Synthesis of \u3cem\u3eN\u3c/em\u3e-Acetyl-ᴅ-quinovosamine in \u3cem\u3eRhizobium etli\u3c/em\u3e CE3 is Completed After Its 4-keto-precursor is Linked to a Carrier Lipid

Bacterial O-antigens are synthesized on lipid carriers before being transferred to lipopolysaccharide core structures. Rhizobium etli CE3 lipopolysaccharide is a model for understanding O-antigen biological function. CE3 O-antigen structure and genetics are known. However, proposed enzymology for CE3 O-antigen synthesis has been examined very little in vitro, and even the sugar added to begin the synthesis is uncertain. A model based on mutagenesis studies predicts that 2-acetamido-2,6-dideoxy-d-glucose (QuiNAc) is the first O-antigen sugar and that genes wreV, wreQ and wreU direct QuiNAc synthesis and O-antigen initiation. Previously, synthesis of UDP-QuiNAc was shown to occur in vitro with a WreV orthologue (4,6-hexose dehydratase) and WreQ (4-reductase), but the WreQ catalysis in this conventional deoxyhexose-synthesis pathway was very slow. This seeming deficiency was explained in the present study after WreU transferase activity was examined in vitro. Results fit the prediction that WreU transfers sugar-1-phosphate to bactoprenyl phosphate (BpP) to initiate O-antigen synthesis. Interestingly, WreU demonstrated much higher activity using the product of the WreV catalysis [UDP-4-keto-6-deoxy-GlcNAc (UDP-KdgNAc)] as the sugar-phosphate donor than using UDP-QuiNAc. Furthermore, the WreQ catalysis with WreU-generated BpPP-KdgNAc as the substrate was orders of magnitude faster than with UDP-KdgNAc. The inferred product BpPP-QuiNAc reacted as an acceptor substrate in an in vitro assay for addition of the second O-antigen sugar, mannose. These results imply a novel pathway for 6-deoxyhexose synthesis that may be commonly utilized by bacteria when QuiNAc is the first sugar of a polysaccharide or oligosaccharide repeat unit: UDP-GlcNAc → UDP-KdgNAc → BpPP-KdgNAc → BpPP-QuiNAc

Experimental scheme for qubit and qutrit symmetric informationally complete positive operator-valued measurements using multiport devices

It is crucial for various quantum information processing tasks that the state of a quantum system can be determined reliably and efficiently from general quantum measurements. One important class of measurements for this purpose is symmetric informationally complete positive operator-valued measurements (SIC-POVMs). SIC-POVMs have the advantage of providing an unbiased estimator for the quantum state with the minimal number of outcomes needed for full tomography. By virtue of Naimark's dilation theorem, any POVM can always be realized with a suitable coupling between the system and an auxiliary system and by performing a projective measurement on the joint system. In practice, finding the appropriate coupling is rather non-trivial. Here we propose an experimental design for directly implementing SIC-POVMs using multiport devices and path-encoded qubits and qutrits, the utility of which has recently been demonstrated by several experimental groups around the world. Furthermore, we describe how these multiports can be attained in practice with an integrated photonic system composed of nested linear optical elements.Comment: 7 pages, 5 figures; v2 published versio

Quinol Oxidase Encoded by \u3cem\u3ecyoABCD\u3c/em\u3e in \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42 is Regulated by ActSR and is Crucial for Growth at Low pH or Low Iron Conditions

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5′ sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions

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

A Quinol Oxidase, Encoded by \u3cem\u3ecyoABCD\u3c/em\u3e, Is Utilized to Adapt to Lower O\u3csub\u3e2\u3c/sub\u3e Concentrations in \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42

Bacteria have branched aerobic respiratory chains that terminate at different terminal oxidases. These terminal oxidases have varying properties such as their affinity for oxygen, transcriptional regulation and proton pumping ability. The focus of this study was a quinol oxidase encoded by cyoABCD. Although this oxidase (Cyo) is widespread among bacteria, not much is known about its role in the cell, particularly in bacteria that contain both cytochrome c oxidases and quinol oxidases. Using Rhizobium etli CFN42 as a model organism, a cyo mutant was analysed for its ability to grow in batch cultures at high (21 % O2) and low (1 and 0.1 % O2) ambient oxygen concentrations. In comparison with other oxidase mutants, the cyo mutant had a significantly longer lag phase under low-oxygen conditions. Using a cyo :: lacZ transcriptional fusion, it was shown that cyo expression in the wild type peaks between 1 and 2.5 % O2. In addition, it was shown with quantitative reverse transcriptase PCR that cyoB is upregulated approximately fivefold in 1 % O2 compared with fully aerobic (21 % O2) conditions. Analysis of the cyo mutant during symbiosis with Phaseolous vulgaris indicated that Cyo is utilized during early development of the symbiosis. Although it is commonly thought that Cyo is utilized only at higher oxygen concentrations, the results from this study indicate that Cyo is important for adaptation to and sustained growth under low oxygen

Compounds Exuded by \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e That Induce a Modification of \u3cem\u3eRhizobium etli\u3c/em\u3e Lipopolysaccharide

Exudates released from germinating seeds and roots of a black-seeded bean (Phaseolus vulgaris cv. Midnight Black Turtle Soup) induce an antigenic change in the lipopolysaccharide (LPS) of Rhizobium etli CE3. By spectroscopic analyses and chromatographic comparisons with derived standards, the chemical structures of the aglycone portions of the major inducing molecules from seed exudate were deduced, and they were identified as delphinidin, cyanidin, petunidin, and malvidin. These anthocyanidins were present in seed exudate mainly as glycosides, the chief inducer being delphinidin 3-glucoside. Also present were 3-glucosides of petunidin and malvidin and glycosides of cyanidin and delphinidin. Seed exudate from a bean variety deficient in anthocyanins did not induce the LPS conversion. The ability of root exudate to induce an antigenic change in the LPS was due to compounds other than anthocyanins

\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

Mutations in \u3cem\u3eRhizobium phaseoli\u3c/em\u3e that Lead to Arrested Development of Infection Threads

Two Rhizobium phaseoli mutants, isolated previously by Tn5 mutagenesis, elicited infection threads which ceased development prematurely, usually within root hairs. These infection threads were wide, globular, and otherwise altered in morphology, compared with normal infection threads. Anatomy and division of the root cortical cells during initial stages of nodule morphogenesis appeared normal. However, later nodule differentiation deviated considerably from normal development, and release of bacteria from infection threads was not observed. In tryptone-yeast extract medium the mutants sedimented during growth in shaken cultures and formed rough colonies on agar. Electrophoresis of washed cultures solubilized in dodecyl sulfate revealed that the major carbohydrate band was absent from the mutants. The behavior of this carbohydrate in phenol-water extraction and gel chromatography, its apparent ketodeoxyoctonate content, and its susceptibility to mild acid hydrolysis suggested that it was a lipopolysaccharide. From the results of genetic crosses or reversion analysis, the defect in synthesizing this carbohydrate material and the defect in infection could be attributed to a single mutation in each mutant

Varying the Abundance of O Antigen in \u3cem\u3eRhizobium etli\u3c/em\u3e and Its Effect on Symbiosis with \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamin