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Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria- Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini- Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation. [ABSTRACT FROM AUTHOR]

Since the maize transposon Ac can move to a new location within the genome, it has been used in removing selectable markers in transgenic plants. In this paper, we developed an inducible transposon system to truncate a selectable marker in transgenic plants. In this system, the marker gene was accompanied by the inducible transposon, but one end of the transposon was located in the intron of the marker gene. As an example of a marker gene, we isolated the rice 5-enolpyruvylshikimate-3-phosphate synthase ( epsps) and modified it for glyphosate tolerance. The transposon contained Ac transposase, which fused with the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). This construct was engineered into an expression vector pCAMBIA1300, harboring a hygromycin-resistant gene. The construct was first transformed into rice calli, and transformed plants were selected on hygromycin-containing medium. The stably transformed calli underwent determination for normal transcripts and tolerance to glyphosate. The results were applied to a rice transformation with the same construct, but using glyphosate as the selective agent. By determining the transformation efficiency, T-DNA copy patterns, we demonstrate that the modified epsps could be a suitable selectable marker to create transgenic rice. Furthermore, after obtaining stable transgenic plants and inducing transposition by salicylic acid, the transposon was excised, the marker gene became truncated, and its expression was terminated. This strategy could be applicable to yield self-stabilizing transposon by locating the transposon’s end in the transposase gene’s intron. [ABSTRACT FROM AUTHOR]

A Ca2+/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184–199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296–324 are essential for calmodulin binding. An oligopeptide designed from residues 297–324 formed a stable peptide–calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca2+/calmodulin signaling in A. dactyloides. [Copyright &y& Elsevier]

A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xpl0-infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE-cellulose, heparin-Sepharose 4B and blue-dextran-Sepharose 4B. It is composed of a single polypeptide of M, 96000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A-T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37°C and 8.0, respectively. The Km of ATP is 26 μM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xpl0 RNA polymerase activity was retained. The enzyme activity was inhibited by KC1, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N-Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xpl0 RNA polymerase upon Xpl0 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xpl0 genome and produce two RNA bands. [ABSTRACT FROM AUTHOR]

Three methods, chromatographic, spectrophotometric and tritium-release assay, were used and compared for the assay of deoxycytidylate methyltransferase. All three methods can be used for assay of this enzyme but the tritium-release assay appears to be the most simple and convenient. With the help of this assay the deoxycytidylate methyltransferase has been isolated and purified from sonically disrupted cells of Xp12-infected Xanthomonas oryzae. Using a procedure that involves fractionation with streptomycin sulfate and ammonium sulfate, filtration through Sephadex G-100 and chromatography on DEAE-cellulose, a 214-fold increase in specific activity was obtained. The enzyme displays a narrow pH optimum at 6.0. Among the buffers tested, 6-morphotino-ethane sulfonate with the addition of Mg2+ is the best. The enzyme can utilize dCMP and CMP as a substrate. The enzyme can also convert tetrahydrofolic acid into dihydrofolic acid. The Km value for dCMP is 31.3 μM and the Km value for tetrahydrofolic acid is 71.4 μM. There is no absolute requirement of ions for the activity of the enzyme; however, the presence of ions causes stimulating or inhibiting effects on enzyme activity that are dependent on the variety and concentration of ions used. [ABSTRACT FROM AUTHOR]

The broad-host-range plasmid RP4 was conjugatively transferred from Escherichia coli to Xanthomonas campestris pv. citri strain XAS4501. Low transfer frequency of RP4 (about 6.2×10) into Xanthomonas and deletion of RP4 plasmid in some of the transconjugants have been observed. The deletion regions have been mapped. RP4 and the deletion mutants were genetically studied in both E. coli and Xanthomonas. Since RP4Δ11, one of the deletion types, was transferred by pRK2013 about 200-fold higher than the self-transmission of RP4 into Xanthomonas, a theoretical transmission frequency in range 10 was postulated for RP4. By using cured RP4-harboring Xanthomonas strains as recipient, the expected transfer frequencies have been obtained. The results revealed (i) mutation(s) in RP4 (deletion or point mutation) or in host may be necessary for the residence of RP4 replicons in Xanthomonas, and (ii) the wild-type RP4 harboring Xanthomonas are, but not restrictionless, mutants. A mutation in transcription or translation associated with the regulation of RP4 genes in Xanthomonas has been suggested. [ABSTRACT FROM AUTHOR]

Background: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon-encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites. Results: The resolvase encoded by transposable element ISXc5 from the Gram-negative phytopathogen Xanthomonas campestris shows two features which distinguish it from other known resolvases. First, it is more closely phylogenetically related to invertases than other resolvases. In particular, two functionally important regions seem highly conserved between this resolvase and members of the invertase subfamily. Second, the enzyme exhibits a large extension of its carboxy-terminal domain with unknown function. We purified ISXc5 resolvase and analysed its resolution reaction in vitro. Our biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by γδ resolvase. However, despite its apparent similarity to invertases, we were unable to detect recombination on standard substrates for DNA inversion, in either the presence or absence of Fis. Conclusions: ISXc5 resolvase employs a reaction mechanism which is common to members of the resolvase family. Its position near the evolutionary borderline to invertases and its high degree of identity within two functionally important regions with members of the DNA invertase subfamily suggest that only a few replacements of c... [ABSTRACT FROM AUTHOR]

Abstract: In order to search for new thermophilic microorganisms and their enzymes, bacterial strains from black liquor of brownstock at washing stage of kraft pulping process were screened. Therein a multiple glycosyl hydrolase-producing strain, BL11, was isolated as a dominant species in the xylan-degrading bacterial population and identified as Paenibacillus campinasensis. The bacterial strain used all kinds of saccharides and polysaccharides, except lignin as carbon source and produced multiple extracellular polysaccharide-degrading enzymes, including one xylanase (41kDa), three cellulases (42, 57 and 86kDa), one pectinase (28kDa) and one cyclodextrin glucanotransferase (38kDa). P. campinasensis BL11 lacked lipase and protease activities and was able to grow over a wide range of pH, but it particularly grew well around neutral pH at 55°C. Based on its physiological characteristics, it has strong potential for industrial application and bioresource utilization. [Copyright &y& Elsevier]