Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

Inoculum size as a factor limiting success of inoculation for biodegradation

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Lactose- and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant DNA technology

Summary Transposon Tn 951-encoded -galactosidase was expressed in Pseudomonas saccharophila and enabled this bacterium to grow on lactose as sole carbon source. In contrast, -galactosidase was not expressed in Alcaligenes eutrophus even if the lacZ gene of Tn 951 was separated from the lacI gene. However, -galactosidase was expressed in A. eutrophus, if a DNA fragment, which was suspected to harbour the promoter of the A. eutrophus poly(3-hydroxybutyric acid)-synthetic genes, was ligated to the promoter probe vector pMC1403, which employs lac Z, Y as reporter genes. Plasmid pPL76, which harboured one of the promoter-lac fusions, enabled A. eutrophus not only to express -galactosidase but also to grow slowly on lactose (doubling time = 25–30 h). Subsequently, the promoter-lac fusion was ligated to Tn5 in pSUP5011 and was inserted into the genome of A. eutrophus H16 and of the glucose-utilizing mutant H16-G⁺1 by applying the suicide plasmid technique. Two recombinant strains, H16-cPL and H16-G⁺1-cPL, which grow with a doubling time of 16–23 h on lactose, were investigated in detail. The cells only utilized the glucose residue of lactose as a carbon source for grouth and excreted galactose into the medium. Only after the Escherichia coli gal operon had been cloned in vector pVK101 and had been mobilized to H16-cPL or H16-G⁺1-cPL, was lactose completely utilized; no galactose was detected in the medium and the growth yields increased twofold. Depending on the orientation of the gal operon in pVK101, the expression of galactokinase seems to be dependent either on the promoter of aminoglycoside phosphotransferase gene (kan) or on the promoter of the tetR gene. Offprint requests to: A. Steinbüchel     

The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii

Summary At water activities (a _w) of 0.998 (no osmoticum) and 0.960 a _w(NaCl), the affinity (K _m) of glycerol transport by Zygosaccharomyces rouxii was 25.6 and 6.4 mmol/l respectively. The maximum uptake rate (V _max) was ca. 2.3 mol/g/min at both a _w's. However, at an a _wof 0.960 using polyethylene glycol (PEG) 400 the K _mand V _max for glycerol transport increased to 61.1 mmol/l and 32.2 mol/g per minute respectively. This suggests that different glycerol transport mechanisms operate during stress by the two osmotica. The addition of uncouplers (2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazine) resulted in the outflow of accumulated [^14C]glycerol from Z. rouxii after on osmotic upshock indicating that an active transport mechanism was operative. The transport mechanism was specific for glycerol since other polyols (mannitol, meso-erythritol and arabitol) had no effect on the uptake rate. During upshock from 0.998 to 0.960 a _w(NaCl), a transient increases in the rate of [¹⁴C]glycerol uptake was observed. However, if PEG 400 was used as osmoticum, the rate of glycerol uptake failed to increase.Offprint requests to: P. J. van Zyl     

Collection and quality of rhesus monkey semen

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.     

The role of phosphatases in signal transduction

The importance of phosphatases in regulating the phosphorylation of proteins involved in cell signaling has been demonstrated by four recent discoveries. First, a new family of receptor-like transmembrane phosphotyrosine phosphatases, highly conserved throughout evolution, was shown to be distributed in a wide variety of tissues. Extensive heterogeneity in the extracellular regions of these molecules points to the existence of a wide diversity of ligands. These ligands are thought to mediate transduction of signals to the cell interior by means of the phosphatase activity occurring within the cytoplasmic domains of the receptor-like transmembrane phosphotyrosine phosphatases. Second, cell-permeable tumor promoters, such as okadaic acid, were shown to be potent phosphatase inhibitors that have multiple effects on signaling pathways. Third, the subunits of the type 2A phosphatase were found to associate with transforming antigens encoded by DNA tumor viruses, indicating a role for phosphatases in mediating abnormal proliferative events. Fourth, several cell-cycle mutants were found to encode phosphatases. This review focuses on the significance of the transmembrane phosphotyrosine phosphatases and on the possible ways in which intracellular phosphatases function in signaling pathways.