Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant

A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil. Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm). An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R. japonicum 3I1b110 that were tested, except one, mutant strain HS123. Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots. Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface. Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin. Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1. A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not. Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage. Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition. These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.     

Host recognition in the Rhizobium-soybean symbiosis: detection of a protein factor in soybean root exudate which is involved in the nodulation process

The mechanism of host-symbiont recognition in the soybean-Rhizobium symbiosis was investigated utilizing mutants of R. japonicum defective in nodulation. Soybeans were grown in clear plastic growth pouches allowing the identification of the area on the root most susceptible to Rhizobium nodulation; the area between the root tip (RT) and smallest emergent root hair (SERH). The location of nodules in relation to this developing zone is an indication of the rate of nodule initiation. Nodules were scored as to the distance from the RT mark made at the time of inoculation. Seventy-eight per cent of the plants nodulate above the RT mark when inoculated with the wild type R. japonicum strain 3I1b110 with the average distance of the uppermost nodule being approximately 2 millimeters above the RT mark. These data indicate that the wild type strain initiates nodulation rapidly within the RT-SERH zone following inoculation. However, inoculation with the slow-to-nodulate mutant strain HS111 resulted in 100% of the plants nodulating only below the RT mark with the average distance of the uppermost nodule being approximately 56 millimeters below the RT mark. Thus, mutant strain HS111 is defective in the ability to rapidly initiate infection leading to nodulation within the RT-SERH zone. The location of the nodules suggest that stain HS111 must `adapt' to the root environment before nodulation can occur. To test this, strain HS111 was incubated in soybean root exudate prior to inoculation. In this case, 68% of the plants nodulated above the RT mark with the average distance of the uppermost nodule being approximately 1 millimeter below the RT mark. Experiments indicated that the change in nodule initiation by strain HS111 brought about by incubation in soybean root exudate was due to a phenotypic, rather than a genotypic change. The half-time of root exudate incubation for strain HS111 necessary for optimal nodulation enhancement was less than 6 hours. Heat sensitivity and trypsin sensitivity of the nodulation enhancement factor(s) in soybean root exudate indicate a protein was involved in the reversal of the delay in nodulation by mutant strain HS111.     

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl

Summary The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

Expression of a subgenomic retroviral messenger RNA

When mRNA for avian retroviral envelope glycoprotein (env) was injected into cells transformed by env-deficient Bryan Rous sarcoma virus, the env deficiency of the injected cells was complemented to allow the release of transforming virus for up to 40 hr. When virus spread within the injected culture was allowed to occur, a second phase of transforming virus production by the injected culture began approximately 2 days following injection, continued for many days and often increased to titers well above those seen soon after injection. The requirement for virus spread, along with the genetic properties of virus released long after injection, supported the hypothesis that the second phase of virus production resulted when injected env mRNA was packaged into virus released by injected cells. When this virus infected other cells within the culture the env mRNA was reverse-transcribed to form a subgenomic, proviral-like molecule able to direct the synthesis of env mRNA. Accordingly, it was shown that neither DNA nor full genomic viral RNA contaminating injected mRNA preparations could account for the results. Evidence that an mRNA can be reverse-transcribed into an active, proviral-like molecule may be of importance in the relationship between retroviruses and their hosts.     

The fine structure of distal receptors on the labium of the aphid,Brevicoryne brassicae L. (Homoptera)

Summary Short peg receptors located at the distal tip of the aphid labium have the structure of mechanoreceptors. Each peg is innervated by a single sensory nerve which is anchored eccentrically to a basal cuticular tube and terminates in electron-dense material in the base of the peg. The arrangement and eccentric insertion of the eight pegs in the labial wall on one side of the stylet groove, with the eccentric insertions of their innervating neurones, provide a mirror image of the receptors on the opposite side. On the basis of a comparison of the structure of these receptors with that of tactile receptors for which electrophysiological data on sensitivity are available, it is possible to predict that the receptors detect both surface contact (pressure) and surface profile; and that the bilateral symmetry in the receptor arrangement facilitates the detection of vein contours which are preferred settling sites on the leaf. The structure of the dendritic terminal and its insertion is that of a well reinforced cytoskeleton designed to transmit tension to the cell membrane, in agreement with the concept that transduction is a membrane related phenomenon. The distal microtubules, fifty per-cent of which originate as well as terminate in the tubular body, are packed in electron-dense material which binds to the cell membrane. The membrane in turn is attached to cuticular components of the receptor. Abrupt changes in dimension of the dendritic outer segment may be designed to modulate the conduction of a membrane potential. On the other hand, lack of continuity in the microtubules makes these organelles poor candidates for the transduction of excitation from a distal site of stimulation to a proximal region.Supported by operating grants Nos. A 6063 and A 9856 from NRCC     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Chemical control of interstrain competition for soybean nodulation by Bradyrhizobium japonicum.

Previous research has shown that a significant limitation to the agricultural use of improved rhizobial inoculant strains is competition from the indigenous soil population. In this work, we sought to test whether chemical inhibitors of flavonoid-induced nod gene expression in Bradyrhizobium japonicum could be identified and utilized to affect interstrain competition for nodulation of soybeans. Approximately 1,000 structural and functional analogs of the known, natural inducers of nod gene expression were tested on six strains of B. japonicum containing a nodY-lacZ fusion. We successfully identified effective inhibitors of nodY expression. The addition of the inhibitor 7-hydroxy-5-methylflavone significantly inhibited nodulation by a sensitive strain and could be used to effectively manipulate the competition between strains for soybean nodulation. However, this work also uncovered significant limitations for the practical use of this methodology. For example, despite the almost universal induction response to the identified natural inducers, there was a wide variability among strains in their response to any specific inhibitor. Given this unexpected variability, the cost of registration of an agronomic chemical, and the potential for the development of resistant field populations, it is unlikely that chemical inhibitors can be successfully applied to a field situation.