Diversity and Dynamics of Indigenous \u3cem\u3eRhizobium japonicum\u3c/em\u3e Populations

A simple method, based upon the separation of cellular proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has been devised for distinguishing between isolates of Rhizobium japonicum. Eleven laboratory strains, previously classified into five serogroups, were analyzed by gel electrophoresis. Groups determined subjectively according to protein patterns matched the serogroups, with one exception. Most strains within serogroups could be distinguished from one another. For studying the ecology of Rhizobium, an important advantage of this technique compared with serology or phage typing is that it discriminates among previously unencountered indigenous bacterial isolates as well as among known laboratory strains. SDS-gels were used to analyze the Rhizobium population of 500 nodules, sampled throughout the growing season, from soybeans at two different Wisconsin localities. Although the soybeans had been inoculated with laboratory strains of R. japonicum, indigenous R. japonicum predominated. At one location, 19 indigenous gel types were distinguished and classified mainly into four groups. At the other location, 18 gel types, falling mainly into three groups, were detected. The predominance of a particular group varied, in some cases dramatically, depending upon the time and depth of nodule formation

\u3cem\u3eRhizobium leguminosarum\u3c/em\u3e CFN42 Lipopolysaccharide Antigenic Changes Induced by Environmental Conditions

Four monoclonal antibodies were raised against the lipopolysaccharide of Rhizobium leguminosarum bv. phaseoli CFN42 grown in tryptone and yeast extract. Two of these antibodies reacted relatively weakly with the lipopolysaccharide of bacteroids of this strain isolated from bean nodules. Growth ex planta of strain CFN42 at low pH, high temperature, low phosphate, or low oxygen concentration also eliminated binding of one or both of these antibodies. Lipopolysaccharide mobility on gel electrophoresis and reaction with other monoclonal antibodies and polyclonal antiserum indicated that the antigenic changes detected by these two antibodies did not represent major changes in lipopolysaccharide structure. The antigenic changes at low pH were dependent on growth of the bacteria but were independent of nitrogen and carbon sources and the rich or minimal quality of the medium. The Sym plasmid of this strain was not required for the changes induced ex planta. Analysis of bacterial mutants inferred to have truncated O-polysaccharides indicated that part, but not all, of the lipopolysaccharide O-polysaccharide portion was required for binding of these two antibodies. In addition, this analysis suggested that O-polysaccharide structures more distal to lipid A than the epitopes themselves were required for the modifications at low pH that prevented antibody binding. Two mutants were antigenically abnormal, even though they had abundant lipopolysaccharides of apparently normal size. One of these two mutants was constitutively unreactive toward three of the antibodies but indistinguishable from the wild type in symbiotic behavior. The other, whose bacteroids retained an epitope normally greatly diminished in bacteroids, was somewhat impaired in nodulation frequency and nodule development

Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB

Genetic Basis for \u3cem\u3eRhizobium etli\u3c/em\u3e CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis

Infection of Soybean and Pea Nodules by \u3cem\u3eRhizobium\u3c/em\u3e spp. Purine Auxotrophs in the Presence of 5-aminoimidazole-4-Carboxamide Riboside

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth

\u3cem\u3eRhizobium japonicum\u3c/em\u3e Mutants Defective in Symbiotic Nitrogen Fixation

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads

Stretching, embeddedness, and scripts in a sociotechnical transition: explaining the failure of electric mobility at Better Place (2007–2013)

Based on field research, interviews, and participant observation, this study explores the failure of Better Place—a now bankrupt company—to successfully demonstrate and deploy battery swapping stations and electric vehicle charging infrastructure. To do so, it draws from concepts in innovation studies, sociotechnical transitions, management science, organizational studies, and sociology. The study expands upon the notion of “fit-stretch”, which explains how innovations can move from an initial “fit” (with existing user practices, discourses, technical form) to a subsequent “stretch” (as the technology further develops, new functionalities are opened up, etc.) in the process of long-term transitions. It also draws from the “dialectical issue life cycle model” or “triple embeddedness framework” to explain the process whereby incumbent industry actors can introduce defensive innovations to “contain” a new niche from expanding. It lastly incorporates elements from design-driven innovation and organizational learning related to schemas and scripts, concepts that illustrate the vision-dependent and discursive elements of the innovation process. It uses the case study of Better Place to test and build upon these concepts. With a market valuation of more than $2 billion, Better Place was poised to become one of the most innovative companies in the electric mobility market. Yet after operating for five years it declared bankruptcy and saw its assets sold off for less than$500,000. We suggest here that Better Place failed because it “stretched” to the point that it “broke;” that it provoked a defensive response from both old automotive manufacturers (such as General Motors) and new ones (such as Tesla); and that the fantastic nature of its visionary scripts convinced its investors and promoters to unrealistically raise expectations and downplay persistent risks

Role of BacA in Lipopolysaccharide Synthesis, Peptide Transport, and Nodulation by \u3cem\u3eRhizobium\u3c/em\u3e sp. Strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions

Effect of Tilt of the Propeller Axis on the Longitudinal-stability Characteristics of Single-Engine Airplanes

Report presents the results of tests of a model of a single-engine airplane with two different tilts of the propeller axis. The results indicate that on a typical design a 5 degree downward tilt of the propeller axis will considerably reduce the destabilization effects of power. A comparison of the experimental results with those computed by use of existing theory is included. A comparison of the experimental results with those computed by use of existing theory is included. It is shown that the results can be predicted with an accuracy acceptable for preliminary design purposes, particularly at the higher powers where the effects are of significant magnitude

Fine-Structure Map of the Histidine Transport Genes in \u3cem\u3eSalmonella typhimurium\u3c/em\u3e

Afine-structure genetic map of the histidine transport region of the Salmonella typhimurium chromosome was constructed. Twenty-five deletion mutants were isolated and used for dividing the hisJ and hisP genes into 8 and 13 regions respectively. A total of 308 mutations, spontaneous and mutagen induced, have been placed in these regions by deletion mapping. The histidine transport operon is presumed to be constituted of genes dhuA, hisJ, and hisP, and the regulation of the hosP and hisJ genes by dhuA is discussed. The orientation of this operon relative to purF has been established by three-point crosses as being: purF duhA hisJ hisP