Search

Transposon-excision revertants with Tn4001 were isolated and analyzed to determine the importance of the Mycoplasma pneumoniae crl gene in cytadherence. Analysis of transposon-excision revertants indicated the direct correlation between the loss of Tn4001, reacquisition of HMW1-HMW3 and restoration of the wild-type phenotype in Mycoplasma pneumoniae. Furthermore, detailed analysis of the crl gene indicated the presence of an open reading frame encoding the HMW2 cytadherence-associated protein.

Three thermotolerant Bacillus isolates from the Philippines - identified as Bacillus subtilis (1280), Bacillus velezensis (1573), and Bacillus amyloliquefaciens (10105) - exhibited cellulase activity in carboxymethylcellulose (CMC) agar plate. Moreover, the expected size (approximately 1,500 bp) putative endoglucanase gene was amplified from all three local isolates. The endoglucanase gene amplified from a Bacillus licheniformis isolate (1320) was also used in the gene sequence analysis. A colorimetric dinitrosalicylic (DNS) acid assay revealed that the total endoglucanase activity of B. velezensis 1573 at 0.168 µM reducing sugar/min is significantly higher than the endoglucanase activity of the B. subtilis 1280 at 0.0673 µM rs/min and B. amyloliquefaciens 10105 at 0.0579 µM rs/min. Endoglucanase gene sequence analysis showed the highest pairwise identity between the endoglucanase gene of B. velezensis 1573 and B. amyloliquefaciens 10105 at 97.4%. The endoglucanase gene of B. licheniformis 1320 has the lowest pairwise identity at 74.6%, 76.2%, and 75.4% with B. amyloliquefaciens 10105, B. subtilis 1280, and B. velezensis 1573, respectively. Predicted amino acid sequences of the endoglucanases revealed variations at the signal peptide, catalytic core (CC) domain, linker, and carbohydrate-binding module (CBM). The endoglucanase of B. subtilis 1280 has a 506D insertion at the CBM domain. None of these variations, however, are located in regions implicated in catalytic reaction and substrate binding. Although the predicted protein structures using MODELLER revealed endoglucanases with good fit and high homology, variations in amino acid sequences may have resulted in the differences in enzyme activity and stability. The study generated gene and protein sequences as well as amplified genes that can be used as a starting template for enzyme improvement by rational design and site-specific mutagenesis or directed evolution using DNA shuffling. [ABSTRACT FROM AUTHOR]

Genes for the hydrocarbon-degrading enzymes - alkane 1-monooxygenase (AlkM) and catechol 1,2-dioxygenase (CatA) - were amplified from three environmental strains of Acinetobacter baumannii from Pasig River and oil sludge sample. Each gene has been previously cloned into a pBAD/Thio-TOPO plasmid expression vector and the recombinant pBAD/Thio-TOPO plasmids were introduced into E. coli TOP10 cells via transformation. This study was conducted to determine if AlkM and CatA proteins will be expressed by the recombinant E. coli TOP10 cells, to determine the optimized conditions for protein expression, and to perform purification of expressed AlkM and CatA fusion proteins. The pilot expression resulted in the production of the putative 62 kDa AlkM-thioredoxin and the 50 kDa CatA-thioredoxin fusion proteins, and the identity of the expressed proteins was verified to be AlkM and CatA using western blotting. The study showed that best AlkM and CatA expression was observed when recombinant cells were grown in Super broth using 6-h post-induction time with at least 0.2% arabinose. Purified AlkM and CatA fusion proteins were obtained using affinity chromatography and subsequent size exclusion chromatography with Sephadex G-75 column. The preliminary activity of the purified CatA fusion enzyme observed at 1.128 units/mg was much lower than activities reported for other species, while an AlkM activity assay could not be performed because AlkM fusion proteins from recombinant E. coli did not solubilize after purification. These suggest the need to perform an extensive study to address AlkM solubility problem and optimize parameters for enzyme activity of expressed CatA and AlkM. Successful expression of AlkM and Cat A genes from a human bacterial pathogen in non-pathogenic E. coli host cells is an initial step to the possible use of recombinant cells in producing AlkM and CatA enzymes from a pathogenic bacterial species in addressing problems of oil contamination. [ABSTRACT FROM AUTHOR]

Accidental fuel oil spills and improper disposal of toxic waste oil into waterways have caused serious pollution problems worldwide. This study is focused on the use of microorganisms and enzymes they produce to address problems of waste oil pollution. Complete genes for enzymes alkane 1-monooxygenase (AlkM) and catechol 1,2-dioxygenase (CatA) implicated in bunker oil degradation were amplified and sequenced from two new strains of Acinetobacter baumannii (S341 and S354) isolated from the polluted Pasig River in the Philippines. Genes for both AlkM and CatA enzymes and predicted proteins exhibited almost identical sequences with the type strain A. baumannii (ATCC 19606). Sequence identity (80%) of alkM genes is lower than the sequence identity (90%) of predicted enzymes between A. baumannii environmental strains and most of the other species under the same genus. Genes for AlkM (or for enzymes with equivalent functions as AlkM like AlkB) and CatA from species that do not belong to the genus Acinetobacter exhibited very low gene and protein sequence identities (49.8-54.6%) with corresponding genes and proteins in strains S341 and S354. Stretches of conserved amino acids in the predicted protein sequences (eight histidine motifs in AlkM/AlkB) and a ferric ion ligated by four amino acids (Tyr164, Tyr200, His224, and His226) in the CatA protein were observed, consistent with previous reports. These conserved regions may be crucial in protein function. The study also reports the successful cloning, in the correct orientation, of complete alkM and catA genes from A. baumannii strains S341 and S354 in pBAD Thio-TOPO plasmid for future expression of the recombinant proteins in non-pathogenic host cells. [ABSTRACT FROM AUTHOR]

The Philippines boasts of diversity in bacterial species that can be explored in producing industrially significant enzymes to help reduce the country's dependence on enzyme importation. This study was focused on four thermotolerant strains of Bacillus pumilus, a species reported to produce serine alkaline protease, lipase, pectate lyase, and laccase. Enzyme gene targeted polymerase chain reaction (PCR) and gene sequence analysis confirmed the presence of four enzyme genes in all strains studied. Preliminary enzyme assays revealed that all strains except 1271, tested positive for protease, pectate lyase, and lipase activities. Only strain 1271, however, exhibited positive laccase activity. These initial results could be the basis for pursuing studies on laccase using strain 1271 and studies on protease, pectate lyase and lipase enzymes from the other three strains. Partial amino acid sequence of the serine alkaline protease gene in strain 1271 revealed five amino acid variations from the other three strains and the variations resulted in protein conformational changes. The translated partial laccase gene sequence of strain 1271 exhibited ten amino acid variations from partial laccase enzymes of the other three strains but the amino acid variations did not result in enzyme conformation change. The amino acid sequences in the complete lipase genes showed five amino acid variations in isolate 1271 compared with enzymes from other three strains but no change in the predicted protein model was observed. Sixteen variations in the amino acid sequences of the pectate lyase enzyme observed among strains resulted in a unique pectate lyase protein conformation for each isolate. The role of enzyme conformation variation as well as other factors that could have led to the enzyme assay results in this study, could be further elucidated with optimized quantitative enzyme assays, gene expression studies and mutagenesis. [ABSTRACT FROM AUTHOR]