Journal of Microbiology

Research Support, Non-U.S. Gov't

In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120: Sonika Kumari , Akhilesh Kumar Chaurasia; J. Microbiol. 2015;53(12):837-846. Published online December 2, 2015; DOI: https://doi.org/10.1007/s12275-015-5281-3

Abstract: Signal peptide (SP) plays a pivotal role in protein translocation. Lipoprotein- and twin arginine translocase (Tat) dependent signal peptides were studied in All3087, a homolog of competence protein of Synechocystis PCC6803 and in two putative alkaline phosphatases (ALPs, Alr2234 and Alr4976), respectively. In silico analysis of All3087 is shown to possess the characteristics feature of competence proteins such as helix-hairpin-helix, N and C-terminal HKD endonuclease domain, calcium binding domain and N-terminal lipoprotein signal peptide. The SP recognition-cleavage site in All3087 was predicted (AIA-AC) using SignalP while further in-depth analysis using Pred-Lipo and WebLogo analysis for consensus sequence showed it as IAA-C. Activities of putative ALPs were confirmed by heterologous overexpression, activity assessment and zymogram analysis. ALP activity in Anabaena remains cell bound in log-phase, but during late log/stationary phase, an enhanced ALP activity was detected in extracellular milieu. The enhancement of ALP activity during stationary phase was not only due to inorganic phosphate limitation but also contributed by the presence of novel bipartite Tat-SP. The Tat signal transported the folded active ALPs to the membrane, followed by anchoring into the membrane and successive cleavage enabling transportation of the ALPs to the extracellular milieu, because of bipartite architecture and processing of transit Tat-SP.

Inhibition of purine nucleoside phosphorylase (PNP) in micrococcus luteus phenylglyoxal: Choi , Hye Seon; J. Microbiol. 1996;34(3):270-273.

Abstract: Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30℃ the bimolecular rate constant for the modification was 0.015 min^-1 mM^-1 in 50 mM NaHCO₃buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope enzyme. Preincuation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

Effect of initial pH and L-arginine on the composition of fatty acids of streptomyces viridochromogenes: Oh, Choong Hun , Jung, Sang Oun , Pyee, Jae Ho , Kim, Jae Heon; J. Microbiol. 1996;34(4):316-319.

Abstract: Mycelia of Streptomyces viridochromogenes grown under different pH were analysed for the fatty acid composition. The low relative proportion of 12-methyltetradecanoic acid and the high relative proportion of palmitic acid were characteristic for the young culture under slight acidic pH that caused delay of the aerial mycelium formation. The addition of L-arginine to the culture medium enabled an arginine auxotroph with bald phenotype to have the fatty acid composition similar to that of the wild type and to develop aerial mycelium. The ratio of 12-methyltetradecanoic acid to palmitic acid might be used as a parameter to explain the optimum growth in the respect of membrane fluidity.

Molecular cloning of the arginine biosynthetic genes from corynebacterium glutamicum: Chun, Jae Yeon , Jung, Sam Il , Ko, Soon Young , Park, Mee Young , Kim, Soo Young , Lee, Heung Shickc , Cheon, Choong Ill , Min, Kyung Hee , Lee, Myeong Sok; J. Microbiol. 1996;34(4):355-362.

Abstract: Complementation cloning of the argC, E, B D, F, and G genes in Corynebacterium glutamicum was done by transforming the genomic DNA library into the corresponding arginine auxotrophs fo Escherichia coli. Recombinant plasmids containing 6.7 kb and 4.8kb fragments complementing the E. coli arg B mutant were also able to complement the E. coli argC, E, A, D, and F mutants, indicating the clustered organization of the arginine biosynthetic genes within the cloned DNA fragments. The insert DNA fragments in the recombinant plasmids, named pRB1 AND pRB2, were physically mapped with several restriction enzymes. By further subcloning the entire DNA fragment containing the functions and by complementation analysis, we located the arg genes in the order of ACEBDF on the restriction map. We also determined the DNA nucleotide sequence of the fragment and report here the sequence of the argB gene. When compared to that with the nutant strain, higherh enzyme activity of N-acetylglutamate kinase was detacted in the extract of the mutant carrying the plasmid containing the putative argB gene, indicating that the plasmid contains a functional argB gene. Deduced amino acid sequence of the argB gene shows45%, 38%, and 25% identity to that from Bacillus strearothermophilus, Bacillus substilus, and E. coli respectively. Our long term goal is genetically engineering C. glutamicum which produces more arginine than a wild type strain does.

Chemical Midification of Purin Nucleoside Phosphorulase in Serratia marcescens: Choi , Hye Seon; J. Microbiol. 1998;36(2):74-79.

Abstract: Serratia marcescens purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been previously described(Choi, H.S. 1998. Biosci. Biotechnol. Biochem. 62, 667-671). Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time dependent manner by phenylglyoxal or diethylpyrocarbonate (DEPC). There was a linear relationship between the observed rate of inactivation and the phenylglyoxal or DEPC concentration. At 30℃ the bimolecular rate constant for the modification was 0.22 mM^-1 min^-1 in 50 mM NaHCO_3 buffer, pH 7.5, for phenylglyoxal and 1.33 mM^-1min^-1 in 50 mM sodium cotrate, pH 6.0, for DEPC. Preincubation with saturated solutions of substrates protected the enzyme from inhibition by kphenylglyoxal and DEPC, indicating that reactions with these reagents were directed at arginyl and histidyl residues, respectively, which are essential for the catalytic function of the enzyme.

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