L-cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase.

Candida albicans is a mucosal commensal organism capable of causing superficial (oral and vaginal thrush) infections in immune normal hosts, but is a major pathogen causing systemic and mucosal infections in immunocompromised individuals. Azoles have been very effective anti-fungal agents and the mainstay in treating opportunistic mold and yeast infections. Azole resistant strains have emerged compromising the utility of this class of drugs. It has been shown that azole resistance can be reversed by the co-administration of a histone deacetylase (HDAC) inhibitor, suggesting that resistance is mediated by epigenetic mechanisms possibly involving Hos2, a fungal deacetylase. We report here the cloning and functional characterization of HOS2 (HighOsmolarity Sensitive), a gene coding for fungal histone deacetylase from C. albicans. Inhibition studies showed that Hos2 is susceptible to pan inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), but is not inhibited by class I inhibitors such as MS-275. This in vitro enzymatic assay, which is amenable to high throughput could be used for screening potent fungal Hos2 inhibitors that could be a potential anti-fungal adjuvant. Purified Hos2 protein consistently deacetylated tubulins, rather than histones from TSA-treated cells. Hos2 has been reported to be a putative NAD+ dependent histone deacetylase, a feature of sirtuins. We assayed for sirtuin activation with resveratrol and purified Hos2 protein and did not find any sirtuin activity.

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Structural and Functional Characterization of Four Novel Fibrinogen Mutations in FGB Causing Congenital Fibrinogen Disorder

International Journal of Molecular Sciences ◽

10.3390/ijms23020721 ◽

2022 ◽

Vol 23 (2) ◽

pp. 721

Author(s):

Eliška Ceznerová ◽

Jiřina Kaufmanová ◽

Žofie Sovová ◽

Jana Štikarová ◽

Jan Loužil ◽

...

Keyword(s):

Functional Characterization ◽

Abnormal Development ◽

Screening Tests ◽

Morphological Properties ◽

Fiber Density ◽

Reverse Phase ◽

Gene Coding ◽

Novel Variants ◽

Fiber Thickness

Congenital fibrinogen disorders are caused by mutations in genes coding for fibrinogen and may lead to various clinical phenotypes. Here, we present a functional and structural analysis of 4 novel variants located in the FGB gene coding for fibrinogen Bβ chain-heterozygous missense BβY416C and BβA68S, homozygous nonsense BβY345*, and heterozygous nonsense BβW403* mutations. The cases were identified by coagulation screening tests and further investigated by various methods. Fibrin polymerization had abnormal development with decreased maximal absorbance in all patients. Plasmin-induced fibrin degradation revealed different lytic phases of BβY416C and BβW403* than those of the control. Fibrinopeptide cleavage measured by reverse phase high pressure liquid chromatography of BβA68S showed impaired release of fibrinopeptide B. Morphological properties, studied through scanning electron microscopy, differed significantly in the fiber thickness of BβY416C, BβA68S, and BβW403*, and in the fiber density of BβY416C and BβW403*. Finally, homology modeling of BβA68S showed that mutation caused negligible alternations in the protein structure. In conclusion, all mutations altered the correct fibrinogen function or structure that led to congenital fibrinogen disorders.

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Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose

Microbial Ecology ◽

10.1007/s00248-018-1247-4 ◽

2018 ◽

Vol 77 (3) ◽

pp. 651-663 ◽

Cited By ~ 8

Author(s):

Wei-fang Xu ◽

Hui-shuang Ren ◽

Ting Ou ◽

Ting Lei ◽

Jun-hong Wei ◽

...

Keyword(s):

Bacillus Subtilis ◽

Biocontrol Agent ◽

Functional Characterization ◽

Mulberry Fruit ◽

Potential Biocontrol Agent

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Transcriptional and functional characterization of the gene encoding acyl carrier protein in Bacillus subtilis

Microbiology ◽

10.1099/mic.0.033316-0 ◽

2010 ◽

Vol 156 (2) ◽

pp. 484-495 ◽

Cited By ~ 8

Author(s):

Mariano A. Martinez ◽

Diego de Mendoza ◽

Gustavo E. Schujman

Keyword(s):

Bacillus Subtilis ◽

Fatty Acid ◽

Molecular Mechanisms ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Functional Characterization ◽

Carrier Protein ◽

Normal Fatty Acid ◽

Gram Positive

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid biosynthesis. The molecular mechanisms of regulation of the acpP structural gene, as well as the function of its gene product, are poorly characterized in Bacillus subtilis and other Gram-positive organisms. Here, we report that transcription of acpP takes place from two different promoters: PfapR and PacpP. Expression of acpP from PfapR is coordinated with a cluster of genes involved in lipid synthesis (the fapR operon); the operon consists of fapR-plsX-fabD-fabG-acpP. PacpP is located immediately upstream of the acpP coding sequence, and is necessary and sufficient for normal fatty acid synthesis. We also report that acpP is essential for growth and differentiation, and that ACP localizes in the mother-cell compartment of the sporangium during spore formation. These results provide the first detailed characterization of the expression of the ACP-encoding gene in a Gram-positive bacterium, and highlight the importance of this protein in B. subtilis physiology.

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Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin

Biochemical Journal ◽

10.1042/bj3000599 ◽

1994 ◽

Vol 300 (2) ◽

pp. 599-603 ◽

Cited By ~ 32

Author(s):

M J O'Donohue ◽

B P Roques ◽

A Beaumont

Keyword(s):

Bacillus Subtilis ◽

Bacillus Stearothermophilus ◽

Biochemical Characterization ◽

Signal Sequence ◽

Cloning And Expression ◽

Bacillus Species ◽

Gene Coding ◽

Strong Homology ◽

Different Strains

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.

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