Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

Sequence and expression of a microspore cDNA clone with homology to a ribosomal protein

A cDNA library was made to RNA from corn anthers containing developing pollen at the uninucleate microspore stage. A randomly selected clone from this library which contained an insert (531 bp) was isolated and sequenced. An open reading frame of 330 bp was located. Computer alignments of the putative amino acid sequence with sequences from GenBank and the SwissProt protein databases indicated homology to L12, an acidic ribosomal protein. RNA blot analysis showed highest levels of this mRNA in mature pollen. The significance of this observation in light of the known biochemistry of ribosome synthesis in developing pollen is discussed.

The Gene CBO0515 from Clostridium botulinum Strain Hall A Encodes the Rare Enzyme N5-(Carboxyethyl) Ornithine Synthase, EC 1.5.1.24

ABSTRACT Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N 5-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum.

Symbiotic “Archaezoa” of the Primitive Termite Mastotermes darwiniensis Still Play a Role in Cellulase Production

ABSTRACT The relictual Mastotermes darwiniensis is one of the world's most destructive termites. Like all phylogenetically basal termites, it possesses protozoa in its hindgut, which are believed to help it digest wood. L. Li, J. Frohlich, P. Pfeiffer, and H. Konig (Eukaryot. Cell 2:1091-1098, 2003) recently cloned the genes encoding cellulases from the protozoa of M. darwiniensis; however, they claimed that these genes are essentially inactive, not contributing significantly to cellulose digestion. Instead, they suggested that the protozoa sequester enzymes produced by the termite in its salivary glands and use these to degrade cellulose in the hindgut. We tested this idea by performing gel filtration of enzymes in extracts of the hindgut, as well as in a combination of the salivary glands, foregut, and midgut. Three major cellulases were found in the hindgut, each of which had a larger molecular size than termite-derived salivary gland enzymes. N-terminal amino acid sequencing of one of the hindgut-derived enzymes showed that it was identical to the putative amino acid sequence of one mRNA sequence isolated by Li et al. (Eukaryot. Cell 2:1091-1098, 2003). The overall activity of the hindgut cellulases was found to be of approximately equal magnitude to the termite-derived cellulases detected in the mixture of salivary gland, foregut, and midguts. Based on these results, we conclude that, contrary to Li et al. (Eukaryot. Cell 2:1091-1098, 2003), the hindgut protozoan fauna of M. darwiniensis actively produce cellulases, which play an important role in cellulose digestion of the host termite.

Antigenic variation of a cysteine-rich protein in Giardia lamblia.

The WB isolate of Giardia lamblia expresses a cysteine-rich 170-kD surface antigen (CRP170) that undergoes antigenic variation. An (6E7), cytotoxic for isolates expressing CRP170, was used in another study to select antigenic variants from clones of the WB isolate of Giardia. CRP170 was replaced by surface-labeled bands ranging in size from approximately 50 to 170 kD. In this study, mAb 6E7 was used to isolate a 1-kb portion of the CRP170 gene (M2-1) from a lambda gt 11 expression library. The M2-1 clone hybridized to a 5.4-kb transcript from isolates expressing CRP170 but did not hybridize to RNA from antigenic variants. Evidence was found for frequent rearrangements at the CRP170 gene locus. DNA sequencing of the M2-1 clone revealed the presence of long tandem repeats. The putative amino acid sequence of M2-1 reveals a 12% cysteine content, and CRP170 is readily labeled in vivo with cysteine.