The atypical organization of the luxI/R family genes in AHL-driven quorum-sensing circuits.

Quorum sensing (QS) is an elaborate regulatory mechanism associated with virulence and bacterial adaptation to the changing environment. QS is widespread in Proteobacteria and acts primarily through N-acylhomoserine lactone (AHL) signals. At the core of the AHL-driven QS systems are the AHL synthase gene (luxI family) and its cognate transcriptional regulator gene (luxR family). Several QS systems display one or more genes intervening between the luxI and luxR, in which gene arrangements are notably different due to the relative position and the transcriptional orientation between the essential luxI/R and the genes of location correlation. These adjacent genes may exert a regulatory impact on the primary QS genes or contribute toward an extension of QS regulatory control. In this review, we describe the organization of AHL-driven QS genes based on previous research and specific genome databases and provide new insights into these atypical QS gene arrangements.

Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.

Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9ppD10A ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.

rpeA, a global regulator involved in mupirocin biosynthesis in Pseudomonas fluorescens NCIMB 10586.

Mupirocin, a polyketide antibiotic produced by Pseudomonas fluorescens, is used as a topical antimicrobial treatment to cure various skin infections. Quorum sensing system plays an important role in regulation of mupirocin biosynthesis in P. fluorescens NCIMB 10586. In Pseudomonas, the RpeA/RpeB two-component signal transduction (TCST) system regulates quorum sensing system. However, the influences of the RpeA/RpeB TCST system on mupirocin production or other cell activities have not been studied. In this work, the homologous genes of rpeA and rpeB in P. fluorescens NCIMB 10586 were identified and inactivated in the chromosome, respectively. The deletion of rpeA reduced the mupirocin production from 160 in the wild-type to 21.3 mg/L along with slightly decreased cell growth, while no significant effected on mupirocin production in the rpeB mutant. Next, it was found that the RpeA/RpeB TCST system regulated the biosynthesis of mupirocin by modulating the quorum sensing system. Furthermore, untargeted metabolomics analysis was employed to detect the influences of RpeA on other cell activities modulated by quorum sensing system. Combined with quantitative real-time PCR, the results demonstrated that RpeA also regulated other cell activities including central carbon, amino acids, fatty acids, and purine metabolism. Overall, this study expands the current understanding of the RpeA/RpeB TCST system and provides several targets for increasing yields of mupirocin. KEY POINTS: • In P. fluorescens, the RpeA/RpeB TCST system regulates the biosynthesis of mupirocin. • RpeA modulates the cell activities through effecting the central carbon metabolism.

Heterologous expression of AHL lactonase AiiK by Lactobacillus casei MCJΔ1 with great quorum quenching ability against Aeromonas hydrophila AH-1 and AH-4.

BACKGROUND: Nowadays, microbial infections have caused increasing economic losses in aquaculture industry and deteriorated worldwide environments. Many of these infections are caused by opportunistic pathogens through cell-density mediated quorum sensing (QS). The disruption of QS, known as quorum quenching (QQ), is an effective and promising way to prevent and control pathogens, driving it be the potential bio-control agents. In our previous studies, AHL lactonase AiiK was identified with many characteristics, and constitutive expression vector pELX1 was constructed to express heterologous proteins in Lactobacillus casei MCJΔ1 (L. casei MCJΔ1). In this study, recombinant strain pELCW-aiiK/L. casei MCJΔ1 (LcAiiK) and wild-type Aeromonas hydrophila (A. hydrophila) were co-cultured to test the QQ ability of LcAiiK against A. hydrophila. RESULTS: A cell wall-associated expression vector pELCW for L. casei MCJΔ1 was constructed. Localization assays revealed that the expressed AiiK was anchored at the surface layer of LcAiiK via vector pELCW-aiiK. LcAiiK (OD600 = 0.5) degraded 24.13 µM of C6-HSL at 2 h, 40.99 µM of C6-HSL at 12 h, and 46.63 µM of C6-HSL at 24 h. Over 50% LcAiiK cells maintained the pELCW-aiiK plasmid after 15 generations of cultivation without erythromycin. Furthermore, LcAiiK inhibited the swimming motility, extracellular proteolytic activity, haemolytic activity and biofilm formation of A. hydrophila AH-1 and AH-4. CONCLUSION: The AHL lactonase AiiK is firstly and constitutively expressed at the surface layer of L. casei MCJΔ1. LcAiiK displayed considerable AHL lactonase activity and great QQ abilities against A. hydrophila AH-1 and AH-4 by attenuating their QS processes instead of killing them. Therefore, the LcAiiK can be exploited as an anti-pathogenic drug or a bio-control agent to control the AHL-mediated QS of pathogenic bacteria.

Pararcticibacter amylolyticus gen. nov., sp. nov., Isolated from a Rotten Hemp Rope, and Reclassification of Pedobacter tournemirensis as Pararcticibacter tournemirensis comb. nov.

A Gram-stain-negative, rod-shaped, non-motile, facultatively anaerobic bacterium, designated FJ4-8T, was isolated from a rotten hemp rope in Chongqing City, PR China. Phylogenetic analysis of 16S rRNA gene sequences indicated that the isolate was closely related to members of the family Sphingobacteriaceae, with the highest similarity to Pedobacter tournemirensis TF5-37.2-LB10T (95.3%) and low similarities to all other species of the genus Pedobacter (90.4-93.9%). Phylogenetic analyses demonstrated that strain FJ4-8T formed a stable subclade with Pedobacter tournemirensis TF5-37.2-LB10T. The clade with these two strains branched adjacent to a clade containing three species of the genus Arcticibacter. MK-7 was detected as the only respiratory quinone. The major fatty acids composed iso-C15:0, iso-C17:0 3-OH and summed feature three. Phosphatidylethanolamine, three aminophospholipids and one unidentified lipid were found as the major polar lipids. The major polyamine was identified as sym-homospermidine. The DNA-DNA hybridization value between strain FJ4-8T and Pedobacter tournemirensis TF5-37.2-LB10T was 42.0 ± 2.5%. Based on its phylogenetic, chemotaxonomic and phenotypic characteristics, the novel strain and TF5-37.2-LB10T were found to be different from members of genera Pedobacter and Arcticibacter. FJ4-8T and TF5-37.2-LB10T represented different species. Therefore, FJ4-8T should be classified as a novel species of a novel genus in the family Sphingobacteriaceae, for which the name Pararcticibacter amylolyticus gen. nov., sp. nov. is proposed. The type strain is FJ4-8T (= KCTC 62640T = CCTCC AB 2018052T). The draft genome sequence is 6290, 449 bp in length, the genomic DNA G+C content was 44.4 mol%. Pedobacter tournemirensis TF5-37.2-LB10T should be transferred to the novel genus as Pararcticibacter tournemirensis comb. nov. (The type strain is TF5-37.2-LB10T (= DSM 23085T = CIP 110085T = MOLA 820T).

Bio-detoxification Bacteria Isolated from Dye-Polluted Soils Promote Lactic Acid Production from Ammonia Pretreated Corn Stover.

Agro-stovers are the most abundant substrates for producing lactic acid, which has great potential application in the production of biodegradable and biocompatible polylactic acid polymers. However, chemical pretreatments on agro-stovers generate inhibitors that repress the subsequent lactic acid fermentation. In this study, three bacterial strains (Enterococcus faecalis B101, Acinetobacter calcoaceticus C1, and Pseudomonas aeruginosa CS) isolated from dye-polluted soils could utilize phenolic inhibitor mimics (vanillin, 4- hydroxybenzaldehyde, or syringaldehyde) from alkaline pretreated corn stovers as a sole carbon source. Lactic acid titer increased from 27.42 g/L (Bacillus coagulans LA204 alone) to 44.76 g/L (CS and LA204) using 50 g/L glucose with 1 g/L 4-hydroxybenzaldehyde added. Lactic acid production from 50 g/L ammonia pretreated corn stover was increased nearly twofold by inoculating phenolic degradation bacteria and lactic acid bacteria (C1& Lactobacillus pentosus FL0421). In the control (FL0421 alone), only 16.98 g/L of lactic acid was produced. The isolated and identified strains degraded the phenolic compounds and increased the lactic acid production from glucose and ammonia pretreated corn stover. These characteristics of the strains support industrial application with efficient in situ detoxification of phenolic compounds during lactic acid production from agro-stovers using simultaneous saccharification and fermentation (SSF).