The unique root-nodule symbiosis between Rhizobium and the aquatic legume,Neptunia natans (L. f.) Druce

We examined the development of the aquatic N₂-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N₂ fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N₂-fixing root-nodule symbiosis with Neptunia natans.We thank J. Whallon for technical assistance, G. Truchet, J. Vasse, S. Wagener, J. Beaman, F. DeBruijn, F. Ewers, and A. Squartini for helpful comments, and N.N. Prasad and G. Birla for assistance in conducting field observations. This work was supported by the Michigan Agricultural Experiment Station and National Science Foundation grants DIR-8809640 and BIR-9120006 awarded to the MSU Center for Microbial Ecology. This study is dedicated to the memory of Dr. Joseph C. Burton, a friend and colleague who made many contributions to the study of the Rhizobiumlegume symbiosis.     

Independent origins of resistance or susceptibility of parasitic wasps to a defensive symbiont

Insect microbe associations are diverse, widespread, and influential. Among the fitness effects of microbes on their hosts, defense against natural enemies is increasingly recognized as ubiquitous, particularly among those associations involving heritable, yet facultative, bacteria. Protective mutualisms generate complex ecological and coevolutionary dynamics that are only beginning to be elucidated. These depend in part on the degree to which symbiont‐mediated protection exhibits specificity to one or more members of the natural enemy community. Recent findings in a well‐studied defensive mutualism system (i.e., aphids, bacteria, parasitoid wasps) reveal repeated instances of evolution of susceptibility or resistance to defensive bacteria by parasitoids. This study searched for similar patterns in an emerging model system for defensive mutualisms: the interaction of Drosophila, bacteria in the genus Spiroplasma, and wasps that parasitize larval stages of Drosophila. Previous work indicated that three divergent species of parasitic wasps are strongly inhibited by the presence of Spiroplasma in three divergent species of Drosophila, including D. melanogaster. The results of this study uncovered two additional wasp species that are susceptible to Spiroplasma and two that are unaffected by Spiroplasma, implying at least two instances of loss or gain of susceptibility to Spiroplasma among larval parasitoids of Drosophila.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Breeding soundness and sex drive by breed and age in beef bulls used for natural mating

A total of 92 range beef bulls (Hereford = 60; Angus = 32) were given a breeding soundness examination (BSE) and two assessments for sex drive prior to their use in 23 breeding trials employing estrous synchronized females. Bulls were in three age groups: yearlings (n=29), two year olds (n=36), and three year olds and older (n=27). All yearling bulls were virgins, but the majority of the older bulls had previous mating experience. Angus bulls were superior (P<0.01) to Herefords in spermatozoal morphology and BSE score. Scrotal circumference increased with age beyond two years in Angus bulls but not in Herefords. Spermatozoal abnormalities generally decreased with age. BSE scores did not differ significantly among age groups. Apart from number of mounts, measures of sex drive did not differ with age or breed of bulls. This represents qualified justification for the current practice of using the same sex-drive assessment procedures for Bos taurus bulls of various ages and breeds.     

Effect of glutamine on penicillin formation in Penicillium chrysogenum

Summary Resting-cell studies in Penicillium chrysogenum have indicated that penicillin formation is inhibited by glutamine concentrations higher than 1 mM. Total inhibition was obtained with 10 mM glutamine. This action was neither reverted by the amino acid precursors of the antibiotic moiety nor glutamin affected the in vitro activity of the first enzyme of the penicillin formation pathway. The inhibition was prevented by 1 mM glutathione by mechanisms not related to limitation in the glutamine incorporation nor connected with degradation of the tripeptide.     

A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase

Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.     

CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial–mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95''s induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.Recent analysis of the cellular heterogeneity within the tumour mass revealed the existence of cells that share characteristics with stem cells of the tissue of origin.¹ These cells are responsible for the tumour''s resistance to current therapies and therefore provide new perspectives in cancer treatment. Cancer stem cells (CSCs) or tumour-initiating cells (TICs) are characterized by their self-renewal and differentiation capacity, which are assessed by their ability to generate a heterogeneous tumour in immunocompromised mice in serial transplantations.² In pancreatic cancer, those properties were initially shown by cells expressing CD24, CD44 and ESA (epithelial surface antigen).³Pancreatic cancer is the fourth leading cause of cancer-related death in the United States of America.⁴ The highly malignant phenotype of pancreatic ductal adenocarcinoma (PDAC) results from aggressive invasion and early metastatic potential. Epithelial–mesenchymal transition (EMT) is considered to be the first step of metastatic spread. During this process, the tumour cells master the ability to detach from their neighbours and gain motile and invasive properties enabling them to spread via blood or lymph vessels.⁵ As cells undergo EMT, they lose their epithelial features including sheet-like architecture, polarity and E-cadherin expression and gradually gain motility and expression of mesenchymal markers such as N-cadherin, fibronectin and vimentin. Recent studies have uncovered a link between the EMT and the acquisition of stem cell characteristics.^{6, 7} Most growth factors such as TGF-β, HGF, EGF, IGF and FGF are known to trigger EMT.⁸ Interestingly, there is growing evidence that the so-called ‘death receptor'' CD95 (Fas/Apo-1) behaves like a growth factor receptor in cancer cells.^{9, 10, 11}CD95 was first discovered as the initiator of programmed cell death by forming death-inducing signalling complex (DISC, including Fas-associated death domain, FADD and caspase-8/10) upon stimulation with CD95 ligand (CD95L).¹² However, mitogen-activated protein kinases (MAPKs), leading to p38, JNK or extracellular signal-regulated kinase (ERK) 1/2 activation, were also reported to be driven by CD95.^{13, 14} In glioblastoma multiforme (GBM), CD95-induced migration depends on the formation of the so-called phosphatidyl-inositol 3-kinase (PI3K) activation complex (PAC),^{11, 12} consisting of the Src family kinase (SFK), Yes and p85, the regulatory subunit of PI3K. PAC components, however, differ between cell types, encompassing also other SFKs or the Syk tyrosine kinase.^{15, 16}Here, we show that the expression of CD95 increases in primary PDACs as compared with non-tumour-bearing pancreas and is higher in metastatic pancreatic cells than in primary PDAC. In CSCs isolated from primary PDAC surgical specimens, the expression of CD95 positively correlates with EMT markers. We also identified Sck as the molecular link between CD95 and activation of the PI3K and MAPK pathways. Neutralization of the CD95L reduces PDAC growth and metastasis. The present study defines CD95 and its downstream signalling pathway components as new targets for PDAC therapy.