OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis.     

Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates.     

Arabidopsis sucrose transporter AtSUC9. High-affinity transport activity, intragenic control of expression, and early flowering mutant phenotype

AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K(0.5) = 0.066 +/- 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alpha-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to beta-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.     

Elevated basal slippage mutation rates among the Canidae

The remarkable responsiveness of dog morphology to selection is a testament to the mutability of mammals. The genetic sources of this morphological variation are largely unknown, but some portion is due to tandem repeat length variation in genes involved in development. Previous analysis of tandem repeats in coding regions of developmental genes revealed fewer interruptions in repeat sequences in dogs than in the orthologous repeats in humans, as well as higher levels of polymorphism, but the fragmentary nature of the available dog genome sequence thwarted attempts to distinguish between locus-specific and genome-wide origins of this disparity. Using whole-genome analyses of the human and recently completed dog genomes, we show that dogs possess a genome-wide increase in the basal germ-line slippage mutation rate. Building on the approach that gave rise to the initial observation in dogs, we sequenced 55 coding repeat regions in 42 species representing 10 major carnivore clades and found that a genome-wide elevated slippage mutation rate is a derived character shared by diverse wild canids, distinguishing them from other Carnivora. A similarly heightened slippage profile was also detected in rodents, another taxon exhibiting high diversity and rapid evolvability. The correlation of enhanced slippage rates with major evolutionary radiations suggests that the possession of a "slippery" genome may bestow on some taxa greater potential for rapid evolutionary change.     

In vitro screening of potato against water-stress mediated through sorbitol and polyethylene glycol

With the objective to develop a practical and effective method of screening potato for drought tolerance, shoot and root growth in microtuber-derived plantlets was studied in vitro in three genotypes with known root mass production under field conditions. Different levels of water-stress were induced using five concentrations of either sorbitol or polyethylene glycol (PEG) in MS medium. Water potential of various media ranged from −0.80 MPa to −2.05 MPa. Water-stress in culture adversely affected plantlet growth, and genotypes differed for their responses. Genotype IWA-1 was less affected than IWA-3 and IWA-5. At the same level of water potential, sorbitol had lower adverse effect than PEG; the latter being sticky. Genotype × sorbitol and genotype × PEG interactions were significant. At 0.2 M sorbitol and 0.003 M PEG, IWA-1 had significantly more roots with higher total root length, root volume, as well as root-dry weight than those of IWA-3 and IWA-5, whereas the latter two genotypes were at par for all these characters. This pattern was similar to the reported pattern of these genotypes for root-dry weight under field conditions. It is concluded that in vitro screening of potato under specific and limited water-stress conditions may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.     

Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening

Background

In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF.

Methods

Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M₁/M₀ and M₂/M₀, respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping.

Results

Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M₂/M₀ but not LCI or M₁/M₀. There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes.

Conclusion

These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.     

Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC-MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as 'activation of plasma proteins involved in acute inflammatory response' and 'positive regulation of cholesterol esterification'. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.