bluuebunny/crossref_metadata_2025 huggingface dataset:
An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum
Authors: Rochelle P A , Ferguson D M , Handojo T J , De Leon R , Stewart M H , Wolfe R L
Published: January 2020

The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lambia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay.
Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii
Authors: Steinert M , Emödy L , Amann R , Hacker J
Published: January 2020

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.
Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum
Authors: Benhamou N , Chet I
Published: January 2020

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.
Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality
Authors: Lee J , Goel A , Ataai M M , Domach M M
Published: January 2020

Volume 63, no. 2, p. 714, Figure 2: panel b should appear as shown below. FIG. 2b [This corrects the article on p. 710 in vol. 63.].
Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production
Authors: Dols M , Chraibi W , Remaud-Simeon M , Lindley N D , Monsan P F
Published: January 2020

The metabolic and energetic properties of Leuconostoc mesenteroides have been examined with the goal of better understanding the parameters which affect dextransucrase activity and hence allowing the development of strategies for improved dextransucrase production. Glucose and fructose support equivalent specific growth rates (0.6 h-1) under aerobic conditions, but glucose leads to a better biomass yield in anaerobiosis. Both sugars are phosphorylated by specific hexokinases and catabolized through the heterofermentative phosphoketolase pathway. During sucrose-grown cultures, a large fraction of sucrose is converted outside the cell by dextransucrase into dextran and fructose and does not support growth. The other fraction enters the cell, where it is phosphorylated by an inducible sucrose phosphorylase and converted to glucose-6-phosphate (G-6-P) by a constitutive phosphoglucomutase and to heterofermentative products (lactate, acetate, and ethanol). Sucrose supports a higher growth rate (0.98 h-1) than the monosaccharides. When fructose is not consumed simultaneously with G-1-P, the biomass yield relative to ATP is high (16.8 mol of ATP.mol of sucrose-1), and dextransucrase production is directly proportional to growth. However, when the fructose moiety is used, a sink of energy is observed, and dextransucrase production is no longer correlated with growth. As a consequence, fructose catabolism must be avoided to improve the amount of dextransucrase synthesized.
Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes
Authors: Kuhnert P , Heyberger-Meyer B , Burnens A P , Nicolet J , Frey J
Published: January 2020

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.
Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose
Authors: Boumerdassi H , Monnet C , Desmazeaud M , Corrieu G
Published: January 2020

Following treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, three mutants of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 that produced diacetyl and acetoin from glucose were isolated. The lactate dehydrogenase activity of these mutants was strongly attenuated, and the mutants produced less lactate than the parental strain. The kinetic properties of lactate dehydrogenase of strain CNRZ 483 and the mutants revealed differences in the affinity of the enzyme for pyruvate, NADH, and fructose-1,6-diphosphate. When cultured aerobically, strain CNRZ 483 transformed 2.3% of glucose to acetoin and produced no diacetyl or 2,3-butanediol. Under the same conditions, mutants 483L1, 483L2, and 483L3 transformed 42.0, 78.9, and 75.8%, respectively, of glucose to C4 compounds (diacetyl, acetoin, and 2,3-butanediol). Anaerobically, strain CNRZ 483 produced no C4 compounds, while mutants 483L1, 483L2, and 483L3 transformed 2.0, 37.0, and 25.8% of glucose to acetoin and 2,3-butanediol. In contrast to the parental strain, the NADH balance showed that the mutants regenerated most of the NAD via NADH oxidase under aerobic conditions and by ethanol production under anaerobic conditions.
Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose
Authors: Formanek J , Mackie R , Blaschek H P
Published: January 2020

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).
Acquired Thermotolerance and Stressed-Phase Growth of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula in Continuous Culture
Authors: Han C J , Park S H , Kelly R M
Published: January 2020

The response of an extremely thermoacidophilic archaeon, Metallosphaera sedula (growth temperature range, 50 to 79(deg)C; optimum temperature, 74(deg)C; optimum pH, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. M. sedula, growing at 74(deg)C, pH 2.0, and a dilution rate of 0.04 hr(sup-1), was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. An abrupt temperature shift from 74 to 79(deg)C resulted in little, if any, changes in cell density and a small increase in total protein per cell. When the culture temperature was shifted further to 80.5(deg)C, cell density dropped to below 5 x 10(sup6) cells/ml from 10(sup8) cells/ml, leading to washout of the culture. Operation at this temperature and slightly higher temperatures, however, could be achieved by exposing the culture to thermal stress more gradually (0.5(deg)C increments). As a result, stable operation could be maintained at temperatures of up to 81(deg)C, and the washout temperature could be increased to 82.5(deg)C. Continuous culture operation at 81(deg)C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for operation at or below 79(deg)C. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (both one and two dimensional) revealed significantly higher levels (sixfold increase) of a 66-kDa stress response protein (MseHSP60), immunologically related to Thermophilic Factor 55 from Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990). If the acclimated culture was returned to a lower temperature (i.e., 74(deg)C), the amount of MseHSP60 returned to levels observed prior to thermal acclimation. Furthermore, when the previously acclimated culture (at 81(deg)C) was shifted back from 74 to 81(deg)C, without going through gradual acclimation steps, the result was the immediate onset of washout, suggesting no residual thermotolerance. This study shows that gradual thermal acclimation of M. sedula could only extend the temperature range of stable growth for this organism by 2(deg)C above its maximal growth temperature, albeit at reduced cell densities. Also, this investigation illustrates the utility of continuous culture for characterizing heat shock response and assessing maximum growth temperatures for extremely thermophilic microorganisms.
Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni
Authors: Junker F , Cook A M
Published: January 2020

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.
Catalytic properties of the cellulose-binding endoglucanase F from Fibrobacter succinogenes S85
Authors: Malburg S R , Malburg L M , Liu T , Iyo A H , Forsberg C W
Published: January 2020

The celF gene from the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes encodes a 118.3-kDa cellulose-binding endoglucanase, endoglucanase F (EGF). This enzyme possesses an N-terminal cellulose-binding domain and a C-terminal catalytic domain. The purified catalytic domain displayed an activity profile typical of an endoglucanase, with high catalytic activity on carboxymethyl cellulose and barley beta-glucan. Immunoblotting of EGF and the formerly characterized endoglucanase 2 (EG2) from F. succinogenes with antibodies prepared against each of the enzymes demonstrated that EGF and EG2 contain cross-reactive epitopes. This data in conjunction with evidence that the proteins are the same size, share a 19-residue internal amino acid sequence, possess similar catalytic properties, and both bind to cellulose allows the conclusion that celF codes for EG2.
Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water
Authors: Miyamoto H , Yamamoto H , Arima K , Fujii J , Maruta K , Izu K , Shiomori T , Yoshida S
Published: January 2020

The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.
Distribution of polyunsaturated Fatty acids in bacteria present in intestines of deep-sea fish and shallow-sea poikilothermic animals
Authors: Yano Y , Nakayama A , Yoshida K
Published: January 2020

The lipid and fatty acid compositions in nine obligate and facultative barophilic bacteria isolated from the intestinal contents of seven deep-sea fish were determined. Phospholipid compositions were simple, with phosphatidylethanolamine and phosphatidylglycerol predominating in all strains. Docosahexaenoic acid (DHA; 22:6n-3), which has not been reported in procaryotes except for deep-sea bacteria, was found to be present in eight strains at a level of 8.1 to 21.5% of total fatty acids. In the other strain, eicosapentaenoic acid (EPA; 20:5n-3) was present at a level of 31.5% of total fatty acids. Other fatty acids observed in all strains were typical of marine gram-negative bacteria. Subcultures from pouches prepared from intestinal contents of five deep-sea fish by the most-probable-number (MPN) method were analyzed for fatty acids, and all subcultures contained DHA and/or EPA. Accordingly, viable cell counts of bacteria containing DHA and EPA were estimated at a maximum of 1.3 x 10(sup8) and 2.4 x 10(sup8) cells per ml, respectively, and accounted for 14 and 30%, respectively, of the total cell counts in the intestinal contents of the deep-sea fish. In the case of 10 shallow-sea poikilothermic animals having bacterial populations of 1.1 x 10(sup6) to 1.9 x 10(sup9) CFU per ml in intestinal contents, no DHA was found in the 112 isolates examined, while production of EPA was found in 40 isolates from cold- and temperate-sea samples. These results suggest that DHA and EPA are involved in some adaptations of bacteria to low temperature and high pressure.
Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8
Authors: Rudi K , Skulberg O M , Larsen F , Jakobsen K S
Published: January 2020

A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.
Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses
Authors: Lee I M , Bartoszyk I M , Gundersen-Rindal D E , Davis R E
Published: January 2020

A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera.
Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation
Authors: Borneman J , Triplett E W
Published: January 2020

Although the Amazon Basin is well known for its diversity of flora and fauna, this report represents the first description of the microbial diversity in Amazonian soils involving a culture-independent approach. Among the 100 sequences of genes coding for small-subunit rRNA obtained by PCR amplification with universal small-subunit rRNA primers, 98 were bacterial and 2 were archaeal. No duplicate sequences were found, and none of the sequences had been previously described. Eighteen percent of the bacterial sequences could not be classified in any known bacterial kingdom. Two sequences may represent a unique branch between the vast majority of bacteria and the deeply branching, predominantly thermophilic bacteria. Five sequences formed a clade that may represent a novel group within the class Proteobacteria. In addition, rRNA intergenic spacer analysis was used to show significant microbial population differences between a mature forest soil and an adjacent pasture soil.
Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells
Authors: Mazzotta A S , Crandall A D , Montville T J
Published: January 2020

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.
Efficiency of indigenous and inoculated cold-adapted soil microorganisms for biodegradation of diesel oil in alpine soils
Authors: Margesin R , Schinner F
Published: January 2020

Biodegradation of diesel oil (5 g(middot)kg [soil dry weight](sup-1)) was investigated in five alpine subsoils, differing in soil type and bedrock, in laboratory experiments during 20 days at 10(deg)C. The biodegradation activities of the indigenous soil microorganisms and of a psychrotrophic diesel oil-degrading inoculum and the effect of biostimulation by inorganic fertilization (C/N/P ratio = 100:10:2) were determined. Fertilization significantly enhanced diesel oil biodegradation activity of the indigenous soil microorganisms. Biostimulation by fertilization enhanced diesel oil biodegradation to a significantly greater degree than bioaugmentation with the psychrotrophic inoculum. In none of the five soils did fertilization plus inoculation result in a higher decontamination than fertilization alone. A total of 16 to 23% of the added diesel oil contamination was lost by abiotic processes. Total decontamination without and with fertilization was in the range of 16 to 31 and 27 to 53%, respectively.
Production and Identification of N-Glucosylrubropunctamine and N-Glucosylmonascorubramine from Monascus ruber and Occurrence of Electron Donor-Acceptor Complexes in These Red Pigments
Authors: Hajjaj H , Klaebe A , Loret M O , Tzedakis T , Goma G , Blanc P J
Published: January 2020

The filamentous fungus Monascus ruber produces water-soluble red pigments in a submerged culture when grown in a chemically defined medium containing glucose as a carbon source and monosodium glutamate as a nitrogen source. Two new molecules with polyketide structures, N-glucosylrubropunctamine and N-glucosylmonascorubramine, constituting under some conditions 10% of the total extracellular coloring matter when glucose as a carbon source was in excess (20 g/liter), were isolated and structurally characterized by high-pressure liquid chromatography, Dionex methods, (sup1)H and (sup13)C nuclear magnetic resonance spectroscopy, and mass spectrometry. The occurrence of the electron donor-acceptor complex effect was demonstrated by UV spectroscopy, polarography, and thin-layer voltammetry. The use of n-butanol as an extraction solvent stabilized the pigments against the effects of daylight for several months, promoting the stability of this type of complex.
Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant
Authors: Stols L , Donnelly M I
Published: January 2020

NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks.
Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis
Authors: Guerchicoff A , Ugalde R A , Rubinstein C P
Published: January 2020

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.
Cyanide Degradation under Alkaline Conditions by a Strain of Fusarium solani Isolated from Contaminated Soils
Authors: Dumestre A , Chone T , Portal J , Gerard M , Berthelin J
Published: January 2020

Several cyanide-tolerant microorganisms have been selected from alkaline wastes and soils contaminated with cyanide. Among them, a fungus identified as Fusarium solani IHEM 8026 shows a good potential for cyanide biodegradation under alkaline conditions (pH 9.2 to 10.7). Results of K(sup14)CN biodegradation studies show that fungal metabolism seems to proceed by a two-step hydrolytic mechanism: (i) the first reaction involves the conversion of cyanide to formamide by a cyanide-hydrolyzing enzyme, cyanide hydratase (EC 4.2.1.66); and (ii) the second reaction consists of the conversion of formamide to formate, which is associated with fungal growth. No growth occurred during the first step of cyanide degradation, suggesting that cyanide is toxic to some degree even in cyanide-degrading microorganisms, such as F. solani. The presence of organic nutrients in the medium has a major influence on the occurrence of the second step. Addition of small amounts of yeast extract led to fungal growth, whereas no growth was observed in media containing cyanide as the sole source of carbon and nitrogen. The simple hydrolytic detoxification pathway identified in the present study could be used for the treatment of many industrial alkaline effluents and wastes containing free cyanide without a prior acidification step, thus limiting the risk of cyanhydric acid volatilization; this should be of great interest from an environmental and health point of view.
Reductive dehalogenation and mineralization of 3-chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer
Authors: Townsend G T , Ramanand K , Suflita J M
Published: January 2020

We investigated the anaerobic biodegradation of 3-chlorobenzoate (3CBz) by microorganisms from an aquifer where chloroaromatic compounds were previously found to resist decay in the presence of sulfate. After a lengthy lag period, 3CBz was degraded in the presence of sulfate and concurrently with sulfate reduction. Chlorine removal from 2,5- or 3,5-dichlorobenzoates and the transient appearance of benzoate from 3CBz confirmed that reductive dehalogenation was the initial fate process for these substrates. Sulfate did not influence 3CBz degradation rates in acclimated enrichment cultures but accelerated the development of 3CBz degradation activity in fresh transfers. Benzoate degradation was more rapid in the presence of sulfate regardless of the enrichment history. Nitrate, sulfite, and a headspace of air inhibited 3CBz dehalogenation, while thiosulfate had no effect. Mass balance determinations revealed that 71 to 107% of the theoretically expected amount of methane was produced from 3CBz and benzoate oxidation in the absence of sulfate. In parallel cultures containing 15 mM sulfate, methanogenesis was reduced to 48 to 71% of that theoretically expected, while sulfate reduction accounted for 12 to 50% of the reducing equivalents. In either the presence or absence of sulfate, steady-state dissolved hydrogen concentrations were similar to those reported for sulfate-reducing or methanogenic environments, respectively. Molybdate inhibited sulfate reduction and 3CBz dehalogenation to a similar extent but did not affect benzoate biodegradation. Sulfate-dependent 3CBz biodegradation was not observed. We conclude that reductive dehalogenation and sulfate reduction occur concurrently in these enrichments and that the sulfate-dependent stimulation in fresh transfers was likely due to the acceleration of benzoate oxidation.
Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis
Authors: Godon J J , Zumstein E , Dabert P , Habouzit F , Moletta R
Published: January 2020

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.
Composition and Biophysical Properties of Lipids in Xenorhabdus nematophilus and Photorhabdus luminescens, Symbiotic Bacteria Associated with Entomopathogenic Nematodes
Authors: Fodor E , Szallas E , Kiss Z , Fodor A , Horvath L I , Chitwood D J , Farkas T
Published: January 2020

Primary and secondary forms of Photorhabdus luminescens Hm and Xenorhabdus nematophilus N2-4 were grown at 18 and 28(deg)C for 24 to 96 h, and we made determinations of the fatty-acid compositions of total lipids and of the fluidity measured by 5-doxyl-stearic acid embedded in liposomes made from total lipids. The levels of the unsaturated fatty acids 16:1 and 18:1 (those with chain lengths of 16 or 18 and one double bond) generally were higher in primary-phase variants of P. luminescens grown at 18(deg)C than in those grown at 28(deg)C. Prolonged culture at 18(deg)C caused the level of 18:1 to fall and reach that observed at 28(deg)C. The ratio of saturated to unsaturated fatty acids rose with prolonged culture times in variants of each species at both phases. When grown at 18(deg)C, the proportion of 16:1 in X. nematophilus was lower than in P. luminescens; the patterns of temperature-induced changes were similar in these species. X. nematophilus contained a greater percentage of short-chain fatty acids (i.e., with chain lengths of <14.0) than P. luminescens. Lipid liposomes from primary and secondary cultures of both bacterial species grown at 18(deg)C were more ordered (i.e., less fluid) than those grown at 28(deg)C. This result suggests the surprising absence of homeoviscous adaptation of membranes to temperature. Also, liposomes from primary cultures were more ordered than those from secondary cultures and membranes from primary cultures of P. luminescens were more ordered at both culture temperatures than membranes from X. nematophilus. The biological significance of the effect of growth conditions on membrane biophysical properties in these bacteria is discussed.
Growth of silicone-immobilized bacteria on polycarbonate membrane filters, a technique to study microcolony formation under anaerobic conditions
Authors: Højberg O , Binnerup S J , Sørensen J
Published: January 2020

A technique was developed to study microcolony formation by silicone-immobilized bacteria on polycarbonate membrane filters under anaerobic conditions. A sudden shift to anaerobiosis was obtained by submerging the filters in medium which was depleted for oxygen by a pure culture of bacteria. The technique was used to demonstrate that preinduction of nitrate reductase under low-oxygen conditions was necessary for nonfermenting, nitrate-respiring bacteria, e.g., Pseudomonas spp., to cope with a sudden lack of oxygen. In contrast, nitrate-respiring, fermenting bacteria, e.g., Bacillus and Escherichia spp., formed microcolonies under anaerobic conditions with or without the presence of nitrate and irrespective of aerobic or anaerobic preculture conditions.
PCR-mediated detection of acidophilic, bioleaching-associated bacteria
Authors: De Wulf-Durand P , Bryant L J , Sly L I
Published: January 2020

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.
Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9
Authors: Techkarnjanaruk S , Pongpattanakitshote S , Goodman A E
Published: January 2020

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.
PCR-based approach for detection of novel Bacillus thuringiensis cry genes
Authors: Juárez-Pérez V M , Ferrandis M D , Frutos R
Published: January 2020

A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences. The first step allows the accurate identification of already-characterized cryI genes by the use of three primers. During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer. The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article.
Diversity and association of psychrophilic bacteria in Antarctic sea ice
Authors: Bowman J P , McCammon S A , Brown M V , Nichols D S , McMeekin T A
Published: January 2020

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature, > 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.
Ether lipids of planktonic archaea in the marine water column
Authors: Hoefs M , Schouten S , De Leeuw J W , King L L , Wakeham S G , Damste J
Published: January 2020

Acyclic and cyclic biphytanes derived from the membrane ether lipids of archaea were found in water column particulate and sedimentary organic matter from several oxic and anoxic marine environments. Compound-specific isotope analyses of the carbon skeletons suggest that planktonic archaea utilize an isotopically heavy carbon source such as algal carbohydrates and proteins or dissolved bicarbonate. Due to their high preservation potential, these lipids provide a fossil record of planktonic archaea and suggest that they have thrived in marine environments for more than 50 million years.
Characterization of the sediment bacterial community in groundwater discharge zones of an alkaline fen: a seasonal study
Authors: Gsell T C , Holben W E , Ventullo R M
Published: January 2020

The cell density, activity, and community structure of the bacterial community in wetland sediments were monitored over a 13-month period. The study was performed at Cedar Bog, an alkaline fen. The objective was to characterize the relationship between the sediment bacterial community in groundwater upwelling zones and the physical and chemical factors which might influence the community structure and activity. DNA, protein, and lipid synthesis were measured at three different upwelling zones by using [3H]thymidine, [14C]leucine, and [14C]glucose incorporation, respectively. The physiological status (apparent stress) of the consortium was assessed by comparing [14C]glucose incorporation into membrane and that into storage lipids. Bacterial cell density was determined by acridine orange direct counts, and gross bacterial community structure was determined by bisbenzimidazole-cesium chloride gradient analysis of total bacterial community DNA. Both seasonal and site-related covariation were observed in all estimates of bacterial biomass and activity. Growth rate estimates and cell density peaked in late July at 2.5 x 10(8) cells/g/day and 2.7 x 10(9) cells/g, respectively, and decreased in December to 2.0 x 10(7) cells/g/day and 1.5 x 10(9) cells/g, respectively. Across sites, membrane-to-storage-lipid ratios were generally highest in late spring and peaked in September for one site. Overall, the data indicate dynamic seasonal differences in sediment bacterial community activity and physiology, possibly in response to changing physical and chemical environmental factors which included the C/N/P ratios of the perfusing groundwater. By contrast, total cell numbers were rather constant, and community structure analysis indicated that the overall community structure was similar throughout the study.