Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

Abstract

The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangiumforming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3=,5=-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5=-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3= as the consensus AmBldDbinding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation.IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).KEYWORDS gene regulation, rare actinomycete, regulon, sporangium formation, transcriptional factor A ctinomycetes are Gram-positive, mainly soil-inhabiting bacteria. Many genera of actinomycetes show filamentous growth and produce spores, and therefore, actinomycetes are generally characterized by a complex morphological development. For

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

“…The amplified DNA fragments were analyzed by sequencing to confirm the absence of undesired mutation. Modified pK19mobsacB, the kanamycin resistance gene of which was replaced with the apramycin resistance gene aac(3)IV , was used as a vector for conjugation.…”

“…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 .…”

Actinoplanes Swims into the Molecular Age

“…In contrast, other filamentous actinomycetes are usually only a focus for research if they produce an antibiotic or other natural product of potential medical relevance, and their developmental biology is not analyzed. It is for this reason in particular that the work reported by Mouri et al (2) on reproduction in Actinoplanes is so refreshing.…”

“…Mouri et al (2) noted that the RXD-X 8 -RXXD c-di-GMP-binding signature observed in the Streptomyces BldD-c-di-GMP crystal structure is conserved in Actinoplanes. As a consequence, they used electrophoretic mobility shift assays (EMSAs) to see if c-di-GMP might also control the activity of Actinoplanes BldD and found that it greatly enhanced the binding of Actinoplanes BldD to its target DNA.…”

“…In Mouri et al (2), starting from a proteomicsbased approach, they show that a transcriptional regulator called BldD plays a critical role in the Actinoplanes life cycle. By conjugating constructs from E. coli into A. missouriensis, they were able to construct a bldD null mutant and found that it entered development prematurely, making abundant sporangia much earlier than the wild type.…”