Nematodes of the family Stilbonematinae are known for their hlghly specific association with ectosymbiotic bacteria. These worms are members of the meiofauna in marine, sulfide-rich sediments, where they migrate around the redox boundary layer. In this study, bacterial ectosymbionts of 2 species of marine nematodes, Stilbonema sp. and Laxus oneistus, were shown to be capable of the respiratory reduction of nitrate and nitrite (denitrification). The use of these alternative electron acceptors to oxygen by the bacteria allows the anlmals to migrate into the deeper, anoxic sedirnents, where they can exploit the sulfide-rich patches of the deeper sediment layers. The accumulation of thiols (sulfide, thiosulfate, sulfate and glutathione) in body tissues of the worms was determined following incubation in the presence of various electron donors (sulfide, thiosulfate) and acceptors (nitrate). In their chemoautotrophic metabolic potential, the ectosymbionts of the 2 nematode species were found to resemble the phylogenetically related, intracellular symbionts of macrofaunal hosts of deep-sea hydrothermal vents and other sulfide-rich habitats.

The nucleotide sequence of a 2.8 kb DNA segment containing an endoglucanase gene (end1) from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from its own regulatory region in Escherichia coli and three putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. The complete amino acid sequence (547 residues) was deduced and homology with the Clostridium thermocellum celE gene product (EGE) was demonstrated. The endoglucanase contained a typical amino-terminal signal sequence and five repeated sequences (PDPTPVD) between amino acids 412-447. The endoglucanase showed relatively high endoglucanase activity against endoglucanase-specific substrates with beta 1-4 linkages but low activity against xylan and an exoglucanase-specific substrate, p-nitrophenyl-beta-D-cellobioside.

The nucleotide sequence of a 2.314 kb DNA segment containing a gene (ced1) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulosa endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Ced1 showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Ced1 enzyme released cellobiose from p-nitrophenyl-beta-D-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Ced1 enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent Mr of approximately 61,000. The calculated Mr of the ced1 gene product was 61,023.

This study relates to the development of an alkaliphilic, thermotolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium continuously over-produces flagellin. To harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene, which codes for flagellin, was inactivated on the chromosome giving rise to the B. halodurans BhFC01 mutant. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-1 subtype C gp120 epitope were, respectively, incorporated into this site as in-frame fusions. The peptides were found to be successfully displayed on the cell surface and functional through metal binding and immunological studies, respectively.

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