Patricia Armshaw

Ultrasonic intensification as a tool for enhanced microbial biofuel yields

Ultrasonication has recently received attention as a novel bioprocessing tool for process intensification in many areas of downstream processing. Ultrasonic intensification (periodic ultrasonic treatment during the fermentation process) can result in a more effective homogenization of biomass and faster energy and mass transfer to biomass over short time periods which can result in enhanced microbial growth. Ultrasonic intensification can allow the rapid selective extraction of specific biomass components and can enhance product yields which can be of economic benefit. This review focuses on the role of ultrasonication in the extraction and yield enhancement of compounds from various microbial sources, specifically algal and cyanobacterial biomass with a focus on the production of biofuels. The operating principles associated with the process of ultrasonication and the influence of various operating conditions including ultrasonic frequency, power intensity, ultrasonic duration, reactor designs and kinetics applied for ultrasonic intensification are also described.

Generation and analysis of an ICE R391 deletion library identifies genes involved in the element encoded UV-inducible cell-sensitising function

ICE R391, a prototype member of the SXT/R391 family of site-specific integrative conjugative elements (ICEs), frequently isolated from enterobacterial pathogens, exhibits an unusual, recA-dependent, UV-inducible, cell-sensitising function. This significantly decreases postirradiation cell survival rates in Escherichia coli host cells, a trait that would at first appear to be counterproductive in terms of adaptation to stress conditions. Construction and screening of a complete ICE R391 deletion library in E. coli identified three ICE R391 genes, orfs90/91, encoding a putative transcriptional enhancer, and orf43, encoding a putative type IV secretion system outer membrane-associated conjugative transfer protein, in the cell-sensitising function. Cloning and complementation of these genes confirmed their involvement in UV sensitising. Expression of both orfs90/91 and orf43 in wild-type E. coli indicated that orf43 encodes a cytotoxic gene product upon up-regulation. Deletion of the orf43 homologue in SXT, s050, also abolished its associated UV sensitisation. We hypothesise that ICE R391 and other members of the SXT/R391 family display decreased survival rates upon exposure to UV irradiation through the induction of orf43.

Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production.

BackgroundThe use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation.ResultsA native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.ConclusionsA significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.

Control of expression of the ICE R391 encoded UV-inducible cell-sensitising function

BackgroundMany SXT/R391-like enterobacterial Integrative Conjugative Elements (ICEs) have been found to express an atypical, recA-dependent, UV-inducible, cell-sensitising phenotype observed as a reduction in post-irradiation cell survival rates in host cells. Characterisation of a complete deletion library of the prototype ICE R391 identified the involvement of three core ICE genes, orfs90/91 encoding a putative transcriptional enhancer complex, and orf43, encoding a putative type IV secretion system, outer membrane-associated, conjugative transfer protein.ResultsIn this study, expression analysis of orf43 indicated that it was up-regulated as a result of UV irradiation in an orfs90/91-dependent manner. Induced expression was found to be controlled from a site preceding the gene which required functional orfs90/91. Expression of orfs90/91 was in turn found to be regulated by orf96, a λ cI-like regulator. Targeted construction of ICE R391 deletions, RT-PCR and qRT-PCR analysis confirmed a regulatory link between orfs90/91 and orf43 while site-directed mutagenesis of orf43 suggested an association with the cell membrane was a prerequisite for the cytotoxic effect.ConclusionsBecause of the recA-dependence of the effect, we hypothesise that UV induction of RecA results in cleavage of the cI-like ICE-encoded repressor protein, the product of orf96. This in turn allows expression of the transcriptional enhancer complex encoded by orfs90/91, which we conclude stimulates transcription of orf43, whose product is directly responsible for the effect.

Analysis and comparative genomics of R997, the first SXT/R391 integrative and conjugative element (ICE) of the Indian Sub-Continent

The aim of this study was to analyse R997, the first integrative and conjugative element (ICE) isolated from the Indian Sub-Continent, and to determine its relationship to the SXT/R391 family of ICEs. WGS of Escherichia coli isolate AB1157 (which contains R997) was performed using Illumina sequencing technology. R997 context was assessed by de novo assembly, gene prediction and annotation tools, and compared to other SXT/R391 ICEs. R997 has a size of 85 Kb and harbours 85 ORFs. Within one of the variable regions a HMS-1 β-lactamase resistance gene is located. The Hotspot regions of the element contains restriction digestion systems and insertion sequences. R997 is very closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of R997 increases the knowledge of the earliest isolated SXT/R391 elements and may provide insight on the emergence of these elements on the Indian sub-continent.

Examination of the cell sensitizing gene orf43 of ICE R391 suggests a role in ICE transfer enhancement to recipient cells

SXT/R391 family of ICEs have been found to express an unusual function that enhances bacterial cell death post-UV irradiation. Previous analysis of ICE R391 found four core SXT/R391 ICE genes to be involved—orf96, orf90, orf91 and orf43. These genes functioned as part of a UV-inducible pathway, where upon exposure to UV, the levels of the Orf43 protein, a TraV homolog which we propose naming TraV(R391), were upregulated, resulting in increased cell sensitization. Here, we examined the effect of orf43 overexpression and found it led to host cell permeabilization. The inducing agent for orf43, UV irradiation, is also known to increase the ICE R391 extrachromosomal form and apparent conjugative transfer rate. We demonstrated, via conjugative transfer deficient mutants, that orf43 overexpression alone restored a small level of ICE R391 transfer to recipient cells via an unknown mechanism other than conjugation. TraV homologs have been reported to function in conjugative transfer. However, TraV(R391) is the first homolog to cause UV-associated cell sensitization. TraV(R391) when overexpressed must contain a unique adaptation or function which results in cell lysis and decreased survival. A hypothesis for retaining such a detrimental effect may be in its role of enhancing ICE survival upon cell damage.

Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry

Highlights • Flow cytometry was used to evaluate the effect of ethanol on Synechocystis strains.• The three Synechocystis strains behaved differently in the presence of ethanol.• UL 004 and UL 030 were more tolerant to the presence of ethanol than the WT strain.• The most efficient ethanol producer (UL030) was also the most tolerant to ethanol.

SXT/R391 Integrative and Conjugative Elements (ICEs) Encode a Novel ‘Trap-Door’ Strategy for Mobile Element Escape

Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

The Potential of the Photoautotroph Synechocystis for Metal Bioremediation

The photoautotrophic cyanobacterium Synechocystis PCC6803 has received much attention as a model photosynthetic cell factory for the production of a range of important biotech products. The biomass remaining from this activity may then have further utility in processes such as metal bioremediation. In addition Synechocystis being an inhabitant of many natural aquatic environments is seen as an environmen‐ tally friendly alternative to using chemical precipitation methodologies for metal remediation. Synechocystis produces a range of extracellular polysaccharide substan‐ ces (EPS) that can undergo modification as a function of culture age and growth nutrients which have been implicated in metal biosorption. Many studies have demonstrated that high levels of charged groups present in EPS are important in forming polymeric matrices with metallic ions allowing their biosorption. Genetic studies has revealed genes involved in such metal binding indicating that EPS can be modified for potential enhancement of binding or modification of the types of metals bound. The utility of metal binding to live and dead biomass of Synechocystis has been demonstrated for a range of metals including Cr(VI), Cd(II), Cu(II), Pb(II), Sb, Ni(II), Mn(II), Mn(IV), As(III), As(V), Cs and Hg. The potential of using Synechocystis as a biosorption platform is discussed.