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Seaweeds represent some of the most important reservoirs of new remedial compounds for humans. The natural products obtained from seaweeds have received extensive consideration on account of their extraordinary dietary and pharmacology applications, having such antiviral, antifungal, antibacterial, and anticancer properties and so on. Above all, there are a number of natural products that have demonstrated attractive value for the development of novel anticancer agents. This book chapter draws attention on colorectal cancer which is one of the main causes of cancer-related demise of human beings. This chapter also illustrates a range of active natural products extracted from seaweeds that have shown to eliminate or slow the progression of cancer. This also covers the mechanism through which these compounds can induce apoptosis in vitro and in vivo. By considering the ability of compounds present in seaweeds to act against colorectal cancers, this chapter highlights the potential use of seaweeds as anticancer agents.

Cyanobacteria are ancient photosynthetic prokaryotes and serve as a model organism for studies such as photosynthesis and earth elements cycling. Cyanobacteria also termed microbial cell factories, because of their ability to utilize carbon dioxide, as well as sunlight absorption in one way, act as primary producers of the aquatic ecosystem while on the other way the diazotrophic forms fix atmospheric nitrogen in paddy fields. Moreover, the successful invention of several fuels as well as fine chemicals from cyanobacteria is indicative of the advancement in synthetic cyanobacteriology in one hand while on the other knocking the door of the augmented application shortly. Unfortunately, the limited availability of genetic manipulation tools for either at single-gene level or pathway to the whole genome in cyanobacteria compared to other organisms handicaps the fundamental biology researches as well as further application and advancement in synthetic cyanobacteriology. However, the role of genetic tools in tuning gene expression, genome-wide manipulations, and carbon flux redirections is available in cyanobacteria. Furthermore, recently a noteworthy headway evolving to familiarizing novel and efficient genetic manipulations tools such as riboswitches, promoters, ribosome-binding site engineering, small RNA regulatory tools, genome-scale modeling strategies, and clustered regularly interspaced short palindromic repeats-associated nuclease has revolutionized the cyanobacteriology. The present chapter disapprovingly recapitulates the recent advancement on the applications and development as well as technical limitations also for the future projections of the toolboxes for genetic manipulation of cyanobacterial genes/genomes. Besides this, the chapter also briefly discusses the toolkits feasible for large-scale cultivation of cyanobacteria.

Chitin is the most copious nitrogenous biopolymer polysaccharide found in the world following cellulose. It has remarkable biocompatibility, nontoxicity, nonimmunogenicity, good biodegradability, antimicrobial efficacy, and strong wound-healing aptitude, leading to its receiving an immense deal of interest in the medical, pharmaceutical, agricultural, and industrial fields. Massive studies and research work have revealed the strong potential of using chitin and its modified types in the area of medical sciences such as wound management, tissue engineering, drug delivery systems, and periodontal and orthopedic applications. In this chapter, we comprehensively discuss the various aspects of the physical, chemical, and biological nature of chitin along with its use in multiple fields such as biomedical, industrial, and agriculture.

Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, Fremyella diplosiphon, was identified by fatty acid methyl esters (FAME) via direct transesterification. Total lipids in wild type (Fd33) and halotolerant (HSF33-1 and HSF33-2) strains determined by gravimetric analysis yielded 19% cellular dry weight (CDW) for HSF33-1 and 20% CDW for HSF33-2, which were comparable to Fd33 (18% CDW). Gas chromatography-mass spectrometry detected a high ratio of saturated to unsaturated FAMEs (2.48-2.61) in transesterified lipids, with methyl palmitate being the most abundant (C16:0). While theoretical biodiesel properties revealed high cetane number and oxidative stability, high cloud and pour point values indicated that fuel blending could be a viable approach. Significantly high FAME abundance in total transesterified lipids of HSF33-1 (40.2%) and HSF33-2 (69.9%) relative to Fd33 (25.4%) was identified using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry, indicating that robust salt stress response corresponds to higher levels of extractable FAME. Alkanes, a key component in conventional fuels, were present in F. diplosiphon transesterified lipids across all strains confirming that natural synthesis of these hydrocarbons is not inhibited during biodiesel production. While analysis of photosynthetic pigments and phycobiliproteins did not reveal significant differences, FAME abundance varied significantly in wild type and halotolerant strains indicating that photosynthetic pathways are not the sole factors that determine fatty acid production. We characterize the potential of F. diplosiphon for biofuel production with FAME yields in halotolerant strains higher than the wild type with no loss in photosynthetic pigmentation.

Cadmium is a non-essential toxic heavy metal for organisms, including plants and cyanobacteria. Cadmium resistance transporters involved in resistance of cells against various toxicants such as drugs and effluxes cytotoxic compounds from cells. However, cadmium resistance-associated protein (CadD) has never been reported from a diazotrophic cyanobacterium Anabaena sp. To test whether the hypothetical protein All3255 of Anabaena sp. PCC7120 a homolog of cadmium resistance-associated protein (CadD) involved in cadmium or heavy metal resistance or not, cloning and heterologous expression analysis of all3255 performed in Escherichia coli BL21 (DE3). Our results revealed that the strain transformed with pGEX-5X-2 + all3255 showed resistant towards not only to cadmium but also other heavy metals such as nickel, copper, zinc, lead and cobalt in addition to arsenic than those of transformed with empty vector (pGEX-5X-2). Furthermore, the results of metal accumulation analysis of these cells unveil a lower accumulation of tested heavy metals in all3255-overexpressing E. coli cells than those transformed with empty vector. This study strongly supports the role of All3255 of Anabaena sp. PCC7120 as a CadD efflux pump of heavy metals in E.coli.

Nanoparticles (NPs) are considered to improve and even revolutionize medicine, optics, and electronics. Most of the physical and chemical processes for NP synthesis require the use of toxic solvents, generation of hazardous byproducts, and high energy consumption. These shortcomings make the biological synthesis a preferred option. Biosynthesis of NPs using cyanobacteria has emerged as a rapidly developing research area in green nanotechnology; selecting the best species and optimizing the reaction conditions are under constant research. NPs can be synthesized from cyanobacteria using cell extracts or induced intracellular synthesis and naturally released in the culture medium. The naturally released NPs are stabilized by algal polysaccharides, which allows easy recovery of NPs. The size of the recovered particles as well as the reaction yield depends on the cyanobacteria genus. This chapter provides a broad understanding of NPs, highlights the disadvantages of conventional synthesis methods and includes evidence that biosynthesized NPs are better for a sustainable environment and has massive scope for pharmaceutical industries and biomedical applications.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 μmol min−1 mg protein−1, and K cat/K m 9.438 × 104 μM−1 min−1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

Metabolic products such as lipids and proteins produced in cyanobacteria represent an excellent source of biomass and do not compete with agricultural land use unlike soybean and corn. Given their potential use as novel materials for biodiesel production, we aimed to explore the effect of cultivation period and nitrogen concentration on the growth rate and lipid content of Fremyella diplosiphon, a model cyanobacterium. In this study, F. diplosiphon grown in BG11/HEPES medium supplemented with 1.5 g L−1 sodium nitrate (NaNO3) for 7, 10, 15, and 20 days were compared to the untreated control in media amended with 0.25, 0.5, and 1.0 g L−1 NaNO3. Cultures were inoculated in liquid media and grown under continuous fluorescent light in an orbital incubator shaker, and extracted lipids subjected to gravimetric analysis and gas chromatography-mass spectroscopy to determine the best culture conditions for lipid production. Our results demonstrated that a reduction in nitrogen concentration had no significant effect on the growth rate across all cultivation periods; however, the accumulation of total lipid content was significantly influenced by nitrogen concentration. A maximum lipid production (40%) with no reduction in growth was observed in 10-day old cultures in a BG11/HEPES medium supplemented with 1.0 g L−1 NaNO3. Fatty acid methyl ester composition of transesterified lipids demonstrated high amounts of methyl palmitate (50–70%) followed by methyl octadecenoate (17–30%) in the accumulated lipids at all treatments. Trace quantities of methyl dodecanoate, methyl hexadecanoate, methyl octadecanoate, and methyl octadecadienoate (1–8%) were also observed in all tested samples, indicating that nitrogen deprivation in culture media increases lipid production without affecting growth.

Cyanobacteria are a natural inhabitant of paddy field and enhance the crop productivity in an eco-friendly manner. Cadmium (Cd) is a perilous trace metal element which not only limits the crop productivity but also inhibits the growth and nitrogen-fixing ability of these diazotrophs as well as the biodiversity of rice field semiaquatic agroecosystems. However, the impact of Cd toxicity in diazotrophic cyanobacteria is yet not adequately addressed. Therefore, in the present study, three diazotrophic cyanobacterial species, i.e., Anabaena sp. PCC7120, Anabaena L31, and Anabaena doliolum were subjected to their LC50 doses of Cd, and their physiological (PSII, Psi, respiration, energy status and nitrogen fixation rate), biochemical variables (such as antioxidant contents and antioxidant enzymes) together with morphological parameters were evaluated. The results of physiological variables suggested that the Cd exposure adversely affects the photosynthesis, respiration, and biological nitrogen fixation ability across three Anabaena species. The results of biochemical variables in terms of accumulation of antioxidants (glutathione, thiol, phytochelatin and proline) content as well as antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), catalase-peroxidase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) revealed that their inter-species stress tolerance behavior may be attributed to the differential accumulation of antioxidants as well as differential antioxidant enzyme activity in three species. Furthermore, the enhanced antioxidant enzymes activity such as GST, GR, CAT, and SOD in Anabaena L31 advocated significantly higher as compared to Anabaena PCC7120 and Anabaena doliolum. In conclusion, Cd-toxicity assessment regarding physiological, biochemical and morphological aspects across three species identified Anabaena L31 as Cd-resistant species than the other two tested species, i.e., Anabaena PCC7120 and Anabaena doliolum.

Cyanobacteria due to their remarkable evolutionary advances such as the presence of oxygenic photosynthesis have been considered as an ideal system for plant-based studies in order to assess fundamental biochemical processes like carbon and nitrogen assimilation and photosynthesis processes. Moreover, the exclusive ability of both photosynthesis and nitrogen fixation together with adaptability to various environmental fluctuations of few genera makes them ubiquitous. Cyanobacterial genes involved in carbon metabolism, fatty acid biosynthesis, and pigment biosynthesis have been exploited as substitute for homologous gene sources, targeting enhanced plant productivity and nutritional values. Present chapter sheds light on key contributions of cyanobacterial biodiversity and biotechnology along with the future prospects for developing transgenic crops of high yield and nutritive value utilizing cyanobacterial genes.

A variety of toxicants such as, metal ions, pesticides, dyes, etc. are continuously being introduced anthropogenically in the environment and adversely affect to the biotic component of the ecosystem. Therefore, the assessment of negative effects of these toxicants is required. However, toxicity assessment anticipated by chemical analysis are extremely poor, therefore the application of the living systems for the same is an excellent approach. Concentration of toxicant as well as cell density both influenced the result of the algal toxicity assay. Here, Scenedesmus sp, a very fast growing green microalgae was selected for study the effects of initial cell densities on the toxicity of Cu(II), Cd(II), Zn(II), paraquat and 2,4-D. Results demonstrated concentration dependent decrease in biomass and specific growth rate of Scenedesmus sp. on exposure of abovesaid toxicants. Paraquat and 2,4-D emerged as extremely toxic to the test alga which reflected from the lowest EC value and very steep decline in biomass was evident with increasing concentration of paraquat and 2,4-D in the medium. Result also demonstrated that initial cell density is a very important parameter than specific growth rate for algal bioassay of various toxicants. Present study clearly illustrated that the use of smaller cell density is always recommended for assaying toxicity of chemicals in algal assays.

The hupL of Anabaena sp. PCC 7120 encodes the large subunit of uptake hydrogenase found in all diazotrophic cyanobacteria and boosts up the nitrogen-fixing potential by catalyzing the removal of the molecular hydrogen produced as a by-product of dinitrogen fixation. Bioinformatics analysis revealed that HupL from Anabaena sp. PCC7120 is a 60.2 kDa, thermostable, glycine-rich protein having highest structural similarity with NiFeSe hydrogenase of Desulfomicrobium baculatumis. Toxicity of selected abiotic stresses like arsenic, cadmium, copper, and salt with HupL was further reconciled by wet-lab approaches like qRT-PCR, hydrogenase and nitrogenase activity assay as hydrogenases unintendedly affect the nitrogenase activity in Anabaena. Down-regulated transcript along with highly inhibited hydrogenase and nitrogenase activities under cadmium stress revealed that cadmium is a potent inhibitor of hydrogenases in Anabaena which indirectly affects its nitrogen-fixing capabilities

Cadmium is a non-essential toxic heavy metal for organisms, including plants and cyanobacteria. Cadmium resistance transporters involved in resistance of cells against various toxicants such as drugs and effluxes cytotoxic compounds from cells. However, cadmium resistance-associated protein (CadD) has never been reported from a diazotrophic cyanobacterium Anabaena sp. To test whether the hypothetical protein All3255 of Anabaena sp. PCC7120 a homolog of cadmium resistance-associated protein (CadD) involved in cadmium or heavy metal resistance or not, cloning and heterologous expression analysis of all3255 performed in Escherichia coli BL21 (DE3). Our results revealed that the strain transformed with pGEX-5X-2 + all3255 showed resistant towards not only to cadmium but also other heavy metals such as nickel, copper, zinc, lead and cobalt in addition to arsenic than those of transformed with empty vector (pGEX-5X-2). Furthermore, the results of metal accumulation analysis of these cells unveil a lower accumulation of tested heavy metals in all3255-overexpressing E. coli cells than those transformed with empty vector. This study strongly supports the role of All3255 of Anabaena sp. PCC7120 as a CadD efflux pump of heavy metals in E.coli.

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.

The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40-60% compared to a 33-40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena.

Alkylhydroperoxide reductase (AhpC), a 1-Cys peroxiredoxin is well known for maintaining the cellular homeostasis. Present study employs proteome approach to analyze and compare alterations in proteome of Anabaena PCC7120 in overexpressing (An + ahpC), deletion (An ∆ ahpC) and its wild type. 2-DE based analysis revealed that the major portion of identified protein belongs to energy metabolism, protein folding, modification and stress related proteins and carbohydrate metabolism. The two major traits discernible from An + ahpC were (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins. Increased accumulation of proteins of light reaction, dark reaction, pentose phosphate pathway and electron transfer agent FDX for nitrogenase in An + ahpC and their simultaneous downregulation in AnΔahpC demonstrates its role in augmenting photosynthesis and nitrogen fixation. Proteomic data was nicely corroborated with physiological, biochemical parameters displaying upregulation of nitrogenase (1.6 fold) PSI (1.08) and PSII (2.137) in An + ahpC. Furthermore, in silico analysis not only attested association of AhpC with peroxiredoxins but also with other players of antioxidative defense system viz. thioredoxin and thioredoxin reductase. Above mentioned findings are in agreement with 33–40% and 40–60% better growth performance of An + ahpC over wild type and An ∆ ahpC respectively under abiotic stresses, suggesting its role in maintenance of metabolic machinery under stress. Significance Present work explores key role of AhpC in mitigating stress in Anabaena PCC7120 through combined proteomic, biochemical and in silico investigations. This study is the first attempt to analyze and compare alterations in proteome of Anabaena PCC7120 following addition (overexpressing strain An + ahpC) and deletion (mutant An ∆ ahpC) of AhpC against its wild type. The effort resulted in two major traits in An + ahpC as (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins.

A variety of toxicants such as, metal ions, pesticides, dyes, etc. are continuously being introduced anthropogenically in the environment and adversely affect to the biotic component of the ecosystem. Therefore, the assessment of negative effects of these toxicants is required. However, toxicity assessment anticipated by chemical analysis are extremely poor, therefore the application of the living systems for the same is an excellent approach. Concentration of toxicant as well as cell density both influenced the result of the algal toxicity assay. Here, Scenedesmus sp, a very fast growing green microalgae was selected for study the effects of initial cell densities on the toxicity of Cu(II), Cd(II), Zn(II), paraquat and 2,4-D. Results demonstrated concentration dependent decrease in biomass and specific growth rate of Scenedesmus sp. on exposure of abovesaid toxicants. Paraquat and 2,4-D emerged as extremely toxic to the test alga which reflected from the lowest EC value and very steep decline in biomass was evident with increasing concentration of paraquat and 2,4-D in the medium. Result also demonstrated that initial cell density is a very important parameter than specific growth rate for algal bioassay of various toxicants. Present study clearly illustrated that the use of smaller cell density is always recommended for assaying toxicity of chemicals in algal assays.

Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

The present study compares protein diversity within three Anabaena species (Anabaena doliolum, Anabaena sp.PCC 7120 and Anabaena L31). 2-DE based analysis of 256 protein spots in control and 1, 3, 5, and 7 days of salt treatment resulted into 96 proteins arching across fourteen functional categories were assigned to biochemical pathways using KOBAS 2.0. While 52.34% of the evaluated protein spots were common across three species, the remaining 47.66% fraction mainly comprised of the hypothetical and unknown proteins. PSORTb, CDD, Motifscan and Pfam revealed function and subcellular localization for 27 of the 31 hypothetical and unknown proteins. The differences in high salt tolerance (LC50) of A. doliolum over A. L31 was reflected by (i) many fold accumulation (as spot volumes) of Alr3090, Alr0803, peptidyl prolyl cis-trans isomerase and modulator of DNA gyrase proteins, and (ii) a better photosynthesis and energy homeostasis as indicated through photosystem activity, respiration, ATP and NADPH contents. Some common noteworthy salt effects include (i) photosystem damage, (ii) DNA damage repair, (iii) upregulated protein synthesis, (iv) enhanced sulphur metabolism, and (v) upregulated pentose phosphate pathway. 34 of the identified protein spots are novel entries to the Anabaena salt proteome. This study reveals the existence of separate strategies even within species to combat stress. Biological significance This study for the first time enumerates protein diversity in three Anabaena species employing their presence/absence and relative abundance. Proteomics integrated with physiology and bioinformatics deciphers differential salt tolerance among the studied species and is the first of its kind to predict the function of hypothetical and unknown proteins. Salt-induced proteomic alterations clearly demonstrate significant metabolic shifts and existence of separate molecular phenome among the species investigated. This may be responsible for niche specificity limiting their application as biofertilizer. Of the 96 identified proteins, a large chunk are new entries to the Anabaena salt proteome while some protein genes may be used as potential candidates for engineering salt tolerant cyanobacteria.

This study examines response of Anabaena sp. PCC 7120 to salt and UV-B stress by combining physiological, biochemical, proteomics and bioinformatics approaches. Sixty five significantly altered protein spots corresponding to 51 protein genes identified using MALDI-TOF MS/MS were divided into nine functional categories. Based on relative abundance, these proteins were grouped into four major sets. Of these, 27 and 5 proteins were up- and downregulated, respectively, both under salt and UV-B while 8 and 11 proteins showed accumulation in salt and UV-B applied singly. Some responses common to salt and UV-B included (i) enhanced expression of FeSOD, alr3090 and accumulation of MDA indicating oxidative stress, (ii) accumulation of PDH, G6P isomerase, FBPaldolase, TK, GAPDH and PGK suggesting enhanced glycolysis, (iii) upregulation of 6-PGD, 6PGL and NADPH levels signifying operation of pentose phosphate pathway, (iv) upregulation of Dps, NDK and alr3199 indicating DNA damage, and (v) accumulation of proteins of ribosome assembly, transcriptional and translational processing. In contrast, enhanced expression of RUBISCO, increased glycolate oxidase activity and ammonium content under salt signify the difference. Salt was found to be more damaging than UV-B probably due to a cumulative effect of ionic, osmotic and oxidative damage. A group of proteins having common expression represent decreased toxicity of salt and UV-B when applied in combination.

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl(2), H(2)O(2), UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl(2) demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.

The karstic limestone terrain poses complex geotechnical problems, as there are many uncertainties in terms of position and dimension of cavities, sinkholes, pinnacles, and overhangs developed in it. The study deals with the 6.5 km long Ampang­ Kuala Lumpur Elevated Highway over the rivers Kelang and Ampang in Malaysia. The large-diameter bored piles and cast in-place piles were constructed for this purpose. The toll plaza area is underlain by karstic limestone. In that area, the piles were located in rows extended as columns connected by crosshead whereas they were provided in pile groups of 2, 3, 4, or 5 connected by a rigid pile cap elsewhere. An instrumented test pile was load tested using static method and calibrated with site investigation results. To design the friction piles and end bearing piles, direct methods (viz. exploratory drilling and rock probes) and indirect methods (viz. resistivity survey and transient electromagnetic survey) were adopted. Based on lithologs, the piles were designed using the safety factor of 2 and 3. The rock probe results were used for designing the rock socket length of the end bearing piles. There was much variation between the designed and encountered rock head levels as revealed during the time of excavation. In such cases, the geotechnical design of end bearing piles was reviewed. As in most of the cases, rock probe holes were inclined (as they slid over a rock cliff, overhang, or pinnacle) giving rise to wrong information of rock head levels. To solve this problem at site, a new integrated approach was evolved considering the 3D subsurface topography of rock head level of the area, position and dimension of karstic features, rock mass classifications, and bearing capacity calculation. Based on the above parameters and other measures (underpinning, micropiling, grouting etc.), the piles were designed and load tested.