Na-Stimulated Transport of l-Methionine in Brevibacterium linens CNRZ 918

The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium levels of its niche.     

Determining [H]Thymidine Incorporation into Bacterioplankton DNA: Improvement of the Method by DNase Treatment

Determination of [H] thymidine incorporation into bacterial DNA versus other macromolecules is usually achieved by NaOH and hot trichloroacetic acid hydrolysis. This procedure was found not to be specific enough. An alternative method founded on DNase treatment is proposed. Under the new method, the fraction of thymidine incorporated into DNA ranged from 10 to 83%.     

Free-living dinitrogen-fixing bacteria isolated from petroleum refinery oily sludge

Dinitrogen-fixing activity (acetylene reduction and N(2) fixation) was found in an oily sludge originating from a petroleum refinery. Two representative dinitrogen-fixing bacterial strains were isolated from this oily waste. Their nitrogenase activity was effective when they were cultivated on sterilized sludge or simple carbon substrates (organic acid salts, sugars). Using the classical methods, these strains could not be unambiguously related to other diazotrophic taxa. The landfarming process is widely used for oily sludge disposal; this study shows that oily sludges are more than a simple carbon input into the soil but that they must also be considered as real sources of dinitrogen-fixing and probably degradative microorganisms.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Rapid characterization of recombinant lambdagt11 clones expressing parasite antigens

Recombinant DNA techniques allow any parasite gene to be isolated, propagated and analysed in great detail. The techniques are now well known and widely used in research on parasite diagnosis, vaccine development and identification of chemotherapeutic targets. There are difficulties, for example in analysing carbohydrate antigens, and in selecting which genes merit the intensive investigation required. In this article john Kelly and Mark Blaxter discuss two simple procedures that allow recombinant clones to be characterized rapidly on the basis of both the parasite DNA sequences that they carry and the parasite antigens that they produce. They take examples from work on antigens of Leishmania donovani.     

Sexual selection and speciation: Issues raised by Hawaiian Drosophila

The Drosophila fauna of Hawaii is extraordinarily diverse, representing about 25% of the world's described species. The most notable characteristics which differentiate the species in Hawaii are morphological and behavioral ones used in courtship and mating. These flies are excellent model species for investigating the evolution of sexually selected traits. Hypotheses regarding the associations between species formation and mating behaviour have been formulated as a result of work on this group, leading to further empirical and theoretical research.     

Physical Analysis of the Campoletis sonorensis Virus Multipartite Genome and Identification of a Family of Tandemly Repeated Elements

This report is an analysis of cross-hybridizing sequences found within the 28 superhelical (SH) DNAs of the multipartite genome of the polydnavirus Campoletis sonorensis virus (CsV). A Southern cross-blot hybridization analysis showed that the majority of CsV EcoRI restriction fragments cross-hybridize to multiple EcoRI fragments. These sequence homologies were analyzed by hybridizing recombinant clones of the CsV SH DNAs B, H, M, and O¹ to Southern blots of undigested CsV DNA, using different hybridization stringencies. The results indicated that homologous regions among the SH DNAs include closely related sequences that are detectable under stringent conditions and related but more diverged sequences which are only detectable under reduced stringencies. A sequence that hybridized to the majority of the CsV SH DNAs was identified and subcloned from the SH DNAs O¹, H, and B. Nucleotide sequence data revealed that these homologous regions contained a family of imperfectly conserved repeated elements. These repeat elements were arranged singly or in direct tandem arrays and had an average length of 540 base pairs. Within the sequenced regions that contained the repeated elements six putative open reading frames were identified. These results show that the CsV genome consists of SH DNAs with complex sequence interrelationships that may have arisen due to multiple recombinational events.     

Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

Summary A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5 ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3 ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.