bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation
Authors: Rich J J , Hirano S S , Willis D K
Published: January 2020

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.
Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor
Authors: Archibald F , Roy B
Published: January 2020

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).
Bacterial migration along solid surfaces
Authors: Harkes G , Dankert J , Feijen J
Published: January 2020

An in vitro system was developed to study the migration of uropathogenic Escherichia coli strains. In this system an aqueous agar gel is placed against a solid surface, allowing the bacteria to migrate along the gel/solid surface interface. Bacterial strains as well as solid surfaces were characterized by means of water contact angle and zeta potential measurements. When glass was used as the solid surface, significantly different migration times for the strains investigated were observed. Relationships among the observed migration times of six strains, their contact angles, and their zeta potentials were found. Relatively hydrophobic strains exhibited migration times shorter than those of hydrophilic strains. For highly negatively charged strains shorter migration times were found than were found for less negatively charged strains. When the fastest-migrating strain with respect to glass was allowed to migrate along solid surfaces differing in hydrophobicity and charge, no differences in migration times were found. Our findings indicate that strategies to prevent catheter-associated bacteriuria should be based on inhibition of bacterial growth rather than on modifying the physicochemical character of the catheter surface.
Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene
Authors: Eldridge R , O'Reilly D R , Hammock B D , Miller L K
Published: January 2020

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.
Measurements of the distribution of adenylate concentrations and adenylate energy charge across Pseudomonas aeruginosa biofilms
Authors: Kinniment S L , Wimpenny J W
Published: January 2020

Adenine nucleotide pools and adenylate energy charge distributions were determined by using a laboratory-generated quasi-steady-state Pseudomonas aeruginosa biofilm. The method used involved freezing and sectioning of the intact biofilm, followed by extraction and assay of the adenylates in the sectioned material. Results indicated an increase in adenylate energy charge of about 0.2 units from the bottom to the surface of the biofilm. However, energy charge values were generally low throughout the biofilm, reaching a maximum of only 0.6 units. Of the adenylates measured, AMP was the predominant nucleotide, especially in the deeper parts of the biofilm profile.
Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin
Authors: Gireesh T , Davidson B E , Hillier A J
Published: January 2020

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).
Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus
Authors: Nicole M , Chamberland H , Geiger J P , Lecours N , Valero J , Rio B , Ouellette G B
Published: January 2020

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.
Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration
Authors: Osawa R
Published: January 2020

Tannin-protein complex (T-PC)-degrading enterobacteria (T-PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T-PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T-PCDE and inhibited the growth of concomitant T-PC-degrading Streptococcus bovis. The T-PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T-PC-degrading S. bovis, suggesting that T-PCDE played a more active role in digesting T-PC in the alimentary tracts of koalas.
Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants
Authors: Stevens K A , Klapes N A , Sheldon B W , Klaenhammer T R
Published: January 2020

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.
Sequential Transhydroxylations Converting Hydroxyhydroquinone to Phloroglucinol in the Strictly Anaerobic, Fermentative Bacterium
Pelobacter massiliensis
Authors: Brune Andreas , Schnell Sylvia , Schink Bernhard
Published: January 2020

The recently isolated fermenting bacterium
Pelobacter massiliensis
is the only strict anaerobe known to grow on hydroxyhydroquinone (1,2,4-trihydroxybenzene) as the sole source of carbon and energy, converting it to stoichiometric amounts of acetate. In this paper, we report on the enzymatic reactions involved in the conversion of hydroxyhydroquinone and pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene). Cell extracts of
P. massiliensis
transhydroxylate pyrogallol to phloroglucinol after addition of 1,2,3,5-tetrahydroxybenzene (1,2,3,5-TTHB) as cosubstrate in a reaction identical to that found earlier with
Pelobacter acidigallici
(A. Brune and B. Schink, J. Bacteriol. 172:1070-1076, 1990). Hydroxyhydroquinone conversion to phloroglucinol is initiated in cell extracts without an external addition of cosubstrates. It involves a minimum of three consecutive transhydroxylation reactions characterized by the transient accumulation of two different TTHB isomers. Chemical synthesis of the TTHB intermediates allowed the resolution of the distinct transhydroxylation steps in this sequence. In an initial transhydroxylation, the hydroxyl group in the 1-position of a molecule of hydroxyhydroquinone is transferred to the 5-position of another molecule of hydroxyhydroquinone to give 1,2,4,5-TTHB and resorcinol (1,3-dihydroxybenzene) as products. Following this disproportionation of hydroxyhydroquinone, the 1,2,4,5-isomer is converted to 1,2,3,5-TTHB, an enzymatic activity present only in hydroxyhydroquinone-grown cells. Finally, phloroglucinol is formed from 1,2,3,5-TTHB by transfer of the 2-hydroxyl group to either hydroxyhydroquinone or resorcinol. The resulting coproducts are again cosubstrates in earlier reactions of this sequence. From the spectrum of hydroxybenzenes transhydroxylated by the cell extracts, the minimum structural prerequisites that render a hydroxybenzene a hydroxyl donor or acceptor are deduced.
Extracellular protease from the antarctic yeast Candida humicola
Authors: Ray M K , Devi K U , Kumar G S , Shivaji S
Published: January 2020

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.
Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen
Authors: Bhunia A K , Johnson M G
Published: January 2020

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.
Reduction of nitroaromatic compounds mediated by Streptomyces sp. exudates
Authors: Glaus M A , Heijman C G , Schwarzenbach R P , Zeyer J
Published: January 2020

Exudates from Streptomyces griseoflavus Tü 2484 effectively mediated electron transfer between hydrogen sulfide and various nitrobenzenes. In general, pseudo-first-order kinetics were observed, except for the initial phase of the reaction at higher pH values. Under fixed pH and Dh conditions, linear free energy relationships were found between the logarithms of the reaction rate constants and the one-electron reduction potentials of the nitroaromatic compounds. No competition was observed between various compounds. Comparison of the results of this study with the results of experiments conducted with model quinones and an iron porphyrin suggest that the secondary metabolites cinnaquinone and dicinnaquinone, excreted by strain Tü 2484 on the order of 100 mg/liter, are responsible for the catalytic activity of the exudate. Further support for this hypothesis comes from the facts that the catalytic activity of the exudate became prominent only after the growth phase of the microorganisms and that the mediating substances have a molecular weight of less than 3,000.
Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats
Authors: Verhagen Frank J. M. , Laanbroek Hendrikus J.
Published: January 2020

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate
Adriamonas peritocrescens
on the competition for limiting amounts of ammonium between the ammonium-oxidizing species
Nitrosomonas europaea
and the heterotrophic species
Arthrobacter globiformis
was studied in the presence of
Nitrobacter winogradskyi
in continuous cultures at dilution rates of 0.004 and 0.01 h
-1
. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM.
A. globiformis
won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h
-1
, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 × 10
5
and 12 × 10
5
flagellates ml
-1
were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h
-1
, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.
Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion
Authors: Kunz D A , Nagappan O , Silva-Avalos J , Delong G T
Published: January 2020

The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS)
Recalcitrant High-Molecular-Weight Material, an Inhibitor of Microbial Metabolism in River Biofilms
Authors: Freeman Christopher , Lock Maurice A.
Published: January 2020

Recalcitrant high (>1,000)-molecular-weight materials (>1K materials) have been proposed to inhibit the metabolic activity of river biofilms. Evidence is presented to support the hypothesis that the mode of action is through occlusion of the surface of the biofilm by recalcitrant >1K materials. The evidence includes the following. (i) The phenomenon was particularly prominent in brown-water rivers, which are rich in recalcitrant >1K material. (ii)Temporal changes in inhibition intensity were observed, which coincided with seasonal changes in the relative recalcitrance of river water >1K materials. (iii) Stores of intracellular carbon (poly-β-hydroxyalkanoates) were progressively compromised by increasing the supply of recalcitrant >1K materials. (iv) Nontoxic synthetic analogs of recalcitrant >1K materials were also demonstrated to be capable of producing an identical inhibitory effect. Finally, it was suggested that the application of recalcitrant >1K materials may represent a novel approach to the control of deleterious microbial communities.
An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition
Authors: Kasbekar D P , Papavinasasundaram K G
Published: January 2020

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.
Evaluation of Chemicals for Restricting Colony Spreading by a Xerophilic Mold,
Eurotium amstelodami
, on Dichloran-18% Glycerol Agar
Authors: Beuchat Larry R. , De Daza Maria Soledad Tapia
Published: January 2020

Twenty chemicals were screened for their effectiveness in restricting colony spreading by four strains of a xerophilic mold,
Eurotium amstelodami
, on dichloran-18% glycerol agar. Triton X-100, Triton X-301, Tergitol NP-7, and Tergitol 15-S-3 (each at 200 μg/ml) and 1,000 μg of sodium deoxycholate, 1 μg of iprodione, 0.1 μg of propiconazole, and 0.01 μg of Maxim per ml were judged to be most effective for restricting the rate of colony spreading.
Inhibition of Settlement by Larvae of
Balanus amphitrite
and
Ciona intestinalis
by a Surface-Colonizing Marine Bacterium
Authors: Holmström Carola , Rittschof Dan , Kjelleberg Staffan
Published: January 2020

In an attempt to isolate bacteria with inhibitory effects against settlement by larvae of sessile invertebrates, 40 marine bacterial isolates were screened for effects against laboratory-reared barnacle larvae (
Balanus amphitrite
) and ascidian larvae (
Ciona intestinalis
). Five isolates displayed non-pH-dependent inhibitory effects against the larvae. The initial characterization of a toxic component released from an isolate, designated D2 (CCUG 26757), and its effect on laboratory-reared barnacle and ascidian larvae were studied. D2 is a facultative, anaerobic, gram-negative bacterium isolated from the surface of
C. intestinalis
from waters off the Swedish west coast at a depth of 10 m. Results suggest that the toxic component is released by D2 during the stationary phase. Aged biofilms were more toxic to the larvae than unaged films. The biologically active compound was in the supernatant of D2 and was heat stable and <500 Da in molecular mass. No evidence of protein or peptide moieties was found. On the basis of two phase and chromatography separations, the component is polar and neutral and contains or binds to carbohydrate moieties. Metaperiodate treatment increased toxicity; undiluted supernatant from a 24-h growth culture of D2 killed barnacle and ascidian larvae within a few hours of exposure, whereas after metaperiodate treatment, the larvae were killed in approximately 30 min.
Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences
Authors: Hicks R E , Amann R I , Stahl D A
Published: January 2020

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.
Reductive Desulfurization of Dibenzyldisulfide
Authors: Miller Kathleen W.
Published: January 2020

Dibenzyldisulfide was reductively degraded by a methanogenic mixed culture derived from a sewage digestor. Toluene was produced with benzyl mercaptan as an intermediate in sulfur-limited medium. Toluene production was strictly associated with biological activity; however, the reducing agent for the culture medium, Ti(III), was partially responsible for production of benzyl mercaptan. Sulfide was not detected. Additions of sodium sulfide did not inhibit toluene production. Additions of 2-bromoethane sulfonic acid prevented methanogenesis but did not adversely affect toluene yields.
Anaerobic Growth of
Thiobacillus ferrooxidans
Authors: Pronk J. T. , de Bruyn J. C. , Bos P. , Kuenen J. G.
Published: January 2020

The obligately autotrophic acidophile
Thiobacillus ferrooxidans
was grown on elemental sulfur in anaerobic batch cultures, using ferric iron as an electron acceptor. During anaerobic growth, ferric iron present in the growth media was quantitatively reduced to ferrous iron. The doubling time in anaerobic cultures was approximately 24 h. Anaerobic growth did not occur in the absence of elemental sulfur or ferric iron. During growth, a linear relationship existed between the concentration of ferrous iron accumulated in the cultures and the cell density. The results suggest that ferric iron may be an important electron acceptor for the oxidation of sulfur compounds in acidic environments.
Numerical taxonomy of gram-negative, nonmotile, nonfermentative bacteria isolated during chilled storage of lamb carcasses
Authors: Prieto M , García-Armesto M R , García-López M L , Otero A , Moreno B
Published: January 2020

A numerical taxonomic study using 75 characters was performed with 132 strains of gram-negative, nonmotile, nonfermentative bacteria selected on the basis of lack of motility and Gram reaction among 1,200 cultures isolated during aerobic storage of lamb carcasses. At the 80% similarity level (SSM), eight clusters were formed. Strains in clusters 1 to 6 could be identified as members of the family Moraxellaceae and, more specifically, as members of the Psychrobacter-[Moraxella] phenylpyruvica subgroup. Of these strains, clusters 1 and 2 (88 strains) were identified as [Moraxella] phenylpyruvica and cluster 3 (15 strains) was identified as Psychrobacter immobilis. Clusters 4, 5, and 6 were not identifiable with any species. Clusters 7 and 8 consisted of 14 strains considered nonmotile variants of Pseudomonas fragi. The highest separation indices corresponded to acid production from certain carbohydrates (melibiose, L-arabinose, and cellobiose). Although strains of Psychrobacter-Moraxella clusters were relatively frequently identified at the completion of slaughter, very few cultures were detected on spoiled carcasses. It appears, therefore, that this group of organisms has only low spoilage potential.
Rapid Method for Analyzing Bacterial Behavioral Responses to Chemical Stimuli
Authors: Nikata Toshiyuki , Sumida Ken , Kato Junichi , Ohtake Hisao
Published: January 2020

A rapid method was developed to analyze bacterial behavioral responses to chemical stimuli. Digital image processing was used to detect the accumulation of bacteria at the mouth of a capillary containing an attractant. The accumulation of bacteria was determined from the total number of cells near the mouth of the capillary per videotape frame. This method was applied to measure the chemotactic response of
Pseudomonas aeruginosa
cells to serine, with results similar to those obtained by the classical capillary plating assay. The videotape method is much less time-consuming and makes it possible to assess the bacterial response to an attractant within a few minutes.
Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms
Authors: Byrd J J , Leahy J G , Colwell R R
Published: January 2020

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.
Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium
Authors: Nicholson D K , Woods S L , Istok J D , Peek D C
Published: January 2020

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.
Utilization of Carbon Substrates, Electrophoretic Enzyme Patterns, and Symbiotic Performance of Plasmid-Cured Clover Rhizobia
Authors: Baldani J. Ivo , Weaver R. W. , Hynes M. F. , Eardly B. D.
Published: January 2020

Plasmids in
Rhizobium
spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of
Rhizobium leguminosarum
bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.
Immunogold and fluorescein immunolabelling of Legionella pneumophila within an aquatic biofilm visualized by using episcopic differential interference contrast microscopy
Authors: Rogers J , Keevil C W
Published: January 2020

Biofilms containing diverse microflora were developed in tap water on glass and polybutylene surfaces. Legionella pneumophila within the biofilms was labelled with monoclonal antibodies and visualized with immunogold or fluorescein isothiocyanate conjugates. Development of a differential interference contrast technique in an episcopic mode enabled simultaneous visualization of the total biofilm flora and gold-labelled legionellae. The legionellae occurred in microcolonies within the biofilm in the absence of amoebae, suggesting that the bacterial consortium was supplying sufficient nutrients to enable legionellae to grow extracellularly within the biofilm.
Vitamin B12-dependent propionate production by the ruminal bacterium Prevotella ruminicola 23
Authors: Strobel H J
Published: January 2020

When Prevotella ruminicola 23 was grown in a defined medium containing a vitamin mixture, significant amounts of propionate were formed. Succinate and acetate were the major fermentation acids produced when vitamins were omitted, and further experiments demonstrated that propionate formation was dependent on vitamin B12. When the organism was grown in continuous culture at dilution rates of less than 0.20 h-1, propionate and acetate were the predominant fermentation products and little succinate was formed when vitamin B12 was present. However, at higher dilution rates, propionate formation declined and succinate accumulated. Since cell protein yields were reduced 15 to 25% in the absence of vitamin B12, the pathway for propionate formation may contain an energy-conserving step.
Degradation of Giardia lamblia cysts in mixed human and swine wastes
Authors: Deng M Y , Cliver D O
Published: January 2020

This study was conducted to determine the persistence of Giardia lamblia cysts in mixed septic tank effluent and swine manure slurry and to correlate fluorescein diacetate-propidium iodide staining of G. lamblia cysts with their morphology under low-voltage scanning electron microscopy. Under field conditions, G. lamblia cysts were degraded more rapidly in the mixed waste than in the control Dulbecco's phosphate-buffered saline (PBS). For total and viable cysts, the mixed waste had D values (time for a 90% reduction in number of cysts) of 18.3 and 15.5 days, and the Dulbecco's PBS control had D values of 41.6 and 26.8 days. The rates of cyst degradation in septic tank effluent and in Dulbecco's PBS were similar. Increasing the proportion of swine manure slurry in the mixed waste favored degradation of the parasite. These results indicate that the mixed waste treatment was the predominant factor affecting the cyst persistence and that it was swine manure slurry that played the role of degrading the parasite. Visualization of viable and nonviable Giardia cysts with low-voltage scanning electron microscopy revealed an excellent correlation between the viability of the cysts determined by fluorescein diacetate-propidium iodide staining and their electron microscopic morphology.
Nitrate and nitrite microgradients in barley rhizosphere as detected by a highly sensitive denitrification bioassay
Authors: Binnerup S J , Sørensen J
Published: January 2020

A highly sensitive denitrification bioassay was developed for detection of NO3- and NO2- in rhizosphere soil samples. Denitrifying Pseudomonas aeruginosa ON12 was grown anaerobically in citrate (30 mM) minimal medium with KClO3 (10 mM) and NaNO2 (3 mM), which gave cells capable of NO2- reduction to N2O but incapable of NO3- reduction to NO2-. Growth on citrate minimal medium further resulted in the absence of N2O reduction. When added to small soil samples in O2-free vials, such cells could be used to convert the indigenous NO2- pool to N2O, which was subsequently quantified by gas chromatography. Cells grown in KClO3-free citrate medium with 10 mM NaNO3 as the electron acceptor were capable of reducing both NO3- and NO2-, and these cells could subsequently be added to the sample to convert the indigenous NO3- pool to N2O. Concentrations of both NO3- and NO2- were thus determined as N2O, with a detection limit of approximately 10 pmol of N. The bioassay could be used to determine NO3- and NO2- pools in 10-mg soil samples taken along a microgradient in the rhizosphere of field-grown barley plants. At both low (10%, wt/wt) and high (18%, wt/wt) water content, relatively high levels of NO2- were found in the rhizosphere compared with bulk soil. Under dry conditions, NO3- was also more abundant in the rhizosphere than in the bulk soil, whereas such a difference was not observed at the high water content. The roles of plant metabolism and bacterial nitrification and denitrification processes for NO3- and NO2- availability in the rhizosphere are discussed.
Moderation of ruminal fermentation by ciliated protozoa in cattle fed a high-grain diet
Authors: Nagaraja T G , Towne G , Beharka A A
Published: January 2020

The objective of this study was to assess the influence of ciliated protozoa on ruminal fermentation in cattle fed high-grain diets. Six ruminally cannulated steers fed a corn-based grain diet (85% concentrate plus 15% alfalfa hay) at 12-h intervals were assigned randomly to two groups, ciliate free and faunated, in a crossover design. Defaunation was by ruminal emptying, omasal flushing, and treatment with sodium sulfosuccinate. Two to 3 weeks after defaunation, the ruminal contents of all steers were sampled before the morning feeding (0 h) and at 1, 2, 4, 6, 8, and 12 h after feeding to measure pH, analyze fermentation products, and monitor counts of ciliated protozoa and lactic acid-producing and -fermenting bacterial groups. Total numbers of ciliated protozoa in the faunated steers averaged 4.3 x 10(5)/g, and the protozoa consisted of nine genera. Ciliate-free steers had lower (P less than 0.01) ruminal pHs (pH 5.97) than faunated cattle (pH 6.45); however, the treatment-time interaction was not significant. Ruminal lactate and ammonia concentrations were similar in both groups. The total volatile fatty acid concentration was higher (P less than 0.05) in the ciliate-free steers than in the faunated steers and exhibited a treatment-time interaction (P less than 0.05). The acetate-to-propionate ratio was higher (P less than 0.05) in the faunated group than in the ciliate-free group and showed a treatment-time interaction (P less than 0.05). Total anaerobic bacterial counts were about fourfold higher in the ciliate-free group than in the faunated group.(ABSTRACT TRUNCATED AT 250 WORDS)
Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus
Authors: Kishishita M , Matsuoka N , Kumagai K , Yamasaki S , Takeda Y , Nishibuchi M
Published: January 2020

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe
Authors: Pal A , Ramamurthy T , Bhadra R K , Takeda T , Shimada T , Takeda Y , Nair G B , Pal S C , Chakrabarti S
Published: January 2020

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from
Chloroflexus aurantiacus
Authors: Ratanakhanokchai Khanok , Kaneko Jun , Kamio Yoshiyuki , Izaki Kazuo
Published: January 2020

A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of
Chloroflexus aurantiacus
J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55°C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71°C, respectively. Metal ions such as Hg
2+
, Zn
2+
, Cu
2+
, Mn
2+
, and Ni
2+
strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca
2+
after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.