Medical bioremediation of age-related diseases

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.     

Temporal effects on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa and Melilotus alba

An assessment was made of the impact of temporal separation on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a single site that had no known history of alfalfa cultivation. Root nodules were sampled on six occasions over two seasons, and a total of 1620 isolates of S. meliloti were characterized on the basis of phage sensitivity using 16 typing phages. Plant infection tests indicated that symbiotic S. meliloti were deficient in the soil at the time of planting and that these bacteria were present at low density during the first season (<10(2)/g of soil); in the second season numbers increased markedly to about 10(5)/g of soil. Overall, 37 and 51 phage types, respectively, were encountered among the nodule isolates from M. sativa and M. alba. The data indicate significant temporal shifts in the frequency and diversity of types associated with the two legume species. Apparent temporal variation with respect to the frequency of types appeared largely unpredictable and was not attributable to any one sampling time. The results indicate an apparent reduction in phenotypic diversity over the course of the experiment. Differential host plant selection of specific types with respect to nodule occupancy was indicated by significant interactions between legume species and either the frequency or diversity of phage types. Isolates from M. sativa that were resistant to lysis by all typing phages (type 14) were unusual in that they were predominant on this host at all sampling times (between 53% and 82% nodule occupancy) and were relatively homogeneous on the basis of DNA hybridization with 98% of the isolates analysed sharing the same nod EFG hybridization profile. In contrast, those isolates from M. alba comprising type 14 were encountered at low total frequency (2%) and were genetically heterogeneous on the basis of Southern hybridization. The implications of the observed temporal and host plant variation for ecological studies are discussed.     

Sinorhizobium meliloti plasmid pRm1132f replicates by a rolling-circle mechanism

pRm1132f isolated from Sinorhizobium meliloti is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a rep gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

Application of immunodiffusion to the identification of Rhizobium meliloti strains competing for nodulation on Medicago sativa

The immunodiffusion technique was successfully used to unambiguously recognize four strains of Rhizobium meliloti in a study of competition for nodulation with Medicago sativa cv. Apollo inoculated with two-, three- and fourstrain mixtures. The serological reactions of all R. meliloti strains revealed no significant changes following plant passage indicating that the antigens involved in immunodiffusion were stable. R. meliloti 102F70 formed 50% or more of the nodules on M. sativa inoculated with two-, three- and four-strain mixtures. The remaining three strains were less competitive and produced similar proportions of nodules (14–20%) on plants inoculated with three- and four-strain mixtures. Cases of mixed-strain occupancy of nodules involving either two of three strains were detected in a sub-sample of nodules. The data also indicated considerable variation in the proportions of strains in the nodules of individual plants.     

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins)

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper- binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine- rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.      

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.