphere . The causative agent, the bacterium Francisella tularensis, is represented by two main types . Type A is found in North America, whereas type B is mainly found in Asia and Europe and to a minor extent in North America . No routine technique for rapid diagnosis of tularemia has been generally applied . We have partially sequenced 16S rRNAs of two F . tularensis strains, as well as the closely related Francisella novicida . Of 550 nucleotides analyzed, only one difference in 16S rRNA primary sequence was found . This 16S rRNA analysis enabled the construction of oligonucleotides to be used as genus- and type-specific probes . Such probes were utilized for the establishment of a method for rapid and selective detection of the organism . This method allowed identification of Francisella spp . at the level of genus and also discrimination of type A and type B strains of F . tularensis . The analysis also permitted the detection of F . tularensis in spleen tissue from mice infected with the bacterium . The results presented will enable studies on the epizootiology and epidemiology of Francisella spp. FEBS Lett, 1990 Mar 26, 262(2), 189 - 93 A repeated decapeptide motif in the C-terminal domain of the ribosomal RNA methyltransferase from the erythromycin producer Saccharopolyspora erythraea; Dhillon N et al.; Re-analysis of the primary structure of the ribosomal RNA N-methyltransferase that confers self-resistance on the erythromycin-producing bacterium Saccharopolyspora erythraea has confirmed the presence of a C-terminal domain containing extensive repeat sequences . Nine tandem repeats can be discerned, with a decapeptide consensus sequence GGRx(H/R)GDRRT, although no single residue is wholly invariant . This highly polar, potentially flexible domain, which is predicted to adopt either a random coil or a structure with beta turns, has a counterpart in the erythromycin methyltransferase of an erythromycin-producing species of Arthrobacter . It also significantly resembles a portion of the C-terminal region of the eukaryotic protein nucleolin, which is unusually rich in dimethylarginine and glycine, and which is also predicted to behave as a random coil in solution . This resemblance, despite the very different roles of these proteins in ribosome biogenesis, strengthens the idea that in both rRNA methyltransferases and nucleolin these C-terminal sequences might contribute to rRNA binding. J Biol Chem, 1990 Mar 15, 265(8), 4364 - 8 Heme-copper and heme-heme interactions in the cytochrome bo-containing quinol oxidase of Escherichia coli; Salerno JC et al.; The cytochrome bo quinol oxidase of Escherichia coli is one of two respiratory O2 reductases which the bacterium synthesizes . The enzyme complex contains copper and 2 mol of b-type heme . Electron paramagnetic resonance (epr) spectroscopy of membranes from a strain having amplified levels of this enzyme complex reveals signals from low- and high-spin b-type hemes, but the copper, now established as a component of the oxidase, is not directly detectable by epr . The high-spin signal from the cytochrome bo complex, which we attribute to cytochrome o, when titrated potentiometrically, gives a bell-shaped curve . The low potential side of this curve is biphasic (Em7 approximately 180 and 280 mV) and corresponds to the reduction/oxidation of the cytochrome(s) . The high potential side of the bell-shaped curve is monophasic (Em7 approximately 370 mV) and is proposed to be due to reduction/oxidation of a copper center which, when in the Cu(II) form, is tightly spin-coupled to a heme, probably cytochrome o, resulting in a net even spin system and loss of the epr spectrum . The low-spin cytochrome b titrates biphasically with Em7 values of approximately 180 and 280 mV, similar to the high-spin component but without the loss of signal at high potentials. Chem Pharm Bull (Tokyo), 1990 Mar, 38(3), 794 - 6 Enzymatic sulfation of quercetin by arylsulfotransferase from a human intestinal bacterium; Koizumi M et al.; A novel type of arylsulfotransferase was partially purified from human intestinal bacteria and its enzymatic properties were examined . Polyphenols such as chalcone, xanthone and flavonoid were found to be sulfated by the bacterial arylsulfotransferase though the sulfation activity varied depending upon the positions of the hydroxyl groups . Quercetin, as an example of a flavonol, was rapidly sulfated when p-nitrophenyl sulfate (PNS) was taken as a donor substrate . At a ten-fold molar excess of PNS over quercetin, two products, the 3,3'-disulfate and 3,3',7-trisulfate derivatives, were formed, but the 4'- and 5-hydroxyl groups were not sulfated . In the case of equimolar or two-fold molar excess of PNS to quercetin, only the 3,3'-disulfate was produced and no monosulfate was formed . The enzymatic procedure is useful as a specific and convenient method for the preparation of polyphenol sulfate esters. Appl Environ Microbiol, 1990 Mar, 56(3), 782 - 7 High diversity in DNA of soil bacteria; Torsvik V et al.; Soil bacterium DNA was isolated by minor modifications of previously described methods . After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons . After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content . No other unusual bases could be detected . The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C . High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C . The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically . Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added . Cuvettes with a 1-mm light path were used, and the A275 was read . DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions . C0t1/2 values were determined relative to that for E . coli DNA, whereas calf thymus DNA was reassociated for comparison . Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1990 Mar, 172(3), 1250 - 5 Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae; Godchaux W 3rd et al.; Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction . Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis . In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components . Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface . Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide . This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content. Indian J Med Res, 1990 Mar, 91, 98 - 105 Cultivation of a nocardioform acid-fast chemoautotrophic bacterium from armadillo tissues infected with Mycobacterium leprae; Dastidar SG et al.; A nocardioform bacterium was isolated from the spleen tissue of an armadillo infected with M . leprae and easily propagated in pure culture in mineral salt medium supplemented with only simple C and N sources (e.g., liquid paraffin, tetradecane, ammonium salts, urea, asparagine, gelatin, xanthin, hypoxanthin etc.) . Complex organic substances, e.g., tyrosin, casein, peptone, meat extract, egg proteins, serum, blood, yeast extract as well as medium 199, did not support the growth of this organism . Microscopically, the organism consisted of acid-fast, long, slender rods which originated from long, fragmented hyphae, or sporulating mycelial tufts; it was acid-fast (at less than 4.0% H2SO4) which was pyridine-susceptible . It produced DOPA-oxidase and Catalase and was lysozyme resistant; this grew best under reduced O2 tension, at pH 7.0 to 8.0 and 28 degrees C . Serologically, it appeared to be only weakly related to the prototype human multibacillary leprosy-derived (reference) nocardioform strain, Nocardia brasiliensis and N . caviae, but was variably related to several mycobacteria strains. J Biol Chem, 1990 Feb 5, 265(4), 1958 - 63 Direct voltammetry of the Chromatium vinosum enzyme, sulfide:cytochrome c oxidoreductase (flavocytochrome c552); Guo LH et al.; The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes . Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme . Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode . Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate . Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin. Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1506 - 10 Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria; Duronio RJ et al.; Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins . Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification . We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships . Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids . Each E . coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity . By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E . coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A) . The fatty acid specificity of N-myristoylation was preserved in this system: {9,10(n)-3H}myristate but not {9,10(n)3H}palmitate was efficiently linked to Gly-1 of the C subunit . {13,14(n)-3H}10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A . Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1) . A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties . The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein . Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E . coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed. Mol Gen Mikrobiol Virusol, 1990 Feb, (2), 29 - 32 {Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245}; Katsy EI et al.; The expressed difference in the plasmid profile of A . brasilense Sp245 is registered as a result of Tn5-Mob-mutability . Integration of the vector pSUP5011 into one of the A . brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported . The properties of A . brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed . The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium . The results suppose 85Md plasmid participation in melaninogenesis. Oral Microbiol Immunol, 1990 Feb, 5(1), 8 - 11 Actinobacillus actinomycetemcomitans mitogenicity for B cells can be attributed to lipopolysaccharide; Eastcott JW et al.; The purpose of this investigation was to determine the component(s) of whole Actinobacillus actinomycetemcomitans bacteria responsible for B cell mitogenic activity . Congenitally athymic "nude" rats were used as a source of B cells devoid of T lymphocyte activity . Spleen cells were cultured with, or without, whole formalin-killed A . actinomycetemcomitans bacteria or with purified LPS from A . actinomycetemcomitans . Dose-response curves to A . actinomycetemcomitans cells or to A . actinomycetemcomitans-LPS showed that responses were dose dependent . If optimal quantities of both A . actinomycetemcomitans and A . actinomycetemcomitans-LPS were added in combination, the proliferative responses were the same as if either was added alone, i.e., the responses were not additive . Polymyxin B at 2 micrograms/well completely abrogated the proliferative response of athymic rat splenocytes to 10(7) A . actinomycetemcomitans cells or to 1.25 micrograms A . actinomycetemcomitans-LPS/well . Therefore, the in vitro early proliferative response of B cells to A . actinomycetemcomitans can be attributed to the presence of LPS on A . actinomycetemcomitans cells . A considerable portion of the in situ lymphocytic gingival response to A . actinomycetemcomitans infection seen in periodontal disease patients may be a B cell mitogenic response to the LPS of this bacterium. Appl Microbiol Biotechnol, 1990 Feb, 32(5), 544 - 9 Oriented immobilization of bacterial photosynthetic membrane; Hara M et al.; We have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction . Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate . Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate . Light-induced potential and current responses could be measured when the membrane immobilized on the SnO2-coated glass plate was dried and sandwiched with a counter electrode of Hg . The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces. Toxicon, 1990, 28(6), 707 - 14 Production of paralytic shellfish toxins by a bacterium Moraxella sp . isolated from Protogonyaulax tamarensis; Kodama M et al.; A bacterium Moraxella sp . isolated from Protogonyaulax tamarensis was cultured in various conditions . Changes of toxicity and toxin components of the cells during culture were analyzed by bioassay and HPLC-fluorometric analysis . Toxin productivity of Moraxella sp . increased when it was cultured in nutrition-deficient environments . The main toxins produced by Moraxella sp . in these conditions were gonyautoxins (GTXs), mainly GTX 1 and 4 which are major toxins of P . tamarensis. Soc Sci Med, 1990, 30(6), 675 - 91 Cow dung, rock salt, and medical innovation in the Hindu Kush of Pakistan: the cultural transformation of neonatal tetanus and iodine deficiency; Mull DS et al.; In mountain villages of Chitral District in northwestern Pakistan, dried cow dung is used as Westerners would use talcum powder when babies are swaddled and rock salt is consumed in tea and other foods . Both substances are esteemed as conveying beneficial 'heat' and 'strength' . Unfortunately, however, cow dung sometimes contains a bacterium that causes neonatal tetanus, and the resulting toxin may enter through the baby's unhealed umbilical cord and cause death . Further, rock salt contains no iodine, and Chitral's soil is so iodine-deficient that goiter is very common . Thus local health workers advocate use of talcum powder rather than cow dung, immunization against tetanus, and replacement of rock salt by powdered iodized salt . The present report documents widespread community acceptance of these innovations despite the fact that the biomedical model of tetanus and goiter was not well understood and indigenous concepts of the causes of the diseases remained virtually undisturbed . Most of the villagers were Ismaili Muslim followers of the Aga Khan; their receptivity to such health messages was influenced by the high value that their religion places on advancement through 'education' and was correlated with their proximity to Ismaili health workers whom they trusted . A major implication of this research for primary health care programs is that when one is attempting to change existing health practices, explication of biomedical models should not be the only focus of concern . Attentiveness to the context in which behavior changes are introduced and interpreted is at least equally important . Further, the fact that new knowledge was added to the old without replacing it illustrates the complexity of human cognition and points to limitations in the KAP (knowledge-attitude-practice) model of health belief and behavior . This report adds to a small but important body of literature documenting the dynamic nature of medical pluralism in the developing world. Digestion, 1990, 47(1), 29 - 34 Presence of Helicobacter pylori in patients with non-ulcer dyspepsia revealing normal antral histological characteristics; Loffeld RJ et al.; Two hundred consecutive patients suffering from non-ulcer dyspepsia were studied for the presence of Helicobacter pylori in antral gastritis and normal antral mucosa, using the combination of culture, modified Giemsa stain and a sensitive immunoperoxidase stain as means of detection . H . pylori gastritis was present in 56% of the cases . The bacterium was present in 75% of cases of normal antral mucosa, however, in low numbers . It is concluded that 87% of patients with non-ulcer dyspepsia are H . polory-positive implying a larger role for the micro-organism as initially thought. J Fr Ophtalmol, 1990, 13(6-7), 333 - 8 {Torpid endophthalmitis in pseudophakia: diagnostic and therapeutic difficulties . Apropos of 4 cases}; Salvanet-Bouccara A et al.; The authors report four cases of chronic endophthalmitis following extracapsular cataract extraction with intraocular lens implantation in the posterior chamber . The first attack of intraocular inflammation occurred in the form of recurrent iridocyclitis two to six months after surgery . The first attack usually responded well to local corticosteroids . After many relapses of inflammation, increasingly resistant to medical treatment, a severe attack occurred leading to the decision to treat such endophthalmitis surgically: endocular fluid aspiration, vitrectomy, intraocular injection of antibiotics combined with systemic antibiotics and corticosteroids . It may be very difficult to prove the infectious origin of torpid endophthalmitis . Growing bacteria from endocular fluid aspirates is much more difficult than in acute endophthalmitis . No organisms were found in this series by this method, despite the fact that fluid aspiration was often performed many times in these 4 cases . A bacterium was identified in 3 cases, twice on the implant itself and once after culture of an iridectomy specimen . Intraocular antibiotic injections resulted in the complete recovery of one patient . Following failure of standard treatment, intraocular lens explantation resulted in the disappearance of infectious inflammatory signs in 3 cases . The treatment of chronic endophthalmitis is governed by the same rules as acute endophthalmitis but is not always as successful . Intraocular lens removal is often the only solution and confirms the pathogenic hypothesis that slime production by organisms, their adherence to the intraocular lens and their quiescent state make them less vulnerable to antibiotics and host defences. Bull Mem Acad R Med Belg, 1990, 145(3-4), 184 - 8; discussion 189-92 {Lyme disease: clinical and sero-epidemiological study of Borrelia burgdorferi infections in Belgium}; Bigaignon G; Lyme disease is a multi-systemic infection caused by the spirochaete Borrelia burgdorferi: this bacterium, discovered in 1982 in the United States, is mainly transmitted by a tick bite, Ixodes ricinus in Europe . In Belgium, a first seroepidemiological study of 3 years has revealed 190 patients and the whole spectrum of clinical pictures was observed, including the early stage of this infection in the skin (erythema chronicum migrans), neurological involvement and arthritis . The Lyme borreliosis is endemic in our country: the incidence ranges from low near the coast to high in the south-eastern part of Belgium. Reprod Nutr Dev, 1990, Suppl 2, 203s - 204s {O-demethylation and metabolism of the methoxyl group of vanillic acid, monomer model of lignin, by the rumen bacterium Syntrophococcus sucromutans}; Dore J et al.; The O-demethylation of a lignin monomer by washed cells of the rumen bacterium S sucromutans was studied using chemically synthesized O-{methyl-14C}vanillate . During cometabolism of pyruvate and vanillate, the label from the methoxyl group was incorporated into acetate, the sole product, which was singly labeled in the methyl group. J Bacteriol, 1990 Jan, 172(1), 419 - 23 Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1; Groenewegen PE et al.; The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated . Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells . Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1 . At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH) . At higher pH values, inhibition was observed only with valinomyci