SignificanceNatural products biosynthesized by cryptic gene clusters represent a largely untapped source for drug discovery. However, mining of these products by promoter engineering is restricted by the lack of streamlined genetic tools, especially in nonmodel biosynthetic gene cluster (BGC)-rich bacteria. Here, we describe the discovery of a pair of bacteriophage recombinases and application of recombinase-assisted promoter engineering to rapidly identify and activate several cryptic biosynthetic gene clusters in two Burkholderiales strains that currently lack effective genetic tools. Construction of an efficient genome engineering platform in a natural product producer expedites mining of cryptic BGCs in their native backgrounds, and host melioration for yield or structure optimization. This strategy enables potentially scalable discovery of novel metabolites with intriguing bioactivities from many other bacteria.

Complex environmental conditions can significantly affect bacterial genome size by unknown mechanisms. The So0157-2 strain of Sorangium cellulosum is an alkaline-adaptive epothilone producer that grows across a wide pH range. Here, we show that the genome of this strain is 14,782,125 base pairs, 1.75-megabases larger than the largest bacterial genome from S. cellulosum reported previously. The total 11,599 coding sequences (CDSs) include massive duplications and horizontally transferred genes, regulated by lots of protein kinases, sigma factors and related transcriptional regulation co-factors, providing the So0157-2 strain abundant resources and flexibility for ecological adaptation. The comparative transcriptomics approach, which detected 90.7% of the total CDSs, not only demonstrates complex expression patterns under varying environmental conditions but also suggests an alkaline-improved pathway of the insertion and duplication, which has been genetically testified, in this strain. These results provide insights into and a paradigm for how environmental conditions can affect bacterial genome expansion.

Myxococcus xanthus DK1622 contains two paralogous groEL gene loci that possess both different sequences and different organizations within the genome. Deletion of either one of these two genes alone does not affect cell viability. However, deletion of both groEL genes results in cell death unless a complemented groEL1 or groEL2 gene is present. The groEL1 gene was determined to be essential for cell survival under heat shock conditions; a strain with mutant groEL2 caused cells to be more sensitive than the wild-type strain to higher temperatures. Mutants with a single deletion of either groEL1 (MXAN_4895) or groEL2 (MXAN_4467) had a growth curve similar to that of the wild-type strain DK1622 in medium containing hydrolyzed proteins as the substrate. However, when cells were cultured on medium containing either Escherichia coli cells or casein as the substrate, deletion of groEL2, but not groEL1, led to a deficiency in cell predation and macromolecular feeding. Furthermore, groEL1 was found to play an indispensable role in the development and sporulation of cells, but deletion of groEL2 had no visible effects. Our results suggest that, although alternatively required for cell viability, the products of the two groEL genes have divergent functions in the multicellular social life cycle of M. xanthus DK1622.

c Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and productyielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ⌬G units. While interestingly, the tri-and tetrasaccharide fractions each contain higher proportions of ⌬G to ⌬M ends, the larger final products contain only ⌬M ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.A lginate is a linear polysaccharide composed of alternating residues of ␤-D-mannuronic acid (M) and its C-5 epimer, ␣-Lguluronic acid (G) (1). The uronic acid residues are arranged into homopolyuronic blocks of M residues (M block), G residues (G block), or heteropolyuronic blocks (MG or GM blocks). Alginate has been identified as a cell wall component of seaweeds belonging to Phaeophyta, such as kelp and sargassum (2, 3). Due to its ability to form a strong gel after absorbing water, algal alginate has been widely used as a supporting material in food, medical, and industrial applications (4, 5). Alginates containing acetyl modifications at the O-2 or O-3 positions have been purified from the extracellular matrix of some bacteria, such as the pathogen Pseudomonas aeruginosa and Azotobacter soil bacteria (6, 7). Understanding how to prevent Pseudomonas pathogens from synthesizing and secreting extracellular high-molecular-weight alginates is important for clinical therapy protocols (8, 9).Alginate lyase can break the 1 to 4 O linkages between the uronic acid residues of alginate via a ␤-elimination mechanism. The reaction forms C-4ϭC-5 double bonds within the s...

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 16S 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 16S 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.