Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes ( iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iif-mediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation. IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N -heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism.

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.

A plant auxin hormone indole-3-acetic acid (IAA) can be assimilated by bacteria as an energy and carbon source, although no degradation has been reported for indole-3-propionic acid and indole-3-butyric acid. While significant efforts have been made to decipher the Iac (indole-3-acetic acid catabolism)-mediated IAA degradation pathway, a lot of questions remain regarding the mechanisms of individual reactions, involvement of specific Iac proteins, and the overall reaction scheme. This work was aimed at providing new experimental evidence regarding the biodegradation of IAA and its derivatives. Here, it was shown that Caballeronia glathei strain DSM50014 possesses a full iac gene cluster and is able to use IAA as a sole source of carbon and energy. Next, IacE was shown to be responsible for the conversion of 2-oxoindole-3-acetic acid (Ox-IAA) intermediate into the central intermediate 3-hydroxy-2-oxindole-3-acetic acid (DOAA) without the requirement for IacB. During this reaction, the oxygen atom incorporated into Ox-IAA was derived from water. Finally, IacA and IacE were shown to convert a wide range of indole derivatives, including indole-3-propionic acid and indole-3-butyric acid, into corresponding DOAA homologs. This work provides novel insights into Iac-mediated IAA degradation and demonstrates the versatility and substrate scope of IacA and IacE enzymes.

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and temperature (5–85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.