Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge

Abstract

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282(T). The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1' were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450(mor) system remained unaltered. In addition, we found a spontaneous deletion of P450(mor) from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.

Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

“…This is in contrast to the case in KTR9, where xplB and xplA occur downstream of glnA. The nucleotide sequence repeats present in the mor/pip loci (29,39,50) are absent in the xpl cluster.…”

“…Approximately 1.5 kb upstream of this cluster is xplR, encoding a GntR-type transcriptional regulator. Cyp151C shares up to 71% amino acid sequence identity with the mycobacterial pipA/morA-encoded cytochromes P450 that are involved in the utilization of the secondary amines piperidine, pyrrolidine, and morpholine (29,39,50). The glnA-xplB fusion is predicted to encode the first 85% of the residues of a glutamine synthetase fused to the entire XplB reductase.…”

“…PCR analysis of KTR9 DNA genomic template with primers that flank the gene fusion point confirmed that there is no gap between these genes (data not shown). Interestingly, the glutamine synthase portion of the glnA-xplB fusion and XplR share up to 70% and 49% sequence identity, respectively, with the glutamine synthases and GntR-type regulators encoded by genes associated with the mycobacterial cyp151 genes (29,39,50). The organization of the xpl gene cluster suggests that it may constitute an operon that is regulated by XplR.…”

“…More particularly, xplR, cyp151C, and the truncated glnA domain of pGKT2 are remarkably similar in sequence and order to their homologs in the mor and pip gene clusters responsible for the degradation of morpholine, piperidine, and related compounds in mycobacteria and rhodococci (29,39,50). Indeed, a recent comparison of the mor/pip loci in six strains suggests that this locus is subject to a relatively high frequency of recombination events.…”

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

“…1) constructed with 16S rDNAs strongly suggested strain B-009 to be a member of the genus Mycobacterium and closely related to M. gilvum (99.5% similarity) which is known to be a rapidly growing and opportunistic species. 38) Nonclinical strains of the genus Mycobacterium are widespread in various environments, [39][40][41] and strain B-009 is supposed to be one such member. Mycolic acids were extracted and defined to ensure that the strain belonged to the genus Mycobacterium in terms of its chemotaxonomy.…”