tRNA, tmRNA and some small RNA genes are recognized as general integration hotspots of genomic islands (GIs). The GMP synthase gene (guaA) has been firstly identified as one insertion hotspot of foreign DNA fragments. Thirty four islands integrated into the guaA genes were identified in the 987 completely sequenced archaeal and bacterial genomes. These alien islands were widely distributed within the host strains belonging to Proteobacteria, Firmicutes and Actinobacteria. The analysis of structural characteristics of these GIs is important for further determination of the island mobility and transference into suitable hosts. The putative functional integrases encoded by guaA-associated islands were mainly composed of phage P4 integrases, and followed by phage PhiLC3 integrases. Interestingly, island-encoding AlpA is close to P4 integrase and is deduced to be the positive transcriptional regulatory factor of P4 integrase while the XRE protein is close to PhiLC3 integrase and may be the negative transcriptional regulatory factor of PhiLC3 integrase. An 8-bp consensus sequence (5′-GAGTGGGA-3′) within the direct repeats of these GIs is the cutting site of the P4 integrases encoding by guaA-associated islands, in which the third nucleotide (G) is the key site. The large-scale investigation of the content of GMP synthase gene hotspots may be useful to find important functional islands within members of many key bacterial species and to transfer useful islands into more suitable hosts.Graphical abstractHighlights► Thirty-four genomic islands integrated into the guaA genes were identified in Proteobacteria, Firmicutes and Actinobacteria. ► Most of the integrases are phage P4 integrase. ► AlpA is the positive transcriptional regulatory factor of P4 integrase. ► An 8-bp consensus sequence (5′-GAGTGGGA-3′) is the cutting site of the P4 integrases, in which the third nucleotide (G) is the key site.

Thailandepsin A is natural product of Burkholderia thailandensis E264 with potent histone deacetylase inhibitory activities and promising anticancer activities. The titer of thailandepsin A is very low (less than 10 mg/l) from limited empirical fermentation. To facilitate preclinical evaluations and potentially clinical development of thailandepsin A, systematic optimization and extractive fermentation of thailandepsin A from B. thailandensis E264 culture in flasks were investigated in this pilot study. The main fermentation parameters—28°C, pH 7.0, inoculum ratio 1% (v/v), incubation duration 60 h, medium volume 26%, shaking speed 170 rpm, and chloroform as extracting solvent—were determined by single factor experiments. Polyaromatic adsorbent resin Diaion HP-20, when added at a concentration of 4% (w/v), was most effective to reduce feedback inhibition of thailandepsin A and to significantly increase the titer of target product. Central composite design was used to further optimize the fermentation medium for B. thailandensis E264. The optimized medium contains glucose 17.89 g/l, tryptone 34.98 g/l, potassium phosphate 24.84 g/l, and sodium citrate 0.01 g/l, which resulted in a large increase of the titer of thailandepsin A to 236.7 mg/l. Finally kinetic models based on the modified logistic and Luedeking–Piret equations were developed, delivering a good description of temporal variations of biomass, product, and substrate in the fermentation process, which could be used as references for developing large-scale fermentation.

A slow-release formula of potential biological pesticide Pyoluteorin (Plt) was prepared by using nanophase material of silicon dioxide loading drugs. The final experimental formula was m(Plt:Brij56:TMOS:HCl(aq)) = 0.04:1.4:2:1, synthesized by a highly ordered monolith (HOM) method. This formula can continuously release 85.13 ± 2.03 % of Plt within 28 days. A characterization study showed the formula formed a well-ordered mesoporous structure, with a surface area of 822 m2 g−1, with a measured mesoporous volume of 0.41 cm3 g−1 and a narrow distribution for the pore size centered at 2.4 nm. A bioactivity experiment showed it authentically prolonged the antifungal effects. This study is the first to report mesoporous formulations for biological pesticides and indicates a potentially interesting drug carrier. The association of a nanostructured silica to the molecular state of the drug holds great interests for field applications as it overcomes the rapid loss of biological function during drug utilities.

Mobile genomic islands (GIs) can be excised from the chromosome, then form a circular intermediate and be reintegrated into the chromosome by the GI internal integrase. Some mobile GIs can also be transferred into a new receptor cell by transformation, conjugation, or transduction. The action sites of the integrase are usually flanked direct repeats (DRs) of the GIs. Accurate localization of the flanking sequences is a precondition for determining the mobility of the GI. Mobile GIs are generally associated with transfer RNAs (tRNAs). Based on the correlation between flanking sequences and tRNA sequences, the flanking sequences of 11 putative mobile GIs in Pseudomonas aeruginosa PAO1, P. aeruginosa PA14, P. fluorescens Pf-5 and P. fluorescens Pf0-1 were identified. Among the 11 GIs, Pf0-1GI-1 is responsible for benzoate degradation. PAO1GI-1, Pf5GI-2, Pf5GI-3, and Pf5GI-4 were confirmed experimentally to be excised from a chromosome to form a circular intermediate. The action sites of the integrases are these GIs direct repeats. Due to distinct DRs, cutting sites for the internal integrase of PAO1GI-1, Pf5GI-2, Pf5GI-3 and Pf5GI-4 were determined outside the T-loop of the tRNAGly gene, outside the anticodon loop of the tRNASer gene and tRNALys gene, and at the asymmetric 3′-end of the tRNALeu gene, respectively. PAO1GI-1 and other mobile GIs may be transferred into many different strains that belong to different phyla because of the clear flanking sequences. This study describes basic information about the action sites of the integrases, assesses the mobility of GIs, and can help design and transfer mobile GIs to candidate strains.

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 μg ml−1, compared with just 5 μg ml−1 for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.

tmRNA, a combination of a tRNA-related fragment and a small mRNA fragment, was confirmed as the integration site of genomic islands (GIs). Using sequence alignment and comparative genomics, 68 GIs associated with tmRNA genes were identified among 13 genera of Enterobacteriaceae. Among them, 53 GIs were found in Escherichia coli and Salmonella enterica. Among these 53 GIs, tandem GIs were verified in eight S. enterica and two E. coli chromosomes. The downstream regions of the tmRNA genes in most of the E. coli and S. enterica chromosomes include one GI or tandem GIs region and a remnant variable region distal to the tmRNA. The chronology of integration of tandem GIs into the genome indicated that GIs farther from the tmRNA were incorporated into the genome earlier than those nearer from the tmRNA. The integrases of the tmRNA gene-associated GIs can be further categorized into three subtypes: HP1 integrases, PhiCTX integrases, and P4 integrases, which are the most predominant. The GIs were first integrated into the chromosome by the P4 integrase, subsequently by the PhiCTX integrase, and finally by the HP1 integrase. Thus, the tmRNA gene is an important site for investigating the genetics and evolution of tandem GIs.

Based on three distinct traits of genomic islands, a novel approach was developed to search for and determine genomic islands in special strains. Two genomic islands in Pseudomonas aeruginosa PAO1 and 7 genomic islands in Pseudomonas aeruginosa PA14 were defined with this method. Among the 9 genomic islands, 4 islands had been characterized before, while the other 5 islands were initially determined. The insert sites of 6 genomic islands are tRNA sequences, direct repeats of PA14GI-3 are relative to tRNALeu, and direct repeats of PA14GI-2 are at the 3′ end of bifunctional GMP synthase/glutamine amidotransferase. Only direct repeats of PA14GI-4 are not clear. Among the 5 newly-found genomic islands, it was supposed that PA14GI-2 is a genomic island related to Hg2+ uptake, PA14GI-3 is a secretory activity genomic island, PA14GI-6 is a pathogenicity island, and functions of PA14GI-1 and PA14GI-5 are not clear. Finally, the tyrosine type integrases in PAO1GI-1, PA14GI-5 and PA14GI-7 were analyzed, and their binding and restriction sites were predicted.

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l−1 of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l−1), glucose (12.7 g l−1), soy peptone (10.9 g l−1), and ethanol (13.8 ml l−1) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l−1, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l−1 PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.

The solubility of pyoluteorin (Plt) in water, dichloromethane, chloroform, and carbon tetrachloride was determined using an analytical method over the temperature range of (278.2 to 333.2) K. The solubility of Plt in chloroform is much higher than that in water, dichloromethane, and carbon tetrachloride. The solubility of Plt increases with increasing temperature in the four solvents, and all measurements were correlated with the Apelblat equation.

•The comparative genomic analysis showed that P. chlororaphis strains have 80% conserved genes.•Its competitive colonization indicates that P. chlororaphis can adapt well to its environment.•P. chlororaphis can synthesize different phenazine compounds and insecticidal proteins.•The plant growth-promoting activities and lack of virulence factor make P. chlororaphis suitable for applications.Pseudomonas chlororaphis HT66, a plant growth-promoting rhizobacterium that produces phenazine-1-carboxamide with high yield, was compared with three genomic sequenced P. chlororaphis strains, GP72, 30–84 and O6. The genome sizes of four strains vary from 6.66 to 7.30 Mb. Comparisons of predicted coding sequences indicated 4833 conserved genes in 5869–6455 protein-encoding genes. Phylogenetic analysis showed that the four strains are closely related to each other. Its competitive colonization indicates that P. chlororaphis can adapt well to its environment. No virulence or virulence-related factor was found in P. chlororaphis. All of the four strains could synthesize antimicrobial metabolites including different phenazines and insecticidal protein FitD. Some genes related to the regulation of phenazine biosynthesis were detected among the four strains. It was shown that P. chlororaphis is a safe PGPR in agricultural application and could also be used to produce some phenazine antibiotics with high-yield.