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Expression of bacterial genes in plant cells.

Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.

Aminoglycosides↗

Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2.

The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described. Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated. Transfections were carried out with DNA from corynephage 782, a member of the beta family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin-resistant strain of C. diphtheriae and carries the resistance gene. Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA. Transfection frequencies were in the range of 3-8 X 10(3) plaque-forming units/micrograms of phage DNA, and transformation frequencies were in the range of 0.2-150 colony-forming units/micrograms of plasmid DNA. Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin. Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram-negative bacteria without the intervention of genetic engineering. pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains. The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin.

Bacteriophages↗

Microbial studies in the Canadian nuclear fuel waste management program.

Atomic Energy of Canada Limited (AECL) has developed a concept for permanent geological disposal of used nuclear fuel in Canada. This concept, based on a multibarrier system, would involve disposal of nuclear fuel waste in titanium or copper containers, surrounded by compacted clay-based buffer and backfill materials, in a vault 500-1000 m deep in granitic rock of the Canadian Shield. Subsurface environments will not be sterile and an experimental program was initiated in 1991 by AECL to address and quantify the potential effects of microbial action on the integrity of the disposal vault. This microbial program focuses on answering specific questions in areas such as the survival of bacteria in compacted clay-based buffer materials under relevant radiation, temperature and desiccation conditions; mobility of microbes in compacted buffer materials; the potential for microbially influenced corrosion of containers; microbial gas production in backfill material; introduction of nutrients as a result of vault excavation and operation; the presence and activity of microbes in deep granitic groundwaters; and the effects of biofilms on radionuclide migration in the geosphere. This paper summarizes the results to date from the research activities at AECL.

Bacterial Physiological Phenomena↗

Metabolism of lactic acid bacteria studied by nuclear magnetic resonance.

The complexity of metabolic and regulatory networks presents a great scientific challenge to an integrated view of how individual components contribute to the overall function. Nuclear magnetic resonance (NMR) spectroscopy is undoubtedly a suitable technique for global investigations of microbial metabolism, since it allows a view into living cells without disturbing the cellular organisation. Therefore, metabolic processes can be monitored in real time under physiological conditions. In the present paper, examples of the application of NMR to study the metabolism of lactic acid bacteria will be given. These include the analysis of labelling patterns in end-products using 13C as a tracer, thereby establishing metabolic pathways, the detection and quantification of intermediates in the pathway of exopolysaccharide biosynthesis, and on line monitoring of glycolytic kinetics to assess the effect of metabolic engineering strategies.

Bioreactors↗

Reductive dechlorination of polychlorinated biphenyls in sediment from the Twelve Mile Creek arm of Lake Hartwell, South Carolina, USA.

Lake Hartwell is a U.S. Army Corps of Engineers reservoir system located on the state line between South Carolina and Georgia, USA. The lake was contaminated with an estimated 200 metric tons of polychlorinated biphenyls ([PCBs]; mainly Aroclor 1016 and 1254), and the entire Twelve Mile Creek watershed and the Seneca River arm of Lake Hartwell were placed on the National Priorities List. Monitored natural attenuation was chosen as a remedy for the contaminated sediment. The relatively warm temperature of Lake Hartwell and lack of significant cocontaminants along with the PCBs distinguish this site from others that have been studied for microbially mediated reductive dechlorination. Microcosm studies were conducted with sediment from two locations in the Twelve Mile Creek arm and confirmed the presence of indigenous microorganisms capable of reductively dechlorinating Aroclor 1254, which contains predominantly tetra-, penta-, and hexachlorobiphenyl. The average number of total chlorines per biphenyl decreased from 4.8 to 4.9 to 2.9 to 3.0, following 250 to 260 d of incubation. The maximum observed dechlorination rates were 0.29 to 0.87 microg-atoms Cl- per gram sediment dry weight per week. The onset of dechlorination activity correlated strongly with maximum methanogenesis, which occurred without a lag in samples from the site that showed signs of in situ fermentation activity. Dechlorination occurred primarily at the meta and para positions (58-63% removal), with no apparent decrease in ortho chlorines. This most closely resembles pattern M, characterized by preferential removal of unflanked and flanked meta chlorines. The microcosm results are consistent with sediment cores analyzed from the same locations, which indicate accumulation with depth of the same ortho- and para-substituted congeners. It therefore appears that the success of monitored natural attenuation for Lake Hartwell will hinge on covering the recalcitrant PCBs with a sufficient amount of uncontaminated sediment to isolate them from the food chain.

Aroclors↗

Microbial enzymes for oxidation of organic molecules.

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.

Bacteria↗

Parallel solid-phase synthesis of nucleoside phosphoramidate libraries.

Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).

Amides↗

Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi.

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-kappaB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

Animals↗

A real-time approach to detect seal defects in food packages using ultrasonic imaging.

The microbial integrity of many types of flexible food packages depends on a zero defect level in the fused seam seal. Human inspection for defects in these seals is marginal at best, and secondary incubation protocols are often used to spot packages with compromised integrity before releasing product for sale. A new type of inspection method has been developed and is being evaluated for robustness. The purpose of the study was to evaluate a new raster scanning geometry to simulate continuous motion, online ultrasonic inspection of the seal region in flexible food package seals. A principal engineering tradeoff of scanning inspection systems is between increased line speed that results from decreased spatial sampling (less acquired data to process) and decreased image quality. The previously developed pulse-echo Backscattered Amplitude Integral (BAI) mode imaging technique is used to form ultrasound images using the new scanning geometry. At an ultrasonic frequency of 22.9 MHz, 38- and 50-microm-diameter air-filled channel defects in all-plastic transparent trilaminate are evaluated. The contrast-to-noise ratio (CNR) of the processed BAI-mode image is used to quantify image quality as a function of spatial sampling. Results show seal defects (38- and 50-microm diameter) are still detectable for undersampled conditions, although image quality degrades as spatial sampling decreases. Further, it is concluded that the raster scanning geometry is feasible for online inspection.

Food Contamination↗

Comparative costs of microbial identification employing conventional and prepackaged commercial systems.

The accuracy of commercially prepackaged kits for microbial identification has become well established. Laboratory workers may encounter increasing demands for production of objective data to justify replacement of systems using individual biochemical tests in tubes. The authors present a system of cost analysis in which materials and labor costs are separately computed, and to which are added the effects of (1) fringe benefits, (2) decreased productivity resulting from administration, quality control, education and development, and (3) the additional expense of indirect costs that are allocated to laboratory procedures by accepted and standardized hospital accounting methods. Labor costs should be based on time-engineered studies conducted in individual laboratories. Alternatively, various published "unit values" may be used. The result may present several alternative differences in cost, depending on which unit values are accepted as applicable to the individual laboratory. Despite these uncertainties, the method of analysis provides a more objective means of justifying the cost of introduction of prepackaged kits where accuracy and speed of identification have already been proven to have advantages over biochemical tests in tubes.

Bacteria↗

Water: friend and foe.

With a basic knowledge of the origin and transport of moisture, psychometrics, and the use of properly calibrated diagnostic tools, an EM can not only determine the origin of many moisture problems (and indoor air quality complaints), but also aid infection control efforts by minimizing microbial growth. In future issues of HHMM, CHEM will introduce the EM to flood and moisture mitigation procedures, as well as training courses and outside contracting services that should be used as an integral portion of a healthcare facility's emergency preparedness program.

Air Conditioning↗

Salivary enhancement: current status and future therapies.

Saliva provides the principal protective milieu for teeth by modulating oral microbial ecosystems and reversing the initial phases of caries development. Patients with inadequate salivary function are at increased risk for dental decay. Therefore, it is likely that therapies that increase overall fluid output of these individuals will reverse early carious lesions. The most common causes of salivary dysfunction are medication usage, Sjögren's syndrome, and damage of salivary parenchyma during therapeutic irradiation. For patients with remaining functional acinar tissue, treatment with the parasypathomimetic secretogogues pilocarpine and Cevimeline may provide relief. However, these medications do not benefit all patients. The possibilities of using gene therapy and tissue engineering to develop treatments for those with severe salivary dysfunction are discussed.

Animals↗

The evolution of biotransformation technologies.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

Benzopyrans↗

Humans as the world's greatest evolutionary force.

In addition to altering global ecology, technology and human population growth also affect evolutionary trajectories, dramatically accelerating evolutionary change in other species, especially in commercially important, pest, and disease organisms. Such changes are apparent in antibiotic and human immunodeficiency virus (HIV) resistance to drugs, plant and insect resistance to pesticides, rapid changes in invasive species, life-history change in commercial fisheries, and pest adaptation to biological engineering products. This accelerated evolution costs at least $33 billion to $50 billion a year in the United States. Slowing and controlling arms races in disease and pest management have been successful in diverse ecological and economic systems, illustrating how applied evolutionary principles can help reduce the impact of humankind on evolution.

Adaptation, Physiological↗

Arginine-substituted Mastoparan-C derivatives combat dual bacterial pathogens: in vitro mechanistic insights and in vivo efficacy in polymicrobial wounds.

UNLABELLED: The synergistic interactions in multi-pathogen infections compromise wound healing and limit therapeutic efficacy. In this study, we designed and synthesized arginine-substituted derivatives of the antimicrobial peptide Mastoparan-C (MP-C). Among them, Arg²MP-C and Arg4.11.12MP-C exhibited potent, broad-spectrum activity against both Escherichia coli and Staphylococcus aureus. Their enhanced antibacterial activity is associated with increased positive charge and optimized hydrophobicity. Mechanistically, both peptides employ a dual-target strategy, disrupting bacterial membranes and binding genomic DNA; Arg²MP-C acted most rapidly against the E. coli envelope, while Arg4.11.12MP-C caused the strongest membrane damage to S. aureus. In a murine polymicrobial wound model, Arg²MP-C treatment nearly achieved complete wound closure by day 10, significantly reduced bacterial loads, and promoted tissue regeneration. This study demonstrates that arginine engineering can yield peptides with potent, multi-mechanistic action, identifying Arg²MP-C as a promising candidate for combating polymicrobial wound infections. IMPORTANCE: Wounds infected with multiple bacterial species are notoriously difficult to treat, often leading to poor healing and limited effectiveness of existing therapies. In this study, we developed new antimicrobial peptides by introducing arginine substitutions into a natural peptide called Mastoparan-C. Two of our engineered peptides, Arg²MP-C and Arg4.11.12MP-C, showed potent activity against two common wound pathogens, Escherichia coli and Staphylococcus aureus. These peptides work through a dual mechanism: disrupting bacterial membranes and binding to bacterial DNA. In a mouse model of mixed-infection wounds, treatment with Arg²MP-C led to nearly complete wound closure by day 10, drastically reduced bacterial counts, and promoted tissue repair. Our findings highlight arginine engineering as a promising strategy to create next‑generation antimicrobial agents that can effectively combat complex polymicrobial wound infections, addressing a critical unmet need in clinical wound care.

Animals↗

Microbial production of 1,3-propanediol.

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a "green" polyester is a most cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

Adenosine Triphosphate↗

Characterization of fouled membranes from a membrane enhanced biological phosphorus removal system.

Characterization of fouled membranes is the first step towards a good understanding of membrane fouling nature and thus formulating effective engineering measures for fouling prevention and control. In this study, fouled membrane fibres collected from a pilot scale membrane enhanced biological phosphorus removal (MEBPR) process were systematically examined. Several analytical tools, including scanning electron microscopy (SEM), conventional optical microscopy (COM), energy dispersive X-ray (EDX) microanalysis, matrix assisted laser desorption/ionization--mass spectrometry (MALDI-MS) analysis, and conventional chemical analysis techniques were used. The results indicated that membrane fouling in the MEBPR process was mainly of an organic nature, and most extractable foulants were carbohydrates and humic or humic-like substances. Unlike in other wastewater treatment membrane bioreactors, microbial growth on fouled membranes was not substantial, probably due to the vigorous aeration applied and the strong hydrodynamic conditions within the membrane pore structure. After a period of sludge filtration, membrane surfaces became more hydrophobic and the resultant hydrophobic interactions between the fouled membranes and mixed liquor constituents might have accelerated the fouling process.

Bioreactors↗

Exploring biohydrogen producing potential of Arctic ice and water through metagenomics and dark fermentation kinetics.

Cryospheric ecosystems in the high Arctic harbor largely unexplored microbiomes with significant biotechnological potential. The present study evaluates the biohydrogen production capabilities of the indigenous microbiome of Ny-Ålesund, Svalbard, using glacial ice and surface water samples. Dark fermentation batch assays were performed at 4 °C and 20 °C with 2-bromoethanesulfonate (BES), a methanogenic inhibitor, to track the succession of metabolic and taxonomic diversity. Metagenomic and functional analyses revealed that under 20 °C and BES conditions, psychrotolerant microbial communities maximize biohydrogen production to 85% of the total biogas produced, with an acetate-dominant fermentation pathway, as inferred from volatile fatty acid (VFA) analysis. This evolves into a highly coordinated system utilizing a coupled Rnf-nitrogenase route alongside Formate Hydrogenlyase and [FeFe]-hydrogenase pathways. Kinetic modelling using the Modified Gompertz equation, along with Q10 temperature-sensitivity indices, demonstrated a very high latent catalytic potential in these cold-adapted microbiomes. This study indicates that Arctic microbiomes are highly elastic thermodynamically and could serve as highly efficient, manipulatable biocatalysts for the environmental recovery of bioenergy through engineered low-temperature systems.

Fermentation↗