Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Enclosure experiment on the control mechanism of planktonic bacterial standing stock

In order to understand the control mechanisms of a large, stable bacterial standing stock, enclosure experiments were conducted in a eutrophic lake, where both bacterial productivity and grazing pressure were very high. Total bacterial number in the different enclosures ranged from 1.2 to 2.7×10⁷ cells mL⁻¹ throughout the experiment. The average bacterial cell production rate estimated from a grazer eliminating experiment was 6.3×10⁵ cells mL⁻¹ h⁻¹. Difference in the bacterial cell production rate between shaded and unshaded enclosures was not apparent. Bacteria showed a reduction in standing stock of only about 25–30% even after the supply of light was cut to 1%. Bacteria in the shaded enclosures then recovered their production rate in the first 12 days of perturbation. Grazing pressure in the shaded enclosures was not less than that for the control. Thus, it was considered a control mechanism of bacterial stable standing stock that the bacteria shifted their organic substrate from extracellular dissolved organic carbon freshly released from phytoplankton to that already stocked in the water column, though it is not known whether the dominant bacteria were the same.     

Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.     

The emergence of transgenic potatoes as commercial products and tools for basic science

The potato has tremendous potential as a transgenic crop and is a good model system by which to analyse metabolic regulation and gene expression. The potato’s difficult genetics, but ease of genetic transformation and its clonal means of propagation make it ideal for applied agricultural molecular genetics. Thus, the next 4 years promise to put the potato (with a diversity of transgenic constructs expressed) in the limelight as many of the first transgenic agricultural products enter the marketplace.     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

Successful transfer of embryos recovered from a cow with chronic cervicitis of mixed aerobic and anaerobic bacterial aetiology a case report

An imported 13-year-old Simmentaler cow was presented with severe purulent cervicitis and endometritis from which Corynebacterium pyogenes , Fusobacterium necrophorum , Bacteroides melaninogenicus and an untyped Clostridium perfringens were isolated. The endometritis responded to treatment but the cervix did not and remained fibrous, continuously patent and purulent. Natural pregnancy was considered unlikely and the cow was prepared as an embryo transfer donor. Eight embryos were recovered and six transferred, resulting in five confirmed pregnancies and four live births.     

The hemolymph coagulation system in invertebrate animals

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 3)--d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.     

Cell envelope phospholipid changes in a moderate halophile during phenotypic adaptation to altered salinity and osmotic stress

Abstract The increased content of negatively-charged phospholipids in membranes of Vibrio costicola grown at high salinities is mediated by increased phospholipid synthesis of phosphatidylglycerol relative to phosphatidylethanolamine. This phenomenon provides a system for investigating the factors involved in triggering and controlling haloadaptation in this moderately halophilic bacterium. We review recent experiments, which show that when subjected to sudden increases in external salinity, V. costicola senses both the absolute NaCl concentration and the magnitude of the salt shift. We show that the latter is sensed at least in part via osmotic pressure effects, since shift-up into sucrose-containing media triggers comparable changes in growth and in phospholipid composition and synthesis.     

An enzymatic assay for acetate in spent bacterial culture supernatants

A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.