Combined Physical and Genetic Map of the Pseudomonas putida KT2440 Chromosome

A combined physical and genetic map of the Pseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with PmeI, SwaI, I-CeuI, and PacI as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and Escherichia coli, as P. putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the P. putida genome eluted from agarose gels. As an alternative, 10 random mini-Tn5-Km mutants of P. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as oriC, 6 rrn loci (rnnA to -F), recA, ftsZ, rpoS, rpoD, rpoN, and gyrB; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of P. putida KT2440 was compared to those of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.     

Mapping analysis of the Xylella fastidiosa genome

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.fastidiosa sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5′-ATTTAAAT-3′),PacI (5′-TTAATTAA-3′), andPmeI (5′-GTTTAAAC-3′) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.     

Genetic tools for reliable gene expression and recombineering in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

NSIT: Novel Sequence Identification Tool

Novel sequences are DNA sequences present in an individual''s genome but absent in the human reference assembly. They are predicted to be biologically important, both individual and population specific, and consistent with the known human migration paths. Recent works have shown that an average person harbors 2–5 Mb of such sequences and estimated that the human pan-genome contains as high as 19–40 Mb of novel sequences. To identify them in a de novo genome assembly, some existing sequence aligners have been used but no computational method has been specifically proposed for this task. In this work, we developed NSIT (Novel Sequence Identification Tool), a software that can accurately and efficiently identify novel sequences in an individual''s de novo whole genome assembly. We identified and characterized 1.1 Mb, 1.2 Mb, and 1.0 Mb of novel sequences in NA18507 (African), YH (Asian), and NA12878 (European) de novo genome assemblies, respectively. Our results show very high concordance with the previous work using the respective reference assembly. In addition, our results using the latest human reference assembly suggest that the amount of novel sequences per individual may not be as high as previously reported. We additionally developed a graphical viewer for comparisons of novel sequence contents. The viewer also helped in identifying sequence contamination; we found 130 kb of Epstein-Barr virus sequence in the previously published NA18507 novel sequences as well as 287 kb of zebrafish repeats in NA12878 de novo assembly. NSIT requires 2GB of RAM and 1.5–2 hrs on a commodity desktop. The program is applicable to input assemblies with varying contig/scaffold sizes, ranging from 100 bp to as high as 50 Mb. It works in both 32-bit and 64-bit systems and outperforms, by large margins, other fast sequence aligners previously applied to this task. To our knowledge, NSIT is the first software designed specifically for novel sequence identification in a de novo human genome assembly.     

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly

Background

The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds.

Results

A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed.

Conclusions

By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1196-3) contains supplementary material, which is available to authorized users.     

Species-specific repetitive extragenic palindromic (REP) sequences in Pseudomonas putida

Pseudomonas putida KT2440 is a soil bacterium that effectively colonises the roots of many plants and degrades a variety of toxic aromatic compounds. Its genome has recently been sequenced. We describe that a 35 bp sequence with the structure of an imperfect palindrome, originally found repeated three times downstream of the rpoH gene terminator, is detected more than 800 times in the chromosome of this strain. The structure of this DNA segment is analogous to that of the so-called enterobacteriaceae repetitive extragenic palindromic (REP) sequences, although its sequence is different. Computer-assisted analysis of the presence and distribution of this repeated sequence in the P.putida chromosome revealed that in at least 80% of the cases the sequence is extragenic, and in 82% of the cases the distance of this extragenic element to the end of one of the neighbouring genes was <100 bp. This 35 bp element can be found either as a single element, as pairs of elements, or sometimes forming clusters of up to five elements in which they alternate orientation. PCR scanning of chromosomes from different isolates of Pseudomonas sp. strains using oligonucleotides complementary to the most conserved region of this sequence shows that it is only present in isolates of the species P.putida. For this reason we suggest that the P.putida 35 bp element is a distinctive REP sequence in P.putida. This is the first time that REP sequences have been described and characterised in a group of non-enterobacteriaceae.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Unravelling the thioesterases responsible for propionate formation in engineered Pseudomonas putida KT2440

Pseudomonas putida KT2440 is becoming a new robust metabolic chassis for biotechnological applications, due to its metabolic versatility, low nutritional requirements and biosafety status. We have previously engineered P. putida KT2440 to be an efficient propionate producer from L-threonine, although the internal enzymes converting propionyl-CoA to propionate are not clear. In this study, we thoroughly investigated 13 genes annotated as potential thioesterases in the KT2440 mutant. One thioesterase encoded by locus tag PP_4975 was verified to be the major contributor to propionate production in vivo. Deletion of PP_4975 significantly decreased propionate production, whereas the performance was fully restored by gene complement. Compared with thioesterase HiYciA from Haemophilus influenza, thioesterase PP_4975 showed a faster substrate conversion rate in vitro. Thus, this study expands our knowledge on acyl-CoA thioesterases in P. putida KT2440 and may also reveal a new target for further engineering the strain to improve propionate production performance.     

A versatile Pseudomonas putida KT2440 with new ability: selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid

Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.

     

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.     

Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440

We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.