Efforts to Minimise the Bacterial Genome as a Free-Living Growing System.

Exploring the minimal genetic requirements for cells to maintain free living is an exciting topic in biology. Multiple approaches are employed to address the question of the minimal genome. In addition to constructing the synthetic genome in the test tube, reducing the size of the wild-type genome is a practical approach for obtaining the essential genomic sequence for living cells. The well-studied Escherichia coli has been used as a model organism for genome reduction owing to its fast growth and easy manipulation. Extensive studies have reported how to reduce the bacterial genome and the collections of genomic disturbed strains acquired, which were sufficiently reviewed previously. However, the common issue of growth decrease caused by genetic disturbance remains largely unaddressed. This mini-review discusses the considerable efforts made to improve growth fitness, which was decreased due to genome reduction. The proposal and perspective are clarified for further accumulated genetic deletion to minimise the Escherichia coli genome in terms of genome reduction, experimental evolution, medium optimization, and machine learning.

Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields.

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain.

Experimental Evolution Expands the Breadth of Adaptation to an Environmental Gradient Correlated With Genome Reduction.

Whether and how adaptive evolution adjusts the breadth of adaptation in coordination with the genome are essential issues for connecting evolution with ecology. To address these questions, experimental evolution in five Escherichia coli strains carrying either the wild-type genome or a reduced genome was performed in a defined minimal medium (C0). The ancestral and evolved populations were subsequently subjected to fitness and chemical niche analyses across an environmental gradient with 29 combinations of eight chemical components of the minimal medium. The results showed that adaptation was achieved not only specific to the evolutionary condition (C0), but also generally, to the environmental gradient; that is, the breadth of adaptation to the eight chemical niches was expanded. The magnitudes of the adaptive improvement and the breadth increase were both correlated with genome reduction and were highly significant in two out of eight niches (i.e., glucose and sulfate). The direct adaptation-induced correlated adaptation to the environmental gradient was determined by only a few genome mutations. An additive increase in fitness associated with the stepwise fixation of mutations was consistently observed in the reduced genomes. In summary, this preliminary survey demonstrated that evolution finely tuned the breadth of adaptation correlated with genome reduction.

Immunogenicity and protection efficacy of enhanced fitness recombinant Salmonella Typhi monovalent and bivalent vaccine strains against acute toxoplasmosis.

The proficiency of Salmonella Typhi to induce cell-mediated immunity has allowed its exploitation as a live vector against the obligate intracellular protozoan Toxoplasma gondii. T. gondii vaccine research is of great medical value due to the lack of a suitable toxoplasmosis vaccine. In the present work, we integrated T. gondii antigen into a growth-dependent chromosome locus guaBA of S. Typhi CVD910 strain to form recombinant S. Typhi monovalent CVD910-SAG1 expressed T. gondii SAG1 antigen and monovalent CVD910-GRA2 expressed T. gondii GRA2 antigen. Furthermore, a low-copy stabilized recombinant plasmid encoding SAG1 antigen was transformed into CVD910-GRA2 to form bivalent CVD910-GS strain. An osmolarity-regulated promoter was also incorporated to control the gene transcription, whereas clyA export protein was included to translocate the antigen out of the cytoplasm. Both CVD910-GRA2 and CVD910-GS displayed healthy growth fitness and readily expressed the encoded T. gondii antigens. When administered in vivo, CVD910-GS successfully induced both humoral and cellular immunity in the immunized BALB/c mice, and extended mice survival against virulent T. gondii. In particular, the mice immunized with bivalent CVD910-GS presented the highest titers of IgG, percentages of CD4+ T, CD8+ T, B cells and memory T cells, and total IgG+ memory B cells as compared to the CVD910-GRA2 and control strains. The CVD910-GS group also generated mixed Th1/Th2 cytokine profile with secretions of IFN-É£, IL-2 and IL-10. This study demonstrated the importance of enhancing live vector fitness to sustain heterologous antigen expression for eliciting robust immune responses and providing effective protection against pathogen.

Transcriptomic Analysis of the Cold-Pretreated Larimichthys crocea Showing Enhanced Growth Fitness in Cold Water.

The large yellow croaker Larimichthys crocea is an economically important marine fish species endemic to China and East Asia. Ningde area of Fujian Province is a major L. crocea aquaculture and spawning center in China. L. crocea cultivated at the Zhoushan area appears to be popular but suffered high mortality in cold water during winter seasons. To reduce the mortality rate, we pretreated fish with cold shocks prior to shift to cold water. In this study, we show that cold-pretreated L. crocea 12 days after shift to cold water increase the viability by 5.77-fold compared to the unpretreated (live fish 75 versus 13, p value = 1.775e-06, n = 100). The highest loss of 31 out of 100 fish in the unpretreated group occurred in day 3 after temperature shift. To identify the pretreatment-induced transcriptional changes that may be attributed to cold-resistance and survival, we performed RNA-seq analysis of a total of 48 fish that were prior to and 48 h, 54 h, and 72 h after temperature shift in pretreated and unpretreated groups in sextuplicate. Transcriptomic profiling analysis indicates that pretreatment-induced transcriptional alterations of enzymes involved in FASI, ß-oxidation, PUFA synthesis, oxidative phosphorylation, and molecular chaperones persisted after temperature shift, suggesting that these metabolic pathways may play a role in L. crocea cold-resistance and survival. Our study provides insights on how the pretreatment enhances the L. crocea growth fitness in cold water.

Fitness Restoration of a Genetically Tractable Enterococcus faecalis V583 Derivative To Study Decoration-Related Phenotypes of the Enterococcal Polysaccharide Antigen.

Commensal and generally harmless in healthy individuals, Enterococcus faecalis causes opportunistic infections in immunocompromised patients. Plasmid-cured E. faecalis strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in Galleria mellonella and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the epa locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific epa-variable regions in host adaptation independently of other genetic variations.IMPORTANCEE. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle.

Coupling feedback genetic circuits with growth phenotype for dynamic population control and intelligent bioproduction.

Metabolic engineering entails target modification of cell metabolism to maximize cell's production potential. Due to the complexity of cell metabolism, feedback genetic circuits have emerged as basic tools to combat metabolic heterogeneity, enhance microbial cooperation as well as boost cell's productivity. This is generally achieved by applying social reward-punishment rules to eliminate cheaters and reward winners in a mixed cell population. With metabolite-responsive transcriptional factors to rewire gene expression, metabolic engineers are well-positioned to integrate feedback genetic circuits with growth fitness and achieve dynamic population control. Towards this goal, we argue that feedback genetic circuits and microbial interactions will be a golden mine for future metabolic engineering. We will summarize the design principles of engineering burden-driven feedback control to combat metabolic stress, implementing population quality control to eliminate cheater cell, applying product addiction to reward productive cell, as well as layering dual dynamic regulation to decouple cell growth from product formation. Collectively, these strategies will be useful to improve community-level cellular performance. Encoding such decision-marking functions and reprogramming cellular logics at population level will enable metabolic engineers to deliver robust cell factories and pave the way for intelligent bioproduction. We envision that various cellular regulation mechanisms and genetic/metabolic circuits could be exploited to achieve self-adaptive or autonomous metabolic function for diverse biotechnological and medical applications. Applying these design rules may offer us a genetic solution beyond bioprocess engineering strategies to further improve the cost-competitiveness of industrial fermentation.

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing.

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (∼28%) and well above the predicted error of Sanger sequencing (∼2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

The Escherichia coli transcriptome linked to growth fitness.

A series of Escherichia coli strains with varied genomic sequences were subjected to high-density microarray analyses to elucidate the fitness-correlated transcriptomes. Fitness, which is commonly evaluated by the growth rate during the exponential phase, is not only determined by the genome but is also linked to growth conditions, e.g., temperature. We previously reported genetic and environmental contributions to E. coli transcriptomes and evolutionary transcriptome changes in thermal adaptation. Here, we describe experimental details on how to prepare microarray samples that truly represent the growth fitness of the E. coli cells. A step-by-step record of sample preparation procedures that correspond to growing cells and transcriptome data sets that are deposited at the GEO database (GSE33212, GSE52770, GSE61739) are also provided for reference.

Simultaneous presence of fhs and purT genes is disadvantageous for the fitness of Escherichia coli growth.

In bacteria, alternate mechanisms are known to synthesize N(10)-formyltetrahydrofolate (N(10)-formyl-THF) and formyl glycinamide ribotide (fGAR), which are important in purine biosynthesis. In one of the mechanisms, a direct transfer of one carbon unit from formate allows Fhs to convert tetrahydrofolate to N(10)-formyl-THF, and PurT to convert glycinamide ribotide (GAR) to fGAR. Our bioinformatics analysis of fhs and purT genes (encoding Fhs and PurT) showed that in a majority of bacteria (∼94%), their presence was mutually exclusive. A large number of organisms possessing fhs lacked purT and vice versa. The phenomenon is so penetrating that even within a genus (Bacillus) if a species possessed fhs it lacked purT and vice versa. To investigate physiological importance of this phenomenon, we used Escherichia coli, which naturally lacks fhs (and possesses purT) as model. We generated strains, which possessed fhs and purT genes in singles or together. Deletion of purT from E. coli in the presence or absence of fhs did not confer a detectable growth disadvantage in pure cultures. However, growth competition assays revealed that the strains possessing either of the single genes outcompeted those possessing both the genes suggesting that mutual exclusion of purT and fhs in organisms confers fitness advantage in mixed cultures.