The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

The compatibility between rhizobia and legumes for nitrogen-fixing nodules and the stages of root hair curling, formation of infection thread, and nodulation initiation have been vitally studied, but the factors for the sustainable root surface colonization and efficient symbiosis within chickpea and rhizobia have been poorly investigated. Hence, we aimed to analyze phenotypic properties and phylogenetic relationships of root-nodule bacteria associated with chickpea (Cicer arietinum) in the north-west Indo Gangetic Plains (NW-IGP) region of Uttar Pradesh, India. In this study, 54 isolates were recovered from five agricultural locations. Strains exhibited high exopolysaccharide production and were capable of survival at 15–42 °C. Assays for phosphate solubilization, catalase, oxidase, Indole acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity revealed that all the tested isolates possessed plant growth-promoting potential. Metabolic profiling using Biolog plates indicated that patterns of substrate utilization differed considerably among isolates. A biofilm formation assay showed that isolates displayed a nearly four-fold range in their capacity for biofilm development. Inoculation experiments indicated that all isolates formed nodules on chickpea, but they exhibited more than a two-fold range in symbiotic efficiency. No nodules were observed on four other legumes (Phaseolus vulgaris, Pisum sativum, Lens culinaris, and Vigna mungo). Concatenated sequences from six loci (gap, edD, glnD, gnD, rpoB, and nodC) supported the assignment of all isolates to the species Mesorhizobium ciceri, with strain M. ciceri Ca181 as their closest relative.

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter P_cuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter P_cuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

Myxococcus xanthus DK1622 is a model system for studying multicellular development, predation, cellular differentiation, and evolution. Furthermore, it is a rich source of novel secondary metabolites and is widely used as heterologous expression host of exogenous biosynthetic gene clusters. For decades, genetic modification of M. xanthus DK1622 has mainly relied on kanamycin and tetracycline selection systems. Here, we introduce an alternative selection system based on chloramphenicol (Cm) to broaden the spectrum of available molecular tools. A chloramphenicol-resistant growth phase and a chloramphenicol-susceptible growth phase before and after chloramphenicol-induction were prepared, and later sequenced to identify specific genes related to chloramphenicol-repercussion and drug-resistance. A total of 481 differentially expressed genes were revealed in chloramphenicol-resistant Cm5_36h and 1920 differentially expressed genes in chloramphenicol-dormant Cm_8h. Moreover, the gene expression profile in the chloramphenicol-dormant strain Cm_8h was quite different from that of Cm5_36 which had completely adapted to Cm, and 1513 differentially expression genes were identified between these two phenotypes. Besides upregulated acetyltransferases, several transporter encoding genes, including ABC transporters, major facilitator superfamily transporters (MFS), resistance-nodulation-cell division (RND) super family transporters and multidrug and toxic compound extrusion family transporters (MATE) were found to be involved in Cm resistance. After the knockout of the most highly upregulated MXAN_2566 MFS family gene, mutant strain DK-2566 was proved to be sensitive to Cm by measuring the growth curve in the Cm-added condition. A plasmid with a Cm resistance marker was constructed and integrated into chromosomes via homologous recombination and Cm screening. The integration efficiency was about 20% at different concentrations of Cm. This study provides a new antibiotic-based selection system, and will help to understand antibiotic resistance mechanisms in M. xanthus DK1622.

Tobacco, Nicotiana tabacum L., is produced largely in China (~1/3 of the global market). In the monsoon summer of 2020, tobacco plant petioles, where axillary buds were removed, became black-rotten, and thick ooze appeared, when squeezed. Lesions encompassed more than half of petiole circumference. Ten tobacco fields (100 plant/field) were investigated in Liuyang, China and 5% disease severity founded in each infected field (Fig. 1A, B, C). Six infected stalks leave of different tobacco were sampled from severe field in Liuyang (N28°21', E113°52') and were surface sterilized (1% sodium hypochlorite for 3 min.), rinsed thrice in sterile distilled water, grounded, and streaked on Luria Bertani agar (LBA). After 24 hours at 28ºC, circular and convex colonies appeared. Hundred colony from ten plates were picked, amplified, and sequenced with the primer 16S-27F/16S-1492R by colony PCR (Lane et al. 1991). 16S rRNA sequence from 100 colony were assembled and fell into two sequences, either similar to Leclercia sp. (86%), or Pantoea sp. (14%). Identification and homology search was done by BLASTn analysis against NCBI and the EzBio Cloud database (Yoon et al. 2017). The Pantoea isolate HN-23 (1,408 bp, MW405831) and the other 16S sequence of 13 Pantoea showed 99.57% identity to the type strain P. endophyitca 596T (PJRT0100022) based on the EzBio Cloud database to identify novel bacteria. Colonies of HN-23 were smooth, translucent, convex with entire margin on LBA, and 1mm and 3mm (diameter), white to yellow, after 24h and 48h (Fig.1 H, I), respectively but white (Fig.1 J, K) on Nutrient Agar (NA). Phenotype of HN-23 (S-1) was performed using API 20E and API ZYM system (bioMérieux, France) and found identical to P. endophytica 596T (Gao et al. 2019). Draft genome of HN-23 (size 4.96Mbp, total Scaffold 79, Scaf N50 218,098bp and Scaf N90 61,041bp) was studied by Illumina sequencing (JAFLWX000000000) and was found to have 98.24% nucleotide identity with the genome of P. endophytica type strain 596. Average nucleotide identity (ANI) values were calculated using Ortho ANIu algorithm (Yoon et al 2017a). HN-23 had 83.89% and 83.65% ANI with P. rodasii LMG26273T and P. dispersa CCUG25232T, respectively (S-2). Six tobacco seedlings (cultivar K326, 30cm height plants grown at greenhouse at 28℃ and 70-80% humidity) were injected by 20μl of culture (109 CFU/ml) of HN-23 and three with dominant species Leclercia sp. HN-7, and reisolated from infected tissues. Pathogenic tissue extract and sterile water were also used as positive and negative control, respectively and experiments were performed in triplicate. After 20h, symptoms of water-soaked decay appeared in the injected leaf axils (Fig. 1D). After 2 days, a severe rot is developed (Fig.1 E). Though, the controls were symptomless (Fig.1 F, G). The bacterium was then isolated from the rotten tissues and identity was confirmed by 16S rDNA sequencing, thus fulfilling Koch's postulates. This species was also reported as endophytes to be isolated from root, stem and leaf of maize planted in diverse parts of China and identified as P. endophytica. To our knowledge, this is the first report of P. endophytica as a plant pathogen, which was firstly isolated from Tobacco planted in southern China.