A calcium-stimulated serine peptidase from a true-branching cyanobacterium, Westiellopsis ramosa sp. nov.

Unbranched heterocytous cyanobacteria produce a number of serine peptidases. We have characterized several peptidases in the cell-free extracts of a true-branched N2-fixing cyanobacterium, Westiellopsis ramosa sp. nov. Upon substrate-gel zymography of intact filaments and heterocytes, five peptidase bands were resolved, whereas in vegetative cells, a single band was discernible. No band was detected in [Formula: see text]-grown cultures suggesting that the peptidases were present under diazotrophic conditions with much of them confined to heterocytes. Using salt precipitation and chromatography, a caseinolytic peptidase, called Wrp49, was purified which also demonstrated fibrinolytic activity. In SDS-PAGE, the purified peptidase was resolved into 17 and 27 kDa fragments. The enzyme in its native state exhibited Mr ≈ 49 kDa, and digested gelatin in a substrate gel at a corresponding position. The enzyme showed amidolytic activity on a plasmin specific substrate, D-Val-Leu-Lys p-nitroanilide. Moreover, a trypsin specific substrate, N-benzoyl-DL-Arg p-nitroanilide was hydrolyzed at an apparent Km = 0.195 mM and Vmax = 5 × 10-7 M s-1. The enzyme was stable in a wide pH and temperature range. While Ca2+ stimulated the activity; phenylmethane sulfonyl fluoride, leupeptin, EDTA and chelants were inhibitory. The activity of the EDTA-inactivated enzyme was completely restored upon adding Ca2+, suggesting that both compounds competed with each other in modulating the enzyme activity. The enzyme showed similarities with a Ca2+ stimulated subtilisin-like serine peptidase of Anabaena variabilis ATCC 29413, but also presented several unique features of metallopeptidases, such as the chelant's response. Moreover, the N-terminal sequence (MTVENLARTGVGPGWR) did not match with any of the known peptidases.

Phylogenetically distant clade of Nostoc-like taxa with the description of Aliinostoc gen. nov. and Aliinostoc morphoplasticum sp. nov.

Nostoc is a complex and tough genus to differentiate, and its morphological plasticity makes it taxonomically complicated. Its cryptic diversity and almost no distinguishable morphological characteristics make this genus incredibly heterogeneous to evaluate on taxonomic scales. The strain NOS, isolated from a eutrophic water body, is being described as a new genus Aliinostoc with the strain showing motile hormogonia with gas vesicles as an atypical feature, which is currently considered as the diacritical feature of the genus but should be subjected to critical evaluation in the near future. The phylogenetic placement of Aliinostoc along with some other related sequences of Nostoc clearly separated this clade from Nostoc sensu stricto with high bootstrap support and robust topology in all the methods tested, thus providing strong proof of the taxa being representative of a new genus which morphologically appears to be Nostoc-like. Subsequent phylogenetic assessment using the rbcL, psbA, rpoC1 and tufA genes was done with the aim of facilitating future multi-locus studies on the proposed genus for better taxonomic clarity and resolution. Folding of the 16S-23S internal transcribed spacer region and subsequent comparisons with members of the genera Nostoc, Anabaena, Aulosira, Cylindrospermum, Sphaerospermopsis, Raphidiopsis, Desmonostoc and Mojavia gave entirely new secondary structures for the D1-D1' and box-B helix. Clear and separate clustering from Nostoc sensu stricto supports the establishment of Aliinostoc gen. nov. with the type species being Aliinostoc morphoplasticum sp. nov. in accordance with the International Code of Nomenclature for algae, fungi and plants.

Role of bacterioferritin comigratory protein and glutathione peroxidase-reductase system in promoting bentazone tolerance in a mutant of Synechococcus elongatus PCC7942.

In this article, we describe the modifications in the antioxidant system of Synechococcus elongatus PCC7942 mutant Mu2 capable of growing at five times higher concentration of bentazone than wild type. Nevertheless, in both the strains, bentazone almost identically induced light-dependent H(2)O(2) production and its extracellular release. However unlike the wild type, peroxide produced upon prolong bentazone incubation was immediately degraded in Mu2. Consequently, the lipid peroxidation activity was also kept low. With prolong incubation of bentazone the mutant displayed a steady increase in glutathione peroxidase-reductase enzyme activities and reduced glutathione content, respectively, by 60% and 130%, favoring an efficient detoxification of bentazone-produced H(2)O(2). Catalase-peroxidase and glutathione S-transferase, though present, remained ineffective in rendering bentazone tolerance. In-gel assays of glutathione S-transferase and glutathione reductase revealed presence of between four and five oligomeric states with mobility shifts. One oligomeric form each enzyme in wild-type strain disappeared upon bentazone treatment. Upon two-dimensional electrophoresis and MALDI-TOF/TOF, a bacterioferritin comigratory protein (peroxiredoxin Q) was found to be already highly expressed in Mu2; whereas in wild type, its level increased only upon bentazone exposure. The bcp transcript pool in WT was relatively low but increased with bentazone, whereas Mu2 exhibited high bcp mRNA even without herbicide. Bacterioferritin comigratory protein and glutathione peroxidase-reductase appear to be responsible for detoxification of bentazone-derived peroxide in Mu2.

A bentazone-resistant mutant of cyanobacterium, Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stress.

A bentazone-resistant mutant of Synechococcus elongatus PCC7942, called Mu2, tolerated elevated NaCl concentrations. As bentazone and bromoxynil exhibit similar mechanism of action, we investigated whether the mutant also toleratedbromoxynil and found it to be true. The line of investigation was then whether the acclimation strategy for the three stressors, bentazone, bromoxynil and NaCl was same or different. The cellular contents of malondialdehyde, hydrogen peroxide and superoxide increased in wild type strain following all the treatments suggesting their toxicities due to oxidative response. Notwithstanding, there were apparently different anti-oxidative measures pertaining to the herbicide and salinity stress. Glutathione contents and activities of superoxide dismutase, catalase-peroxidase, glutathione S-transferase and glutathione reductase decreased under NaCl, whereas bromoxynil affected only glutathione S-transferase reductase. Moreover, in-gel assays revealed that bromoxynil promoted appearance of isozymes of catalase-peroxidase, while NaCl induced such response only for superoxide dismutase. On the other hand, in Mu2, glutathione peroxidase-reductase and glutathione showed upward trend after bromoxynil exposure, whereas NaCl raised peroxidase and superoxide dismutase. Proteome comparison revealed peroxiredoxin Q to be highly expressed in wild type strain under bromoxynil, whereas NaCl favoured flavodoxin over-expression. Their amounts were already high in Mu2. We suggest that Mu2 acclimatized to bromoxynil in a manner similar to bentazone by upgrading peroxiredoxin Q and glutathione peroxidase-reductase. Conversely, for NaCl it devised another mechanism involving peroxidase and superoxide dismutase, and flavodoxin.

Partial Purification and Characterization of an Elastase-inhibitory Lipid Derivative from a Cyanobacterium, Microcystis Ku2.

A unicyanobacterial isolate of cyanobacterium, identified as Microcystis Ku2, produced a mammalian elastase-inhibitory lipid derivative. Protease inhibitors in cyanobacteria are unequivocally peptides. Since this metabolite appeared in lipid phase, we worked on a hypothesis that whether metabolite other than peptides could be responsible for the characteristic inhibition. It was purified by saponification and reverse phase column chromatography. The resulting compound was tentatively characterized as a glycolipid with structure of sulfated di-pentose derivative of ceramide (MW = 956 Da). The apparent IC(50) for elastase was 1.3 µM.

PCR-based detection of microcystin-producing cyanobacterial blooms from Central India.

Microcystin synthetase-gene-specific primers were used to identify hepatotoxic microcystin producing genotypes in six Microcystis spp.-dominant water blooms. Four blooms gave positive PCR reaction. They produced microcystin-RR and -LR amounting to 0.037 to 0.095% of the dry mass.

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806.

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.