ABSTRACT
The primary pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile, recently identified as a novel strain (NW4327) of Pseudoalteromonas agarivorans, produced collagenase which degraded R. odorabile skeletal fibers. We now report the collagenase of P. agarivorans as a metalloprotease which required Ca2+ and Zn2+ as cofactors. The collagenase was a TonB dependent receptor (TBDR) having a carboxypeptidase regulatory like domain (CRLD) in the N-terminal along with an outer membrane (OM) channel superfamily domain. The genes for TBDR sub-components and collagenase formed one unified entity in the genome of P. agarivorans NW4327. This association of a collagenase with a TBDR distinguished it from all known functional collagenases till date and for the first time, established the enzymatic capability of TBDRs. Predicted TBDR model demonstrated only 15% identity with ferripyoverdin receptor and the CRLD displayed merely 24% identity with carboxypeptidase catalytic chain. Presence of signal peptide, lack of transmembrane helices, absence of N-terminal in the cytoplasmic side, extracellular localization and recovery from the culture supernatant implicated that the TBDR was secreted. Stronger binding of the collagenase with marine sponge type IV collagen than type I collagen, revealed through molecular docking, indicated higher specificity of the enzyme towards type IV collagen.
Subject(s)
Aquatic Organisms/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Collagenases/chemistry , Collagenases/metabolism , Gammaproteobacteria/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Aquatic Organisms/enzymology , Chemical Phenomena , Collagenases/isolation & purification , Gammaproteobacteria/enzymology , Ions/chemistry , Metals/chemistry , Models, Molecular , Molecular Conformation , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity RelationshipABSTRACT
The existing techniques for detection of polyhydroxyalkanoates (PHA) in halophilic archaea/bacteria are either imprecise or require prior PHA production before screening. The proposed method involves amplification of the approximately 280-300 bp conserved region of Class III PHA synthase (phaC) gene of halophiles using the primers codehopCF and codehopCR (Han et al. Appl Environ Microb 76:7811-7819, 2010). In this study, the best reaction condition was ascertained after repeated trials. This developed method was tested on nine haloarchaeal and halobacterial type strains and 28 environmental halophilic archaea and bacteria isolated from the salt pans of the east and west coasts of India. 29 strains were found to be phaC-positive, while eight were found to be phaC-negative although they appeared PHA positive through conventional Nile Red staining. 16S rRNA-based phylogenetic analysis identified 9 haloarchaeal and 9 halobacterial species as novel PHA producers. Multiple sequence alignment of the phaC gene-derived amino acid sequences showed that only 7 amino acid residues were conserved within all four classes of phaC enzymes, whereas 61 amino acids were identical among the phaC enzyme specific to the haloarchaeal and halobacterial strains presently investigated. All phaC-positive strains produced PHA in standard nutrient deficient medium, whereas the phaC-negative strains did not accumulate any PHA as detected by gas chromatography and nuclear magnetic resonance analyses, thus proving the precision of the developed method and elimination of false positives seen with the traditional Nile Red staining procedure.
Subject(s)
Acyltransferases/genetics , Archaea/genetics , Archaeal Proteins/genetics , Bacterial Proteins/genetics , Euryarchaeota/genetics , Acyltransferases/chemistry , Amino Acid Sequence , Archaea/enzymology , Archaea/metabolism , Archaeal Proteins/chemistry , Bacterial Proteins/chemistry , Euryarchaeota/enzymology , Euryarchaeota/metabolism , Industrial Microbiology/methods , Molecular Sequence Data , Polyhydroxyalkanoates/biosynthesis , Polymerase Chain Reaction/methodsABSTRACT
Heterotrophic carbon utilizing microbes were acclimatized in the laboratory by inoculating sludge collected from the waste discharge pond of a small-scale rural abattoir in India in a nutrient solution intermittently fed with glucose and ammonium chloride. Cultures of 10 well-developed isolates were selected and grown in a basal medium containing glucose and ammonium chloride. Culture supernatants were periodically analyzed for ammonium nitrogen (NH(4)(+)-N) and chemical oxygen demand (COD). Polyphasic taxonomic study of the most active nitrifier (S18) was done. Half saturation concentration (K(s)), maximum rate of substrate utilization (k), yield coefficient (Y) and decay coefficient (K(d)) were determined from the Lineweaver-Burk plot using the modified Monod equation. S18 was able to remove 97 ± 2% of (NH(4)(+)-N) and 88 ± 3% of COD. Molecular phylogenetic study supported by physiological and biochemical characteristics assigned S18 as Achromobacter xylosoxidans. Nitrification activity of A. xylosoxidans was demonstrated for the first time, while interestingly, the distinctive anaerobic denitrification property was preserved in S18. K (s) values were determined as 232.13 ± 1.5 mg/l for COD reduction and 2.131 ± 1.9 mg/l for NH(4)(+)-N utilization. Yield coefficients obtained were 0.4423 ± 0.1134 mg of MLVSS/mg of COD and 0.2461 ± 0.0793 mg of MLVSS/mg of NH(4)(+)-N while the decay coefficients were 0.0627 ± 0.0013 per day and 0.0514 ± 0.0008 per day, respectively. After a contact period of 24 h, 650 ± 5 mg/l solids were produced when the initial concentration of COD and NH(4)(+)-N were 1820 ± 10 mg/l and 120 ± 5.5 mg/l, respectively. This is the first report on the kinetic coefficients for carbon oxidation and nitrification by a single bacterium isolated from slaughterhouse wastewater.
