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Covering: 2017.01 to 2023.11 Natural products biosynthesized by myxobacteria are appealing due to their sophisticated chemical skeletons, remarkable biological activities, and intriguing biosynthetic enzymology. This review aims to systematically summarize the advances in the discovery methods, new structures, and bioactivities of myxobacterial NPs reported in the period of 2017–2023. In addition, the peculiar biosynthetic pathways of several structural families are also highlighted. [ABSTRACT FROM AUTHOR]

Background: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. Results: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. Conclusions: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in micro-organisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites. [ABSTRACT FROM AUTHOR]

Background: Microbial secondary metabolism is regulated by a complex and mostly-unknown network of global and pathway-specific regulators. A dozen biosynthetic gene clusters for secondary metabolites have been reported in myxobacteria, but a few regulation factors have been identified. Results: We identified a transcription regulator Esi for the biosynthesis of epothilones. Inactivation of esi promoted the epothilone production, while overexpression of the gene suppressed the production. The regulation was determined to be resulted from the transcriptional changes of epothilone genes. Esi was able to bind, probably via the N-terminus of the protein, to an inverted repeat sequence in the promoter of the epothilone biosynthetic gene cluster. The Esi-homologous sequences retrieved from the RefSeq database are all of the Proteobacteria. However, the Esi regulation is not universal in myxobacteria, because the esi gene exists only in a few myxobacterial genomes. Conclusions: Esi binds to the epothilone promoter and down-regulates the transcriptional level of the whole gene cluster to affect the biosynthesis of epothilone. This is the first transcription regulator identified for epothilone biosynthesis. [ABSTRACT FROM AUTHOR]

Myxobacteria, phylogenetically located in the delta division of the Proteobacteria, are well known for characterized social behaviors and large genomes of more than 9 Mb in size. Myxococcus fulvus is a typical species of the genus Myxococcus in the family Myxococcaceae. M. fulvus 124B02, originally isolated from a soil sample collected in Northeast China, is the one and only presently known myxobacterial strain that harbors an endogenous autonomously replicating plasmid, named pMF1. The endogenous plasmid is of importance for understanding the genome evolution of myxobacteria, as well as for the development of genetic engineering tools in myxobacteria. Here we describe the complete genome sequence of this organism. M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. Comparative genomic analyses suggest that pMF1 has a longstanding sustention within myxobacteria, and probably contributes to the genome expansion of myxobacteria. [ABSTRACT FROM AUTHOR]

Although many molecular ecological surveys have been conducted, there is little concerning the details of specific bacterial groups, resulting in an incomplete understanding of the microorganismal composition and community structures in the environment. Myxobacteria are micropredators that are metabolically active in the soil microbial food web and have typically been considered minority components of soil bacterial communities. In this study, we surveyed the percentage of myxobacteria in a single soil sample via pyrosequencing on combined universal libraries of the V3- V4 and V6- V8 hypervariable regions of the 16 S rRNA gene. Surprisingly, myxobacteria accounted for 4.10% of the bacterial community and 7.5% of the total operational taxonomic units at the 3% similarity level in the soil, containing almost all of the cultivated myxobacterial families or genera. To testify the appearance of myxobacteria in soil niches, we retrieved myxobacteria-related 16 S rRNA gene sequences of 103 high-throughput sequencing data sets obtained from public databases. The results indicated that myxobacteria-related sequences were among the predominant groups in these data sets accounting for 0.4-4.5% of bacterial communities. The abundance of myxobacterial communities were correlated with site temperature, carbon-to-nitrogen ratio and pH values. Based on these results, we discussed the survival strategies of myxobacterial community in soil. [ABSTRACT FROM AUTHOR]

Myxobacteria are widely distributed in soil and oceanic sediment with a phylogeographic separation at high levels of classification. However, it is unclear whether freshwater environments, from which there has been no isolation report of myxobacteria since 1981, are habitats for myxobacteria. In this study, we investigated the presence of myxobacteria in lake mud using a two-step strategy. First, we constructed two universal bacterial libraries from the V3- V4 ( V34) and V6- V8 ( V678) hypervariable regions of 16 S rRNA gene sequences. High-throughput 454 pyrosequencing revealed that myxobacteria were one of the major bacterial groups in the lake mud. They accounted for 5.77% of the total sequences and 7.52% of the total operational taxonomic units ( OTUs) at a phylogenetic distance of 0.03. The community composition and taxonomic structure of the mud myxobacterial community were further analysed using myxobacteria-enriched libraries targeting the V34 and V678 regions, which were amplified with Cystobacterineae- and Sorangineae-specific primer pairs respectively. Phylogenetic analysis showed that the limnetic myxobacteria exhibited closer relationships to their soil than their marine relatives, but there were also exclusive taxa of limnetic myxobacteria detected. These results, together with a survey on available Gen Bank data, indicate that lake mud is a primary habitat for myxobacteria. [ABSTRACT FROM AUTHOR]

Microorganisms are globally dispersed and are able to proliferate in any habitat that supports their lifestyles, which, however, has not yet been explored in any specific microbial taxon. The social myxobacteria are considered typical soil bacteria because they have been identified in various terrestrial samples, a few in coastal areas, but none in other oceanic environments. To explore the prevalence of marine myxobacteria and to investigate their phylogenetic relationships with their terrestrial counterparts, we established myxobacteria-enriched libraries of 16S rRNA gene sequences from four deep-sea sediments collected at depths from 853 to 4675 m and a hydrothermal vent at a depth of 204 m. In all, 68 different myxobacteria-related sequences were identified from randomly sequenced clones of the libraries of different samples. These myxobacterial sequences were diverse but phylogenetically similar at different locations and depths. However, they were separated from terrestrial myxobacteria at high levels of classification. This discovery indicates that the marine myxobacteria are phylogeographically separated from their terrestrial relatives, likely because of geographic separation and environment selection. [ABSTRACT FROM AUTHOR]

One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 105–106 colony-forming units per μg DNA, which was about 103–105 fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation. [ABSTRACT FROM AUTHOR]

Abstract: The diversity of type I polyketide synthases (PKSs) in cellulolytic myxobacterium Sorangium was explored by assaying the ketoacyl synthases (KSs) in 10 Sorangium strains with two degenerate primer sets and 64 different KS fragments were obtained. For their deduced amino acid sequences, eight were identical to three known KSs from Sorangium and Magnetospirillum, while the others showed 54–83% identities to the modular KS domains reported from various microorganisms. Parts of the Sorangium KSs tightly share the clade with Actinobacteria excluding any other analyzed myxobacterial KSs, or with Cyanobacteria /Myxobacteria. Parts are widely located in the three functional groups – “Loading”, “NRPS/PKS” and “Trans-AT”. Sorangium KSs in the Actinobacteria, Cyanobacteria/Myxobacteria, or “Loading” clade further evolved independently on its own genus. Notably, the modular KSs from other Myxobacteria genera, i.e. Myxococcus, Stigmatella, Melittangium, Cystobacter and Angiococcus are often distributed crosswise and form non-Sorangium blend subgroups. “NRPS/PKS” and “Trans-AT” are two rather diverse groups and the Sorangium KSs in these clades evolved crosswise with other taxa lineages. The results presented in this paper suggest that the inherent genetic strategies, together with frequent gene importing from many organisms (HGT) have contributed to the evolution of modular PKSs in Sorangium. These findings reinforce that Sorangium strains are really excellent creators for novel and diverse polyketides. [Copyright &y& Elsevier]

The diversity of myxobacteria in a soil niche was explored using culture-dependent and -independent methods. Conventional cultivation for bacteriolytic myxobacteria produced six types of myxobacteria, which were identified as two Myxococcus spp., two Corallococcus spp., a Cystobacter sp. and a Nannocysts sp. Hybridization analysis of the soil bacterial 16S rRNA gene library with myxobacteria-specific probes revealed that myxobacteria accounted for less than 1% in the bacterial community. A Cystobacterineae 16S rRNA genes-rich library was further established from the soil DNA by polymerase chain reaction amplification with a Cystobacterineae-specific primer combined with a universal bacterial primer. Screening of the special library using Cystobacterineae- and Sorangineae-specific probes produced approximately 45% and 3% positive signals respectively. Sixty-four positive clones were randomly selected for sequencing. Except three repeats, the sequences were diverse ranging from 0.3% to 21.3%, and homologous with the known myxobacteria at 77.6–99.8%, including 57 in Cystobacterineae, one close to Nannocystis and three much more distant from the known myxobacteria. The sequences in the Cystobacterineae can further be divided into at least 12 groups, of which most were unreported. The results suggest that myxobacteria in nature are much more diverse than were ever known, even in one soil niche. [ABSTRACT FROM AUTHOR]

Summary: The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color. [Copyright &y& Elsevier]

This paper describes a simple method for the isolation of salt-tolerant myxobacteria from marine conditions. As the results show in this paper, salt-tolerant myxobacteria are found to be able to grow, but unable to form fruiting bodies at high salt concentrations. The fruiting body structures of the salt-tolerant strains were all formed in conditions with lower seawater content, i.e. lower than 60% seawater (about 2.0% salt content) or distilled water supplemented with MgCl2. The method picked up the fruiting bodies for isolation. [Copyright &y& Elsevier]

A correction to the article "Exploring the diversity of myxobacteria in a soil niche by myxobacteria-specific primers and probes" that was published in the 2005 issue is presented.

Abstract: Thus far, pMF1 is the only endogenous myxobacterial plasmid whose replication mechanism is unclear. In this study, we determined that the plasmid replicates via the theta-mode. The pMF1.14 gene, located in the pMF1.13–pMF1.15 operon (repABC), encodes an essential replication initiation protein that was predicted to have no typical DNA/protein binding motifs but contains rich disordered regions. The pMF1 replication-related essential cis-acting DNA region, approximate 370bp, was located within pMF1.14, and was found to contain several directly and inverted atypical repeats. The unique characteristics of the pMF1 replicon are suggested to be the reason for its strict narrow host range in Myxococcus cells. [Copyright &y& Elsevier]

Abstract: The diversity of myxobacteria present in campus garden soil was surveyed by both cultivation-based and cultivation-independent methods. Detailed phylogenetic analysis of cultured and uncultured myxobacteria 16S rRNA gene sequences revealed that many undescribed relatives of the myxobacteria exist in nature. Molecular systematic analyses also revealed that myxobacterial genera described to date on the basis of the morphology of multi-cellular fruiting bodies were mostly monophyletic. However, these known taxa comprised only in a small part of the sequences recovered directly from soil in a cultivation-independent approach, indicating that the group is much more diverse than previously thought. We propose that the myxobacteria exist in two forms: the fruiting and the non-fruiting types. Most of the uncultured myxobacteria may represent taxa which rarely form fruiting bodies, or may lack some or all of the developmental genes needed for fruiting body formation. In order to identify non-fruiting myxobacteria, new morphology-independent cultivation and isolation techniques need to be developed. [Copyright &y& Elsevier]

Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution. [ABSTRACT FROM AUTHOR]