Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli

In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Generic Business Process Model for SMEs in M-Commerce Based on Talabat’s Case Study

SMEs face a variety of challenges in their attempts to keep up with the cyber revolution, even though SMEs are a major part of the world economy. In a previous publication, the authors established that ‘B2C’ model does not accurately represent or support SMEs in M-Commerce. Instead, the authors reviewed SMEs and SME supporting apps from mobile app marketplaces and suggested a model called ‘B2i2C’. In this model, the ‘i’, in the form of intermediary business entity are playing a vital role in SMEs breakthrough into M-Commerce. Following on, this paper reviews business processes to generate a generic model adaptable to a variety of SME related products and services. This paper presents the case study of Talabat, one of the most successful GCC e-business models that supports SMEs to have come out from Kuwait. The information collected from online resources, student placements and feedback from operation managers attempt to emulate the business process model for a variety of ‘B2i2C’ business models. The generic model is then tested against three different scenarios to identify the level of similarity. The results demonstrate a high degree of adaptability of the model and a major opportunity to explore in the area of SME supporting app in M-Commerce

The champion of urban water resources management in the Chinese city — the case of Ningbo

Ningbo is a coastal megacity located at the East Coast of China and developing rapidly with proactive trading and export economic activities. The city owns a ranked top ten international port and it is the major strategic spot of 21st century maritime Silk Road from the “Belt and Road” policy established for promoting further international trades and developments. In future, populations and economy in Ningbo are expected continuously growing in the next few decades. The demand of quality freshwater resources thus is enormously increasing. Ningbo municipal government has established the “Five water management” (五水共治) policy in 2013 that aims to manage (i) sewage discharge; (ii) flooding; (iii) surface water; (iv) water conservation and (v) freshwater supply. Indeed, the municipal government also liaised and initiated the “Sponge City Program” after 2015 that Ningbo was selected as one of the pilot city; these policies and practices are successful up to now. This article adopts the case study of Ningbo to investigate the reasons of municipal government to promote the policy, to understand the public perception of this water management policy in Ningbo through conducted semi-structured interviews. During the 2017 and 2019, we conducted a questionnaire (N = 110) and interviews (N = 10) that follow up for justification of the public perception with the local communities. Our findings indicated that the communities had not been engaged closely with these practices, but generally supporting these two urban water management practices; and agreed that the urban water conditions (urban floods and pollution) had been improved. Also, the article discusses whether these (5 Water and SCP) practices can be extensively applied in other Chinese cities. We will provide recommendations at the end of the article

Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. [Image: see text