Insight into Different Stages of Steroid Degradation in Thermophilic Saccharopolyspora hirsuta VKM Ac-666T Strain.

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT-qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.

ß-elimination of hyaluronate by red king crab hyaluronidase.

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40-50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the [Formula: see text]-elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

The Naphthalene Catabolic Genes of Pseudomonas putida BS3701: Additional Regulatory Control.

Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the "MicroBak" preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation.