Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

Calcineurin responsive zinc-finger-1 binds to a unique promoter sequence to upregulate neuronal calcium sensor-1, whose interaction with MID-1 increases tolerance to calcium stress in Neurospora crassa.

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca2+ stress in Neurospora crassa. Increasing concentration of Ca2+ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca2+ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca2+ permeable channel MID-1 for tolerance to Ca2+ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca2+ stress.

Phospholipases play multiple cellular roles including growth, stress tolerance, sexual development, and virulence in fungi.

Phospholipases are ubiquitous enzymes that hydrolyze phospholipids. Based on the cleavage site of the ester linkage in the substrate phospholipids, phospholipases are classified into four major types, phospholipase A (PLA), phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD), which are further classified into various subtypes. Phospholipases hydrolyze phospholipids into various signaling products including phosphatidic acid (PA), diacylglycerol (DAG), free fatty acids (FFAs), and lyso-phospholipids (LPLs). These signaling products regulate numerous processes such as cytoskeletal dynamics, growth, homeostasis, membrane remodeling, nutrient acquisition, secretion, signal transduction, stress tolerance, sexual development, and virulence in various organisms including fungi. Due to these key cellular roles, phospholipases are also promising targets in diagnostic and therapeutic applications. In this review, we discuss current knowledge about the cellular roles of different classes of phospholipases in fungi.

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

A novel melt stable and high tensile strength biopolymer (polyhydroxyalkanoates) from Bacillus megaterium (MTCC10086) and its characterization.

In the present investigation, we have defined a novel biopolymer from Bacillus megaterium strain with novel melt stability, high tensile strength, and elongation to break properties higher to polypropylene and similar to polyethylene the polymers available commercially. The polymer was characterized with FTIR and XRD. The percent crystalinity was found to 44.09% with tensile strength 42 (Mpa) and elongation to break (%) 142 higher than polypropylene. The polymeric properties were confirmed by differential scanning calorimeter and universal testing.