J. P. M. de Vrind

Identification and molecular analysis of the Leptothrix discophora SS‐1 mofA gene, a gene putatively encoding a manganese‐oxidizing protein with copper domains

A small amount of a manganese‐oxidizing protein was purified from spent culture medium of Leptothrix discophora strain SS‐1 and used to raise antibodies (αMOF). Expression libraries of L. discophora were constructed in λgtll and screened with αMOF. DNA inserts from the resulting αMOF‐positive λgt11 clones were used to isolate the corresponding gene (called mofA) from a genomic library. The mofA gene was mapped in the center of a NcoI‐EcoRI restriction fragment of 7262 bp. This fragment was cloned in a low‐copy broad‐host vector. The resulting plasmid (pGBM31) was used in an in vitro transcription‐translation experiment with an Escherichia coli S30 extract. Transcription of the mofA gene could be initiated on its own putative promoter region. Translation resulted in a protein of approximately 180 kD as determined by gel electrophoresis. DNA sequence analysis revealed an open reading frame of 4986 bp preceded by a potential promoter region and a ribosome binding site. The translation product, consisting of ...

Stimulation of Mn2+ Oxidation in Leptothrix discophora SS-1 by Cu2+ and Sequence Analysis of the Region Flanking the Gene Encoding Putative Multicopper Oxidase MofA

The effect of Cu2+ on the Mn2+-oxidizing activity secreted by Leptothrix discophora strain SS-1 was studied. Cu2+ stimulated the activity of spent medium from stationary cultures when added during growth of the bacteria, although cell yield decreased with increasing Cu2+ concentrations. The stimulation was tentatively explained by involvement of the putative multicopper oxidase MofA in Mn2+ oxidation. Cu2+ did not enhance the activity when added directly to spent medium, possibly because the ions had to be incorporated during synthesis of the oxidizing factor. Sequence analysis of the region downstream from the mofA gene resulted in identification of mofB, which encodes a protein with a potential peptidyl-prolyl-cis-trans isomerase site, and mofC, which encodes a protein with a potential heme-binding site. The possibility that mofA, mofB, and mofC belong to one operon, and the possible functions of MofB and MofC, are discussed.The effect of Cu2+ on the Mn2+-oxidizing activity secreted by Leptothrix discophora strain SS-1 was studied. Cu2+ stimulated the activity of spent medium from stationary cultures when added during growth of the bacteria, although cell yield decreased with increasing Cu2+ concentrations. The stimulation was tentatively explained by involvement of the putative multicopper oxidase MofA in Mn2+ oxidation. Cu2+ did not enhance the activity when added directly to spent medium, possibly because the ions had to be incorporated during synthesis of the oxidizing factor. Sequence analysis of the region downstream from the mofA gene resulted in identification of mofB, which encodes a protein with a potential peptidyl-prolyl-cis-trans isomerase site, and mofC, which encodes a protein with a potential heme-binding site. The possibility that mofA, mofB, and mofC belong to one operon, and the possible functions of MofB and MofC, are discussed.

Immunology and organic geochemistry

Abstract Two “conventional” antibody preparations directedn against fractions of soluble macromolecules from shells of the recent bivalve Ensis ensis were allowed to react with shell fragments of a large variety of invertebrates, mainly molluscs. Use was made of a special adaptation of the Enzyme-Linked ImmunoSorbent Assay (ELISA). One antibody preparation, aE1, reacted with a broad variety of taxa, including representatives of nonmolluscan phyla, while the other, aE3, only recognized determinants in taxa closely related to Ensis . Reactions with fossils were obtained with both antisera. By incubation of antibody solutions with etched shell powders of non- Ensis shells more specific preparations were obtained that gave more meaningful systematic results. In an artificial diagenesis experiment carried out with shell fragments of Mercenaria mercenaria the degradation of individual determinants of the soluble matrix could be followed with monoclonal antibodies. These data are related with the amino acid racemization profile. In the light of the available information the possible significance of immunology for organic geochemical research is discussed.

Immunological Studies on Macromolecules from Invertebrate Shells — Recent and Fossil —

The study of macromolecules that are contained in biominerals is relevant for our understanding of the biomineralization process. Moreover, comparative investigations are likely to give valuable systematic information. In our laboratory it was found on two occasions that the antigenic properties of soluble macromolecules from 70 million year old cephalopod shells are still preserved (1,2). Also biochemical analyses carried out elsewhere have revealed that macromolecules from calcified tissues may be preserved over considerable periods of geological time (3–5). Because they are protected from degradation by the mineral phase, these macromolecules are an attractive, albeit hardly explored, source of paleontological and geological information. Both our previous findings and the results of the present study support the view that immunology may become an important tool for the retrieval of the information contained in the macromolecules from biominerals, both fossil and recent. The present investigation concentrates on recent shells of invertebrates and is mainly concerned with the systematic specificity of such immunological reactions.

Calcification in Coccolithophorids

Coccolithophorids, a group of monocellular, photosynthetic marine algae belonging to the class of Haptophyceae Parke and Dixon, are characterized by their ability to form calcified bodyscales, so-called coccoliths. These coccoliths are fine round or oval structures which, on analysis, are composed of CaCO3. The morphology of coccoliths is species-specific and enormous variations in sizes and shapes has been described (cf. Black, 1965; Okada and Mclntyre, 1977). Fossil coccoliths are important biostratigraphical tools. Geographically, nowadays, these coccolithophorids have a wide area of distribution: they have been found in tropical, subtropical as well as in arctic waters (Klaveness and Paasche, 1979) and contribute to a considerable extent to the formation of extensive lime ores in the deepsea. Also in the geological past the coccolithophorids have been partly responsible for mass accumulations of CaCO3, especially in the Cretaceous.