Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.     

好氧细菌对多环芳烃降解机制的研究进展

多环芳烃(PAHs)是指两个或两个以上的苯环以线性排列、弯接或簇聚方式构成的一类碳氢化合物。这类化合物广泛分布于环境中, 具有潜在的致畸性、致癌性和遗传毒性。在自然环境中, 好氧细菌对PAHs的生物降解是一种很重要的方式, 凸显其在清除环境PAHs污染物中具有广阔的应用前景。在过去二十多年中, 科学家们已经从基因水平上对好氧细菌降解PAHs的机制进行了深入的研究, 其中包括PAHs降解基因的多样性、与PAHs降解有关的基因以及细菌群体PAHs遗传适应机制等。在此, 就好氧细菌对多环芳烃降解机制的研究进展进行了综述和讨论。     

好氧细菌对多环芳烃降解机制的研究进展

多环芳烃(PAHs)是指两个或两个以上的苯环以线性排列、弯接或簇聚方式构成的一类碳氢化合物.这类化合物广泛分布于环境中,具有潜在的致畸性、致癌性和遗传毒性.在自然环境中,好氧细菌对PAHs的生物降解是一种很重要的方式,凸显其在清除环境PAHs污染物中具有广阔的应用前景.在过去二十多年中,科学家们已经从基因水平上对好氧细菌降解PAHs的机制进行了深入的研究,其中包括PAHs降解基因的多样性、与PAHs降解有关的基因以及细菌群体PAHs遗传适应机制等.在此,就好氧细菌对多环芳烃降解机制的研究进展进行了综述和讨论.     

Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Comparison of 26 Sphingomonad Genomes Reveals Diverse Environmental Adaptations and Biodegradative Capabilities

Sphingomonads comprise a physiologically versatile group within the Alphaproteobacteria that includes strains of interest for biotechnology, human health, and environmental nutrient cycling. In this study, we compared 26 sphingomonad genome sequences to gain insight into their ecology, metabolic versatility, and environmental adaptations. Our multilocus phylogenetic and average amino acid identity (AAI) analyses confirm that Sphingomonas, Sphingobium, Sphingopyxis, and Novosphingobium are well-resolved monophyletic groups with the exception of Sphingomonas sp. strain SKA58, which we propose belongs to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible for their ability to degrade various recalcitrant aromatic compounds and polysaccharides, respectively. Many of these enzymes are encoded on megaplasmids, suggesting that they may be readily transferred between species. We also identified enzymes putatively used for the catabolism of sulfonate and nitroaromatic compounds in many of the genomes, suggesting that plant-based compounds or chemical contaminants may be sources of nitrogen and sulfur. Many of these sphingomonads appear to be adapted to oligotrophic environments, but several contain genomic features indicative of host associations. Our work provides a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling.     

Construction of Broad Host Range Cloning Vectors for Gram-negative Bacteria

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Ap^r, Cm^r, and Tc^r genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cm^r gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.     

Presence of Culturable Bacteria in Cocoons of the Earthworm Eisenia fetida

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10⁸ CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10⁹ cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10⁸ CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.     

The Growth of Gram-Negative Bacteria in the Hen's Egg

Summary: Bacteriological and chemical methods were used to follow the course of infection in eggs, incubated at 27°, the air cells of which had been inoculated with a suspension of washed bacteria. In the 3–4 days following inoculation, limited bacterial multiplication occurred in the inner membrane of the air cell but very few organisms entered the albumen. These populations then remained static or decreased slightly until renewed multiplication occurred 12–30 days after inoculation. This was induced by contact of the yolk and the shell membranes: it occurred on the 12–20th day in eggs in which the yolk moved towards the site of inoculation, but later when the yolk moved in the opposite direction. At this time there was a general infection of the egg contents and significant changes occurred in the pH and glucose concentration in the albumen. In eggs that had been inoculated with chromogenic and/or proteolytic bacteria, the first macroscopic changes of the contents were seen at this time. The rate and extent of the initial multiplication was influenced by the composition of the fluid used to suspend the washed bacteria and, in all instances, the fastest multiplication occurred when iron was added to the inoculum. Moreover, renewed multiplication occurred when iron was added to the albumen of eggs in which the bacteria were in the stationary phase.     

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 10⁶ and 7.1 × 10⁶ transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

Method for Investigation of Competition between Bacteria as a Function of Three Environmental Factors Varied Simultaneously

Competition between microorganisms as affected by temperature, pH, and the sodium chloride (NaCl) concentration was investigated by selective replication from gradient plates. Salmonella typhimurium was inhibited by Pseudomonas putida at 20 and 23°C but not 30 and 35°C. P. putida no longer grew at the extremes of pH and NaCl concentration, particularly at 30 and 35°C.     

Egg color as an adaptation for thermoregulation

ABSTRACT.   Avian embryos are incubated at temperatures only 2–6 °C below that at which hyperthermia begins to influence survival. In habitats where sunlight directly strikes the eggs, even for short periods, heat gain may be a substantial threat to survival, and reflective pigmentation may reduce the rate of heat gain. The results of previous studies suggest that light-colored eggs acquire heat slower than dark eggs, but artificial pigments were used to create differences in egg coloration. This approach is problematic because natural eggshell pigments have low absorbance in the near-infrared waveband that encompasses about half of incident solar radiation. We used naturally-pigmented eggs to measure the influence of egg coloration on heat gain. Triads ( N = 18) of eggs from Brewer's ( Euphagous cyanocephalus ), Red-winged ( Agelaius phoeniceus ), and Yellow-headed ( Xanthocephalus xanthocephalus ) blackbirds were crossed with six nests of each species and either exposed to full sunlight or placed under a diffusing umbrella. Thermisters recorded internal egg temperature every minute until an asymptotic temperature was reached. Eggs in full sunlight acquired heat more rapidly than eggs in the shaded environment, but heat gain did not vary with egg color in either environment. Eggs placed in Yellow-headed Blackbird nests took longer to reach asymptotic temperature, but there was no significant egg-by-nest interaction. Thus, it appears that differences in reflectivity of eggshell pigments in the visible range (400–700 nm) do not result in different rates of heat acquisition. The thermoregulation hypothesis was not supported.     

Egg yolk lipoproteins as substrates for lipases

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.     

The Egg Yolk Reaction Produced by Several Species of Bacteria

S ummary . A study of the egg yolk reaction produced by various species of bacteria has shown that it is caused by the hydrolysis of phosphatidylcholine. Staphylococcus aureus and Serratia marcescens degrade phosphatidylcholine by a series of hydrolytic reactions which are initiated by phosphatidylcholine: cholesterol o -acyltransferase. The degradation of phosphatidylcholine results in the formation of a sub-surface, opaque homogeneous zone around colonies growing on egg yolk agar and a collar of lipid material floating on the surface of egg yolk broth. This reaction may not be shown by all species growing in egg yolk media but it can be demonstrated if enzyme extracts are used.     

Earthworm egg capsules as vectors for the environmental introduction of biodegradative bacteria.

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2, 4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10(2) or 10(5) CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10(4) CFU R. eutropha (pJP4) g-1 of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2, 4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.