Pseudomonas strain NCIMB10586, in the P. fluorescens subgroup, produces the polyketide antibiotic mupirocin, and has potential as a host for industrial production of a range of valuable products. To ...underpin further studies on its genetics and physiology, we have used a combination of standard and atypical approaches to achieve a quality of the genome sequence and annotation, above current standards for automated pathways. Assembly of Illumina reads to a PacBio genome sequence created a retrospectively hybrid assembly, identifying and fixing 415 sequencing errors which would otherwise affect almost 5% of annotated coding regions. Our annotation pipeline combined automation based on related well-annotated genomes and stringent, partially manual, tests for functional features. The strain was close to P. synxantha and P. libaniensis and was found to be highly similar to a strain being developed as a weed-pest control agent in Canada. Since mupirocin is a secondary metabolite whose production is switched on late in exponential phase, we carried out RNAseq analysis over an 18 h growth period and have developed a method to normalise RNAseq samples as a group, rather than pair-wise. To review such data we have developed an easily interpreted way to present the expression profiles across a region, or the whole genome at a glance. At the 2-hour granularity of our time-course, the mupirocin cluster increases in expression as an essentially uniform bloc, although the mupirocin resistance gene stands out as being expressed at all the time points.
Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and ...Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.
Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.
P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.
Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging ...to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin.
High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via "mutasynthesis" that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates.
Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.
Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional ...polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged “in series” to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.
Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, ...sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate‐responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high‐level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single‐chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.
Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, such as IPTG, which can be expensive and chemically unstable. To cut the cost of RPP, Hothersall and co‐workers have developed new expression systems, which use nitrate, a cheap, stable and abundant inorganic ion, to induce high‐level RPP. As nitrate levels are high in many commercial fertilizers, they demonstrate that controlled RPP can even be achieved using inexpensive garden products purchased from any local store.
The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates ...transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions -44.5 and -77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position -44.5 or position -77.5. We show that NarL can also activate the ogt promoter when located at position -67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the -44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.
We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. ...Experiments using the expression of β-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1β and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production.
•Urea-induced recombinant protein production using the P. mirabilis ureD promoter.•Production of high value biopharmaceuticals with the uti101 expression system.•Fine tuning of expression levels by inducer concentration and thermoregulation.•Inexpensive recombinant protein production induced by urea, fertilisers and urine.
•The PAR promoter system can express recombinant proteins to many different levels.•The system is versatile, functioning in many E. coli strains and growth regimes.•Promoters are tightly regulated, ...allowing low-level expression of toxic proteins.
Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific “promoter activity ratings” (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.
The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. ...In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with ΔmupC and ΔmupO, ΔmupU, ΔmupV, or ΔmacpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V.
Type I polyketide synthases often use programmed β-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the ...terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where β-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.
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