- Volume 163, Issue 6, 2017

Multiple interacting factors affect the performance of engineered biological systems in synthetic biology projects. The complexity of these biological systems means that experimental design should often be treated as a multiparametric optimization problem. However, the available methodologies are either impractical, due to a combinatorial explosion in the number of experiments to be performed, or are inaccessible to most experimentalists due to the lack of publicly available, user-friendly software. Although evolutionary algorithms may be employed as alternative approaches to optimize experimental design, the lack of simple-to-use software again restricts their use to specialist practitioners. In addition, the lack of subsidiary approaches to further investigate critical factors and their interactions prevents the full analysis and exploitation of the biotechnological system. We have addressed these problems and, here, provide a simple‐to‐use and freely available graphical user interface to empower a broad range of experimental biologists to employ complex evolutionary algorithms to optimize their experimental designs. Our approach exploits a Genetic Algorithm to discover the subspace containing the optimal combination of parameters, and Symbolic Regression to construct a model to evaluate the sensitivity of the experiment to each parameter under investigation. We demonstrate the utility of this method using an example in which the culture conditions for the microbial production of a bioactive human protein are optimized. CamOptimus is available through: (https://doi.org/10.17863/CAM.10257).

An endophytic fungus, MC_25L, has been isolated from the leaves of MonardacitriodoraCerv. ex Lag., a medicinal and aromatic herb from the northwestern Himalayas. It produces a fruity fragrance while growing on potato dextrose agar, suggesting that it is producing volatile organic compounds (VOCs). The endophyte inhibited the growth of plant pathogens such asSclerotiniasp. and Aspergillusflavus by virtue of VOCs. Identification of MC_25L based on morphological and microscopic features, as well as ITS-based rDNA sequence analysis, revealed that it is a Fusariumsp. GC–MS analysis revealed that this endophyte produces a unique array of VOCs, in particular hexanal, p-fluoroanisole, pentafluoropropionic acid 2-ethylhexyl, (5E)-5-ethyl-2-methyl-5-hepten-3-one, 2-butyl-2-hexanol, (7E)-2-methyl-7-hexadecene and acoradiene. Three major compounds were hexanal, (5E)-5-ethyl-2-methyl-5-hepten-3-one and acoradiene, and they account for around 84.57 % of the total VOCs. Moreover, of interest was the presence of hexanal, which has applications in the food and cosmetic industries, as well as in mycofumigation. This is the first report of a fungal endophyte producing the industrially important plant-like VOC hexanal. Hexanal is also active biologically. Thus this study indicates that Fusariumsp. (MC_25L) is a potential candidate for the up-scaling of hexanal.

Escherichia coli has two machineries for the synthesis of FeS clusters, namely Isc (iron–sulfur cluster) and Suf (sulfur formation). The Isc machinery, encoded by the iscRSUA-hscBA-fdx-iscXoperon, plays a crucial role in the biogenesis of FeS clusters for the oxidoreductases of aerobic metabolism. Less is known, however, about the role of ISC in the maturation of key multi-subunit metalloenzymes of anaerobic metabolism. Here, we determined the contribution of each iscoperon gene product towards the functionality of the major anaerobic oxidoreductases in E. coli, including three [NiFe]-hydrogenases (Hyd), two respiratory formate dehydrogenases (FDH) and nitrate reductase (NAR). Mutants lacking the cysteine desulfurase, IscS, lacked activity of all six enzymes, as well as the activity of fumaratereductase, and this was due to deficiencies in enzyme biosynthesis, maturation or FeS cluster insertion into electron-transfer components. Notably, based on anaerobic growth characteristics and metabolite patterns, the activity of the radical-S-adenosylmethionine enzyme pyruvate formate-lyase activase was independent of IscS, suggesting that FeS biogenesis for this ancient enzyme has different requirements. Mutants lacking either the scaffold protein IscU, the ferredoxin Fdx or the chaperones HscA or HscB had similar enzyme phenotypes: five of the oxidoreductases were essentially inactive, with the exception being the Hyd-3 enzyme, which formed part of the H2-producing formate hydrogenlyase (FHL) complex. Neither the frataxin-homologue CyaY nor the IscX protein was essential for synthesis of the three Hyd enzymes. Thus, while IscS is essential for H2 production in E. coli, the other ISC components are non-essential.

Isolates of Escherichia coli, except Shigella, are generally prototrophic; they do not require any growth factors to grow in mineral medium. However, a nicotinic acid requirement is common among B2 phylogroup STc95 O18 E. coli clone strains. Nicotinic acid is a precursor of nicotinamide adenine dinucleotide (NAD), an essential molecule that plays central role in cellular metabolism. The defect in NAD synthesis of these strains is due to alterations in de novo biosynthesis pathway nadB gene. Here, by studying growth on minimal medium with glycolytic (glucose) or gluconeogenic (pyruvate or succinate) substrates as the carbon supply in a large panel of E. coli natural isolates representative of the species diversity, we identify an absolute nicotinic acid requirement in non-STc95 strains due in one case to a nadA inactivation. The growth on glucose medium of some extraintestinal pathogenic E. coli strains belonging to various non-O18 B2 phylogroup STc95 clones is restored either by aspartate or nicotinate, demonstrating that the nicotinic acid requirement can also be due to an intracellular aspartate depletion. The auxotrophic requirements depend on the carbon source available in the environment. Moreover, some strains prototrophic in glucose medium become auxotrophic in succinate medium, and conversely, some strainsauxotrophic in glucose medium become prototrophic in succinate medium. Finally, a partial depletion of intracellular aspartate can be observed in some prototrophic strains belonging to various phylogroups. The observed more or less significant depletion according to isolates may be due to differences in tricarboxylic acid cycle enzyme activities. These metabolic defects could be involved in the adaptation of E. coli to its various niches.

Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacter jejuni this reversible adaptive process is mediated by mutations in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase for this CPS modification, cj1421, and two other phase-variable CPS genes generated phage resistance in C. jejuni. Here we investigate the population dynamics of C. jejuni NCTC11168 when exposed to phage F336 in vitro using a newly described method – the 28-locus-CJ11168 PV analysis. Dynamic switching was observed in the ON/OFF states of three phase-variable CPS genes, cj1421, cj1422 and cj1426, during phage F336 exposure, with the dominant phage-resistant phasotype differing between cultures. Although loss of the phage receptor was predominately observed, several other PV events also led to phage resistance, a phenomenon that increases the chance of phage-resistant subpopulations being present in any growing culture. No other PV genes were affected and exposure to phage F336 resulted in a highly specific response, only selecting for phase variants of cj1421, cj1422 and cj1426. In summary, C. jejuni may benefit from modification of the surface in multiple ways to inhibit or reduce phage binding, thereby ensuring the survival of the population when exposed to phages.

Cyclic-di-GMP is an ubiquitous second messenger in bacteria. Several c-di-GMP receptor proteins have been identified to date, and downstream signalling pathways are often mediated through protein–protein interactions. The photoreceptor Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 comprises three domains related to c-di-GMP metabolism: two GGDEF and one EAL domain. It has been shown that the C-terminal GGDEF domain acts as blue-light triggered c-di-GMP producer thereby inhibiting motility of the cells in blue light. The specific function of the other two c-di-GMP related domains remained unclear. In this study, we test knockout mutants of potential interaction partners of Cph2 for altered phototactic behaviour. Whereas wild-type cells are non-motile under high-intensity red light of 640 nm, the mutant Δslr1143 displays positive phototaxis. This phenotype can be complemented by overexpression of full-length Slr1143, which also results in an increased cellular c-di-GMP concentration. However, the non-motile phenotype of wild-type cells under high-intensity red light appears not to be due to an elevated cellular c-di-GMP content. Using co-precipitation and yeast two-hybrid assays, we demonstrate that the GGDEF domain of Slr1143 interacts with the EAL and the GGDEF domains of Cph2. However, under the test conditions, the interaction of the two proteins is not light-dependent. We conclude that Slr1143 is a new Cph2-interacting regulatory factor which modulates motility under red light and accordingly we propose Cip1 (Cph2-interacting protein 1) as a new designation for this gene product.