Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii.

We have constructed a plasmid to express and secrete dextranase in the oral bacterium Streptococcus gordonii. The dextranase gene from Arthrobacter sp. strain CB-8 was linked to a promoter and a DNA sequence encoding the signal peptide of Streptococcus downei glucosyltransferase I (gtfI) followed by the Escherichia coli rrnBt1t2 terminator and inserted in the shuttle vector pVA838. S. gordonii transformed with this plasmid (pMNK-4) expressed and secreted mature Arthrobacter dextranase. The transformant was found to repress the firm adherence of water-insoluble glucan in a coculture experiment with cariogenic bacteria, Streptococcus sobrinus, in the presence of sucrose. Such genetically engineered oral bacteria could provide a therapy to prevent dental caries.     

Identification of Arthrobacter oxydans, Arthrobacter luteolus sp. nov., and Arthrobacter albus sp. nov., isolated from human clinical specimens

Five Arthrobacter isolates from clinical specimens were studied by phenotypic, chemotaxonomic, and genetic characterization. Two strains had characteristics consistent with those of Arthrobacter oxydans. One strain was related to A. citreus; however, DNA-DNA hybridization and phenotypic characteristics indicated that this strain belongs to a new species, for which the name Arthrobacter luteolus sp. nov. is proposed. Two strains were closely related to A. cumminsii by 16S rRNA gene sequencing, but DNA-DNA hybridization, peptidoglycan type, and some phenotypic features indicated that they should be assigned to a new species, for which the name Arthrobacter albus sp. nov. is proposed. The type strain of A. luteolus is CF25 (DSM 13067). The type strain of A. albus is CF43 (DSM 13068).     

Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: implication in regulation of NAD+/NADP+ balance

NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+-degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7-8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5'-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol.     

Siderophore production by Vibrio vulnificus

Previous studies in our laboratory, as well as clinical evidence, have suggested that increased iron levels in the host may be important in infections caused by the halophilic pathogen Vibrio vulnificus. To study iron acquisition, we induced siderophore production by growth in a low-iron medium, and biochemical testing indicated the production of both hydroxamate- and phenolate-type siderophores. The siderophores were extracted from growth filtrates with ethyl acetate (for phenolates) and phenol-chloroform-ether (for hydroxamates). These extracts enhanced the growth of V. vulnificus when the bacterium was grown in iron-limited medium. The ability of these siderophores to stimulate the growth of Salmonella typhimurium LT-2 enb-7 (a mutant deficient in the biosynthesis of enterochelin) and Arthrobacter flavescens JG-9 (a hydroxamate auxotroph) supported the conclusion that V. vulnificus produces both hydroxamate- and phenolate-type siderophores.     

Phenotypic and phylogenetic characterization of a new Corynebacterium species from dogs: description of Corynebacterium auriscanis sp. nov.

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus Corynebacterium; this subline is close to but distinct from C. falsenii, C. jeikeium, and C. urealyticum. The unknown bacterium from dogs was distinguished from all currently validated Corynebacterium species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species, Corynebacterium auriscanis. The type strain of C. auriscanis is CCUG 39938(T).     

Laribacter hongkongensis gen. nov., sp. nov., a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema.

A bacterium was isolated from the blood and empyema of a cirrhotic patient. The cells were facultatively anaerobic, nonsporulating, gram-negative, seagull shaped or spiral rods. The bacterium grows on sheep blood agar as nonhemolytic, gray colonies 1 mm in diameter after 24 h of incubation at 37 degrees C in ambient air. Growth also occurs on MacConkey agar and at 25 and 42 degrees C but not at 4, 44, and 50 degrees C. The bacterium can grow in 1 or 2% but not 3, 4, or 5% NaCl. No enhancement of growth is observed with 5% CO(2). The organism is aflagellated and nonmotile at both 25 and 37 degrees C. It is oxidase, catalase, urease, and arginine dihydrolase positive, and it reduces nitrate. It does not ferment, oxidize, or assimilate any sugar tested. 16S rRNA gene sequencing showed that there are 91 base differences (6.2%), 112 base differences (7.7%), and 116 base differences (8.2%) between the bacterium and Microvirgula aerodenitrificans, Vogesella indigofera, and Chromobacterium species, respectively. The G+C content (mean and standard deviation) is 68.0% +/- 2.43%, and the genomic size is about 3 Mb. Based on phylogenetic affiliation, the bacterium belongs to the Neisseriaceae family of the beta-subclass of Proteobacteria. For these reasons, a new genus and species, Laribacter hongkongensis gen. nov., sp. nov., is proposed, for which HKU1 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging pathogen.     

Identification of diaminopimelic acid in the Legionnaires disease bacterium.

Diaminopimelic acid was found to be a component of the cell wall of the Legionnaires disease bacterium, thus providing additional evidence that the organism is a bacterium. The presence of this amino acid was determined by gas-liquid chromatography and confirmed by gas chromatography-mass spectrometry.     

Human infection from an unidentified erythrocyte-associated bacterium.

A 49-year-old splenectomized man had an infection from an unidentified, gram-positive, rod-shaped bacterium that adhered to the majority of his peripheral-blood erythrocytes. On transmission electron microscopy, the bacterium was seen to be extra-erythrocytic and was 0.2 micrometer wide by 1.0 to 1.7 micrometer long. It possessed a thick, granular cell wall, a trilamellar membrane external to the cell wall and prominent mesosomes. Attempts to cultivate the organism in vitro or to duplicate the patient's disease in splenectomized animals were unsuccessful. The patient's response suggested that the bacterium was susceptible to cell-wall-active antibiotics and to chloramphenicol but not to tetracycline. This bacterium may be the cause of other chronic, fever-producing, multisystem diseases of unknown origin.     

Molecular epidemiology of Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale is a recently described gram-negative rod-shaped bacterium associated with respiratory tract infections in poultry. In order to determine the molecular epidemiology of this bacterium, we characterized 55 O. rhinotracheale isolates from eight countries on four continents by multilocus enzyme electrophoresis (MLEE), repetitive sequence based-PCR (rep-PCR), and 16S rRNA gene sequencing. MLEE discriminated the O. rhinotracheale isolates into six electrophoretic types (ETs), of which only three ETs were recovered from domesticated poultry. The 16S rRNA gene sequence and rep-PCR analyses confirmed the results obtained by MLEE and indicated limited heterogeneity among isolates of O. rhinotracheale recovered from poultry. Taken together, the results of our analysis demonstrate that the majority of O. rhinotracheale isolates recovered from domesticated poultry throughout the world are represented by a small group of closely related clones and suggest that the bacterium was recently introduced to domesticated poultry from wild bird populations.     

Isolation and characterization of a black-pigmented Corynebacterium sp. from a woman with spontaneous abortion

An unusual black-pigmented coryneform bacterium was isolated from the urogenital tract of a woman who experienced a spontaneous abortion during month 6 of pregnancy. Biochemical and chemotaxonomic analyses demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Phylogenetic analysis based on 16S rRNA sequences (GenBank accession no. AF220220) revealed that the organism was a member of a distinct subline which includes uncultured Corynebacterium MTcory 1P (GenBank accession no. AF115934), derived from prostatic fluid, and Corynebacterium CDC B8037 (GenBank accession no. AF033314), an uncharacterized black-pigmented coryneform bacterium. On the basis of chemotaxonomic and phylogenetic evidence, this organism probably represents a new species and is most closely related to the uncharacterized Centers for Disease Control and Prevention group 4 coryneforms. Our strain is designated CN-1 (ATCC 700975).     

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.     

Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.     

Arthrobacter globiformis — ein neues hefelytisches Bakterium

A bacterium capable to lyse viable yeast cells was isolated from compost enriched with baker's yeast cells and was identified as Arthrobacter globiformis. The yeast lytic enzyme complex produced by shaking culture was precipitated with ammonium sulfate, dialyzed and lyophilized. The crude residue contained β-glucanase and α-mannanase, yet not chitinase. Optimale carbon sources in the culture medium for a high enzyme synthesis were 0.5% β-glucan for the production of β-glucanase, resp., 3% α-mannan for the production of α-mannanase. Addition of 10% whole died baker's yeast cells to the culture medium effected similar results. The crude enzyme residue released among other things spheroplasts from cells of the yeast Pichia membranaefaciens, Metschnikowia pulcherrima, Hansenula anomala and Candida guilliermondii “H”. However, it did not or only weakly lyse viable cells of the yeasts Rhodotorula rubra, Rhodosporidium toruloides and Sporobolomyces roseus. The mutants of Candida guilliermondii “H” with modified glucan, resp., mannan concentrations in the cell walls did not indicate differences in their susceptibility to lysis.     

Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease.

Over a 12-year period, 16 human strains of a gram-negative, catalase-positive, halophilic, aerobic, nonmotile, small coccoid bacterium were received for identification. On the bases of biochemical characteristics and cellular fatty acid profiles, 14 of these strains were similar to the "Philomiragia" bacterium (Yersinia philomiragia, species incertae sedis). Additional characteristics were growth on Thayer-Martin agar but no growth or sparse, delayed growth on MacConkey agar; oxidase positive; acid production, often weak and delayed, from D-glucose, sucrose, and maltose; urease negative; no reduction of nitrates; and H2S produced but often delayed in triple sugar iron agar. Both the human isolates and the "Philomiragia" bacterium contained C10:0, C14:0, C16:0, C18:1 omega 9c, C18:0, 3-OH C18:0, C22:0, and C24:1 as major cellular fatty acids and ubiquinone eight (Q8) as the major isoprenoid quinone. These cellular acids in these relative amounts have been found previously only in Francisella tularensis and Francisella novicida, suggesting a relationship between the "Philomiragia" bacterium and Francisella species. Of the 14 human "Philomiragia"-like isolates, 9 were from blood, 3 were from lung biopsies or pleural fluid, and one each was from peritoneal fluid and cerebrospinal fluid. DNA relatedness studies (hydroxyapatite method, 50 and 65 degrees C) showed that these 14 strains were a single group that was the same species as the "Philomiragia" bacterium. Two other human strains were oxidase negative and H2S negative. They formed a single DNA relatedness group that was indistinguishable from the type strains of both F. tularensis and F. novicida. DNA relatedness of "Philomiragia" bacterium type and other strains to strains of F. novicida and F. tularensis, including the type strains, was 35 to 46%. One of the two F. novicida- and F. tularensis-like strains was isolated from blood, and the other was isolated from a cervical lymph node. On the basis of these findings, we propose transferring Y. philomiragia from the genus Yersinia to the genus Francisella as Francisella philomiragia comb. nov. Having confirmed that F novicida and F. tularensis are the same species and having shown that F. novicida is pathogenic for humans, we further propose eliminating the species F. novicida and demoting it to a biogroup of F. tularensis.     

Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells.

Shigella flexneri causes bacillary dysentery, an invasive disease of colonic epithelial cells in humans. The capacity of bacteria, once they have entered into a cell and escaped the phagocytic vacuole, to spread intracellularly and directly to adjacent cells without further extracellular passage is a key factor in invasion of the epithelial layer. Movement of intracellular bacteria is dependent upon the polymerization of actin; concentration of the formed filaments to one end of the bacterium is associated with initiation of movement. This movement may lead to the formation of a protrusion at the cell surface through which the bacterium passes to an adjacent cell. Development of these protrusions in infected HeLa cells is described, with emphasis on two critical observations. First, initiation of movement is coupled with bacterial division since elongation of the bacterial body is associated with relocalization of the previously uniformly distributed layer of actin to one pole of the bacterium. Second, the actin-bundling protein plastin appears to bundle the actin filaments just posterior to the bacterium, producing an ongoing contraction of the cylindrical actin tail that may be associated with forward movement of the bacterium within the protrusion.     

Legionnaires' disease: description of an epidemic of pneumonia.

An explosive, common-source outbreak of pneumonia caused by a previously unrecognized bacterium affected primarily persons attending an American Legion convention in Philadelphia in July, 1976. Twenty-nine of 182 cases were fatal. Spread of the bacterium appeared to be air borne. The source of the bacterium was not found, but epidemiologic analysis suggested that exposure may have occurred in the lobby of the headquarters hotel or in the area immediately surrounding the hotel. Person-to-person spread seemed not to have occurred. Many hotel employees appeared to be immune, suggesting that the agent may have been present in the vicinity, perhaps intermittently, for two or more years.     

Purification and characterization of Helicobacter mustelae urease.

Helicobacter mustelae is a urease-rich bacterium associated with gastritis in ferrets. The ureases of H. mustelae and Helicobacter pylori, a bacterium implicated in human gastritis, share many characteristics. Helicobacter sp. ureases appear to be unique among bacterial enzymes in exhibiting submillimolar Km values and in being composed of two subunits.     

Exotoxin activity associated with the Legionnaires disease bacterium.

Hemolysis occurred around growth of the Legionnaires disease bacterium on supplemented Mueller-Hinton agar containing sterile defibrinated blood from each of five mammalian species. Hemolysis was most pronounced with guinea pig or rabbit blood, was less intense with horse or sheep blood, and was slight with blood from a human donor. Sterile filtrates of allantoic fluid from embryonated hen's eggs that had been infected with this organism displayed hemolytic activity in a radial hemolysis assay with guinea pig cells in agar. Growth of the Legionnaires disease bacterium on F-G agar with 5% hen's egg yolk was surrounded by a zone of clearing and more circumscribed zones of iridescence and increased opacity on and in the medium. Attempts to detect activity analogous to that of Escherichia coli heat-labile or heat-stable enterotoxin in allantoic fluid from infected eggs or in cultures of the Legionnaires disease bacterium were not successful.     

Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov.

AIMS: To characterise a strain of Gram negative aerobic straight or slightly curved rods (HKU3) isolated from the blood culture of a 9 year old Chinese boy with neutropenic fever and pseudobacteraemia. METHODS: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests, scanning electron microscopy, and transmission electron microscopy. Genotypically, the 16S rRNA gene of the bacterium was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the Genbank by multiple sequence alignment. The G + C content was determined by thermal denaturation. A phylogenetic tree was constructed by the PileUp method. RESULTS: The cells of the bacterial strain were aerobic, sporulating, Gram negative straight or slight curved rods. The bacterium grew on horse blood agar as non-haemolytic, grey colonies of 1 mm in diameter after 24 hours of incubation at 37 degrees C in ambient air. No enhancement of growth was seen in 5% CO(2). It grew at 50 degrees C as pinpoint colonies after 72 hours of incubation, but did not grow at 65 degrees C or on MacConkey agar. It was non-motile. It produced catalase (weakly positive) and cytochrome oxidase. It reduced nitrate, produced beta galactosidase, hydrolysed esculin, and utilised sodium acetate. A scanning electron micrograph of the bacterium showed straight or slightly curved rods. A transmission electron micrograph of the cell wall of the bacterium revealed multiple electron dense layers, including the outer membrane, middle murein layer, and inner cytoplasmic membrane, compatible with its Gram smear appearance. 16S rRNA gene sequencing showed that there were 7.7%, 8.0%, 8.2%, and 8.6% differences between the 16S rRNA gene sequence of the bacterium and those of Paenibacillus macerans, Paenibacillus borealis, Bacillus ehimensis, and Paenibacillus amylolyticus, respectively. The mean (SD) G + C content of the bacterium was 47.6 (2.1) mol%. Phylogenetically, it belongs to the genus paenibacillus (previously called group 3 bacillus). CONCLUSIONS: A bacterium that exhibited phenotypic and genotypic characteristics that are very different from closely related members of paenibacillus was the cause of pseudobacteraemia in a patient with neutropenic fever. A new species, Paenibacillus hongkongensis sp. nov. is proposed, for which HKU3 is the type strain.