NONRANDOM GENOTYPIC ASSOCIATIONS IN A LEGUME—BRADYRHIZOBIUM MUTUALISM

Genetically divergent lineages often coexist within populations of the annual legume Amphicarpaea bracteata. At one site dominated by two such lineages (termed biotypes “C” and “S”), isolates of root-nodule bacteria (Bradyrhizobium sp.) were sampled from both hosts and analyzed by enzyme electrophoresis. Symbiont populations on the two plant biotypes were highly distinct. Out of 15 bacterial multilocus genotypes detected (among 51 isolates analyzed), only one was shared in common by the two plant biotypes. Cluster analysis revealed three bacterial lineages (designated I, II, and III), with lineage I found exclusively on biotype C plants, and the two other lineages almost completely restricted to biotype S hosts. Laboratory inoculation tests indicated that lineage I bacteria were strictly specialized on biotype C hosts, forming few or no nodules on plants of the other host biotype. Bacterial lineages II and III were capable of forming nodules on both kinds of plants, but nodule numbers were often significantly higher on biotype S hosts. The nonrandom association between plant and bacterial lineages at this site implies that genetic diversity of hosts is an important factor in the maintenance of polymorphism within the symbiont population.     

Guidance and Activation of Murine Macrophages by Nanometric Scale Topography

We studied the guidance and activation of macrophages from the P388D1 cell line and rat peritoneum by topographic features on a nanometric scale. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves and steps, 30–282 nm deep. The contact of cells with the patterned surface activated cell spreading and adhesion and increased the number of protrusions of the cell membrane. These changes were accompanied by an increase in the amount of F-actin in cells. The accumulation of F-actin and vinculin in cells was observed along the edges of single steps or grooves. Formation of focal contacts along discontinuities in the substratum was accompanied by the phosphorylation of tyrosine colocalized with F-actin and vinculin. A similar pattern of staining was seen in cells stained for vitronectin receptor, αV integrin, but not for integrins α5β1 or α3β1. Cells cultured on nanogrooves showed a higher phagocytotic activity than cells cultured on plain substrata. We show that macrophages can react to ultrafine features of topography of a size comparable to that of a single collagen fiber and become activated by the contact with topographic features.     

Synergistic and Hierarchical Adhesive and Topographic Guidance of BHK Cells

Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 μm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 μm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-μm-wide adhesive tracks and to 5-μm-wide, 6-μm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-μm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.     

Structural studies of the O-specific polysaccharide from Serratia marcescens N.C.T.C. 1377

The putative O-specific polysaccharide of Serratia marcescens N.C.T.C. 1377 is a partially acetylated glucorhamnan. By means of 1H- and 12C-n.m.r. spectroscopy, methylation analysis, and periodate oxidation, it was shown that the polymer has a disaccharide repeating-unit for which the following structure is proposed: leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-L-Rhap-(1-leads. O-Acetyl groups are probably located at C-2 of the rhamnopyranosyl residues. Except for the extent of O-acetylation, the polysaccharide is identical with the corresponding product from S. marcescens Bizio (A.T.C.C. 264), for which a different structure has previously been proposed.     

Biannual breeding and moult-breeding overlap of the Chestnut-bellied Starling Spreo pulcher

At Kano, Nigeria, the Chestnut-bellied Starling Spreo pulcher has two distinct breeding seasons, a long one from the late dry season to the early rains (February to June) and a short one in September, October and rarely November following the rains. Males have enlarged testes from February to October, so the breeding periods are probably determined by the reproductive condition of the females. Most individuals breed in both periods but some breed only in the pre-rains period. Adult wing-moult begins in March and April, overlapping with any breeding attempts in the latter half of the pre-rains session; some birds continue to moult into the post-rains session. Post-juvenal wing-moult is from August to January for birds reared in the previous pre-rains period and is simultaneous with adult moult for birds reared in the post-rains period. An increase in ambient temperature may be the most important proximate cue determining the pre-rains and also the post-rains breeding periods. A correlation between moult-breeding overlap and co-operative breeding for some tropical birds is suggested and discussed briefly.     

Distribution and Physiology of Aerobic Bacteria Containing Bacteriochlorophyll a on the East and West Coasts of Australia

Aerobic heterotrophic bacteria containing bacteriochlorophyll were isolated from specimens from a wide variety of marine environments on the west (Shark Bay, Lake Clifton, Lake Heyward, and Perth) and east (near Townsville and Brisbane) coasts of Australia. The bacteria were found in a high proportion (10 to 30%) of the total heterotrophic bacterial strains isolated from marine algae, seagrasses, stromatolites, the epiphytes on stromatolites, seawater, and sands; in some cases they constituted up to 49% of the total. This is much higher than the previous report of 6% from Japan. A high percentage, 13%, was also found in the seawater of Hamelin Pool, at Shark Bay, where the salinity was 66%. The number of these bacteria was generally low in seawater and sands, with a few exceptions. There were no aerobic bacteriochlorophyll-containing bacteria on sponges or corals. The isolated strains were orange or pink, and most had absorption maxima around 800 and 850 to 870 nm, the latter range being the absorption of bacteriochlorophyll a in vivo. The maximum bacteriochlorophyll content was 1 nmol/mg (dry weight) of bacterial cells. Most of the bacteria did not grow phototrophically under anaerobic conditions in a broth medium containing succinate. Cells and cell extracts grown under aerobic conditions had photochemical activities such as reversible photooxidations of the reaction center and cytochrome(s). Some strains showed denitrifying activity. The optimal salinity for bacterial growth varied between strains.     

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.     

Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants.     

Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.