Regulation of sporangium formation by the orphan response regulator TcrA in the rare actinomycete Actinoplanes missouriensis

Abstract

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.

Involvement of three FliA‐family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis

“…We quantified the transcript levels of fliA1 , fliA2 and fliA3 in the wild‐type and Δ tcrA mutant strains by qRT‐PCR analysis using RNA samples extracted from a mixture of sporangia and vegetative hyphae cultivated on HAT agar for 5 days, which corresponds to the late stage of sporangium formation in the A. missouriensis life cycle. Consistent with the RNA‐Seq analysis in our previous study (Mouri et al, ), the transcript levels of these genes in the Δ tcrA mutant were extremely lower than those in the wild‐type strain (102‐, 222‐ and 402‐fold, respectively; Figure c), showing that TcrA directly activates the transcription of fliA1 , fliA2 and fliA3 . As described below, we demonstrated that the newly identified TcrA box upstream from fliA2 is required for the in vivo function by a gene complementation test (see Figure ).…”

“…In contrast, many squashed sporangia were observed in the Δ fliA1 Δ fliA2 , Δ fliA1 Δ fliA2 Δ fliA3 and Δ fliA1 Δ fliA2 Δ fliA3 Δ fliA4 mutants, indicating that the deficiency of both FliA1 and FliA2 resulted in abnormal sporangium formation (Figures a–d and ). This phenotypic change is quite similar to that observed in the Δ tcrA mutant (Mouri et al, ). In a gene complementation test, the introduction of fliA2 with its own promoter into the Δ fliA1 Δ fliA2 mutant resulted in normal sporangium formation.…”

“…Direct repeat sequences of TcrA boxes in the typical location are shaded in green. Additional direct repeat sequences identified in our previous study are shaded in yellow (Mouri et al, ). Arrows indicate the direction of the direct repeat sequences.…”

“…Furthermore, we found that putative FliA‐dependent promoters are located in the regions upstream from a total of 122 other transcriptional units that are under the indirect control of TcrA and that three FliA‐family sigma factor genes, fliA1 ( AMIS_41310 ), fliA2 ( AMIS_62620 ) and fliA3 ( AMIS_64240 ), were significantly downregulated in the tcrA null (Δ tcrA ) mutant strain. These findings indicate that TcrA regulates over a few hundred genes through the transcriptional activation of some or all of the three FliA‐family sigma factor genes (Mouri et al, ). As mentioned above, the FliA/WhiG‐family sigma factor is involved in the expression of developmental and motility‐related genes in Streptomyces and motile unicellular bacteria respectively.…”

Involvement of β‐Alkylation Machinery and Two Sets of Ketosynthase‐Chain‐Length Factors in the Biosynthesis of Fogacin Polyketides in Actinoplanes missouriensis

“…Actinoplanes missouriensis 431 is a rare actinomycete isolated from barnyard soil near Hamilton, Missouri, USA . It shows interesting morphological features and forms sporangia containing motile sporangiospores . We have shown that this strain produces several secondary metabolites, including alkyl‐ O ‐dihydrogeranyl‐methoxyhydroquinones and 3‐hydroxy‐6‐dimethylallylindolin‐2‐one .…”