Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.     

Extracellular nuclease produced by a marine bacterium. I. Extracellular deoxyribonuclease formation by a marine Vibrio sp

Deoxyribonuclease (DNase) activity was found in the culture fluids of numerous marine bacteria isolated from seawater. Among these ogranisms, marine bacterium, Vibrio sp., strain No. 2, showed the highest deoxyribonucleic acid-hydrolyzing activity. This organism requires salts of seawater for both growth and extracellular DNase formation. The DNase activity could not be detected in the synthetic seawater culture liquid lacking magnesium ion, and DNase activity decreased in a calcium-deficient medium. The optimum temperature for the growth of this organism was between 15 and 25 degrees C. The formation of extracellular DNase was the greatest at 20 degrees C and less activity was found at 10 and 30 degrees C.     

N-type calcium channel blockers from a marine bacterium, Cytophaga sp. SANK 71996

N-(3-Acyloxyacyl)glycines were isolated as N-type calcium channel blockers from a marine bacterium Cytophaga sp. SANK 71996. The identification and fermentation of the producing strain and structure characterization of N-(3-acyloxyacyl)glycines by spectral analyses and chemical syntheses are described together with their antagonistic activities.     

Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303

OBJECTIVE: To examine the relationship of skill, as represented by NCAA division level, to anterior cruciate ligament rupture in collegiate men's and women's basketball and soccer players. DESIGN: Randomized, retrospective. PARTICIPANTS: Women's and men's basketball and soccer players at NCAA Division I, II, and III institutions. MAIN OUTCOME MEASURE: Athletes with or without ACL injury. RESULTS: There was no relationship of ACL injury rate to NCAA division level in men's or women's basketball or soccer. CONCLUSIONS: There are many different variables that contribute to a player's skill level. Although these variables may relate to ACL injury and may be responsible for the differential in injury rate between men and women, skill level as represented by collegiate division does not relate to ACL injury. When considering possible etiologies of the differential in ACL injury rates between men and women, the use of the term "skill" should be avoided, and more specific terms used.     

B-90063, a novel endothelin converting enzyme inhibitor isolated from a new marine bacterium, Blastobacter sp. SANK 71894

We raised mAbs to whole L5178Y leukemia/lymphoma (LL) cells to identify adhesion proteins involved in adherence between LL cells and marrow stromal cells. One mAb, 4C, and its subclones 4C.1 and 4C.2 inhibited adherence of L5178Y LL cells to MLT. a nontransformed murine marrow stromal cell line. These MoAbs are directed against CD45RA. Control anti-CD45 mAbs and isotype mAbs were non-inhibitory. Other anti-CD45 mAbs, M1/9.3, RA3-3A1/6.1 and RA3-2C2/1 do not compete with mAb 4C.1 for binding to the L5178Y cell surface, but mAb 4C.1 competes for binding of mAb RA3-2C2/1. Effects of mAb 4C on tyrosine-phosphatase activity of CD45 in L5178Y cells are minimal, suggesting direct involvement of CD45 as an adhesion protein.     

Cloning of gene responsible for tributyltin chloride (TBTC1) resistance in TBTC1-resistant marine bacterium, Alteromonas sp. M-1

A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp. M-1, was constructed using plasmid vector pUC 19. Three positive clones were obtained from E. coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1. These clones could grow in LB liquid medium containing 100 microM TBTC1. Plasmids harbouring genes of Alteromonas sp. M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively. Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment. The ORF was 324 bp (108 amino acids). The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein. Homology search in amino acids alignment indicated that the product has homology with transport proteins.     

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing L-iduronic acid and D-QuiNHb4NHb

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di[(S)-3-hydroxybutyramido]glucose, respectively. This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature.     

Solution and gel rheology of a new polysaccharide excreted by the bacterium Alteromonas sp. strain 1644

The present series of experiments were designed to examine a potential role for central descending pain facilitatory systems in mediating secondary hyperalgesia produced by topical application of mustard oil and measuring the nociceptive tail-flick reflex in awake rats. Topical application of mustard oil (100%) to the lateral surface of the hind leg produced a facilitation of the tail-flick reflex that was significantly reduced in spinal transected animals. Mustard oil hyperalgesia was also inhibited in animals that had received electrolytic lesions in the rostral ventromedial medulla (RVM). Intrathecal (i.t.) administration of the non-selective cholecystokinin (CCK) receptor antagonist proglumide (10 micrograms) prior to mustard oil application completely blocked both the lesser and greater hyperalgesic responses observed in spinal transected and normal animals, respectively, and produced an inhibition of the tail-flick reflex in normal animals. Administration of the selective CCKB receptor antagonist L-365260 i.t. dose-dependently inhibited mustard oil hyperalgesia (ID50 = 364 ng) at doses approximately 5-fold less than the CCKA receptor antagonist devazepide (ID50 = 1760 ng). Similar to spinal proglumide, microinjection of the neurotensin antagonist SR48692 (3.5 micrograms) into the RVM blocked mustard oil hyperalgesia and inhibited the tail-flick reflex. These data suggest that secondary hyperalgesia produced by mustard oil is mediated largely by a central, centrifugal descending pain facilitatory system which involves neurotensin in the RVM and spinal CCK (via CCKB receptors). The inhibition of the tail-flick reflex produced by mustard oil following spinal or supraspinal administration of receptor antagonists suggests concurrent activation of central descending facilitatory and inhibitory systems.     

Physiology and ecology of bacteriophages of the marine bacterium Beneckea natriegens: salinity

The effects of variation in ionic levels on the stability and replication of two bacteriophages (nt-1 and nt-6) host specific for the marine bacterium Beneckea natriegens were examined. Monovalent cations influenced the adsorption of the nt-1 but not the nt-6 phage; however, one-step growth studies showed that NaCl was required for replication of both phage. The NaCl optimum for nt-1 production was 0.25 M NaCl, the same as the growth optimum for B. natriegens. However, the optimum for nt-6 production was 0.16 M NaCl. These NaCl optima for host and phage are at estuarine rather than oceanic levels. The nt-1 phage was better suited to replicate at NaCl levels typical of higher salinity areas (18-35%) and the nt-6 phage was better suited to replicate at lower salinities (5-18%). The nt phage were more resistant to low NaCl levels than their host bacterium and appeared limited to marine waters by the lower survival salinity of B. natriegens coupled with phage inactivation processes occurring in natural estuarine waters.     

Thermothrix azorensis sp. nov., an obligately chemolithoautotrophic, sulfur-oxidizing, thermophilic bacterium

A new aerobic, obligately chemolithoautotrophic, thermophilic, sulfur-oxidizing bacterium, Thermothrix azorensis, was isolated from a hot spring on Sao Miguel Island in the Azores. The cells of this organism are gram negative, nonsporulating, and rod shaped. Filament formation appears to occur as a response to nonoptimal growth conditions. Growth occurs at 63 to 86 degrees C, and the optimum temperature is 76 to 78 degrees C. The optimum pH range for growth is 7.0 to 7.5. The G+C content of the DNA of our isolate is 39.7 mol%. This isolate uses thiosulfate, tetrathionate, hydrogen sulfide, and elemental sulfur as energy sources. Of particular interest are the absence of Calvin cycle enzymes and the initial appearance of sulfide during the lag phase of growth of aerobic cultures grown on elemental sulfur. The subsequent formation of thiosulfate is followed by oxidation of the thiosulfate to sulfate. T. azorensis differs from the only other Thermothrix species that has been described, Thermothrix thiopara, by having higher optimum and maximum growth temperatures, by being an obligate chemolithoautotroph, and by its close but separate position on a 16S rRNA sequence-based phylogenetic tree. Our T. azorensis isolate has been deposited in the American Type Culture Collection as strain ATCC 51754T (T = type strain).     

The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases

We report here cloning from the marine gliding bacterium Cytophaga drobachiensis of kappa-carrageenase, a glycoside hydrolase involved in the degradation of kappa-carrageenan. Structural features in the nucleotide sequence are pointed out, including the presence of an octameric omega sequence similar to the ribosome-binding sites of various eukaryotes and prokaryotes. The cgkA gene codes for a protein of 545 aa, with a signal peptide of 35 aa and a 229-aa-long posttranslationaly processed C-terminal domain. The enzyme displays the overall folding and catalytic domain characteristics of family 16 of glycoside hydrolases, which comprises other beta-1,4-alpha-1,3-D/L-galactan hydrolases, beta-1,3-D-glucan hydrolases (laminarinases), beta-1,4-1,3-D-glucan hydrolases (lichenases), and beta-1,4-D-xyloglucan endotransglycosylases. In order to address the origin and evolution of CgkA, a comprehensive phylogenetic tree of family 16 was built using parsimony analysis. Family-16 glycoside hydrolases cluster according to their substrate specificity, regardless of their phylogenetic distribution over eubacteria and eukaryotes. Such a topology suggests that the general homology between laminarinases, agarases, kappa-carrageenases, lichenases, and xyloglucan endotransglycosylases has arisen through gene duplication, likely from an ancestral protein with laminarinase activity.     

Haloanaerobium lacusroseus sp. nov., an extremely halophilic fermentative bacterium from the sediments of a hypersaline lake

A new extremely halophilic chemoorganotrophic bacterium (strain H200T [T = type strain]) was isolated from the hypersaline sediments of Retba Lake in Senegal. This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grew optimally at 40 degrees C in the presence of 180 to 200 g of NaCl per liter. The DNA base composition was 32 mol% guanine plus cytosine. The fermentation products from glucose were ethanol, acetate, H2, and CO2. Yeast extract was required for growth. The fermentable substrates included D-fructose, galactose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, D-mannitol, glycerol, and Casamino Acids. On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species. The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species. We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus.     

Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well

A new thermophilic, xylanolytic, strictly anaerobic, rod-shaped bacterium, strain SEBR 7054T, was isolated from an African oil-producing well. Based on the presence of an outer sheath (toga) and 16S rRNA sequence analysis data, this organism was identified as a member of the genus Thermotoga. Strain SEBR 7054T possessed lateral flagella, had a G + C content of 50 mol%, produced traces of ethanol from glucose but no lactate, and grew optimally in the presence of 0 to 0.2% NaCl at 70 degrees C. Its phenotypic and phylogenetic characteristics clearly differed from those reported for the five previously validly described Thermotoga species. Therefore, we propose that strain SEBR 7054T is a member of a new species of the genus Thermotoga, Thermotoga hypogea sp. nov. The type strain of T. hypogea is SEBR 7054 (= DSM 11164).     

Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium

A cryptic plasmid found at high copy number was isolated from Flavobacterium sp. KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced. The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196. The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. The plasmid contains two open reading frames of significant length, ORFI and ORFII. ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2. The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.     

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.     

Nucleotide sequence of plasmid pAQ1 of marine cyanobacterium Synechococcus sp. PCC7002

A delay in identifying incipient flap failure may inevitably lead to complete pedicle thrombosis and the no-reflow phenomenon. The authors report a clinical case of a lateral arm free flap that suffered complete pedicle thrombosis. They successfully salvaged this flap, a type C fasciocutaneous "flow-through" flap, by manually moving the thrombus from proximal to distal in the main flap artery. This freed the septofasciocutaneous upward-perforating branches, by smoothing and applying firm pressure to the vessel, combined with thrombolytic therapy. Their technique is offered as an alternative procedure for salvaging a failing flow-through flap.     

Identification and characterization of an extracellular protease activity produced by the marine Vibrio sp. 60

Since nitric oxide (NO) has been widely accepted as a novel neuromodulator, which activates soluble forms of guanylate cyclase to increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels, the effect of water-soluble substance in cigarette smoke on cyclic GMP levels were investigated using nerve terminals prepared from rat cerebral cortex. Although the smoke-substance itself failed to affect cyclic GMP levels in the synaptosomes, the smoke-substance significantly inhibited the increases in cyclic GMP levels induced by NO donors. The blocking effect of the smoke-substance was inhibited by concomitant incubation with superoxide dismutase, but not with mannitol. In addition, the effect of smoke-substance was mimicked by products of the xanthine/xanthine oxidase system, but not by nicotine. The effect of smoke-substance was preserved at least 7 days after they were stored at room temperature. Therefore, these results suggest that the smoke-substance may possess long half-lives to produce the radicals which inactivate NO, and to inhibit the increase in cyclic GMP levels in nerve terminals. The interference with NO may explain the part of mechanism in effects of cigarette smoke on neuronal functions.     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 degrees C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons.     

Alterococcus agarolyticus, gen.nov., sp.nov., a halophilic thermophilic bacterium capable of agar degradation

Five strains of facultatively anaerobic moderately thermophilic bacteria were isolated from two hot springs in the intertidal zone of Lutao, Taiwan. They produced extracellular agarase on agar medium, yielding reducing sugars and organic acids as the end products under either aerobic or anaerobic conditions. The growth temperature range was approximately 38-58 degrees C with an optimal temperature of about 48 degrees C. The five strains tolerated a relatively narrow pH range from 7.0 to 8.5. They were Gram-negative halophiles growing optimally at 2.0-2.5% NaCl (ca. 0.34-0.43 M). They were capable of anaerobic growth by fermenting glucose and producing various organic acids such as butyrate, propionate, formate, lactate, and acetate. Cells grown in liquid medium were motile monotrichous cocci, normally 0.8-0.9 micron in diameter. They possessed saturated anteiso-15-carbon acid (anteiso-C15:0) as the most abundant cellular fatty acid (46.0-51.3 mo1%) and had G + C contents ranging from 65.5 to 67.0 mo1%. They are the first thermophiles found to degrade agar and also the first halophilic thermophilic bacteria known to be capable of both aerobic and anaerobic fermentative growth. These bacteria are considered to represent a new genus that we named Alterococcus, and Alterococcus agarolyticus is the type species.     

The melA gene is essential for melanin biosynthesis in the marine bacterium Shewanella colwelliana

The surface-adhering, Gram-negative marine bacterium Shewanella colwelliana synthesizes a red-brow melanin in the late stage of exponential growth in laboratory culture. Previous studies identified a single gene, melA, from S. colwelliana that could impart the ability to produce melanin to an E. coli host. However, these studies did not demonstrate a requirement for melA during melanization in S. colwelliana. In this paper, genetic analyses, using a broad host range conjugation system to generate specific lesions, reveal that melA null mutants fail to synthesize pigment. The wild-type melA gene provided in trans on a low copy number plasmid complemented these null mutations, as well as a spontaneous pigment variant, to wild-type melanin synthesis. Polyclonal antibodies, raised against a MelA-LacZ fusion protein, were used to confirm the presence of the melA gene product in wild-type S. colwelliana and verify its absence in the non-pigmented mutants. In addition, detection of the MelA protein over the course of growth in batch culture revealed a constant steady-state level of MelA protein, suggesting that the timing of melanization and the quantity of melanin synthesized is not controlled at the level of melA expression.