Active transport and energy conservation in Escherichia coli

1. The main aim of this work was to compare the efficiencies of growth of various phenotypes of Escherichia coli. Several strains were grown in arithmetic (Wallace, 1975), chemostat and batch cultures. Values for maintenance energies were derived in order to calculate maximum growth yields in terms of carbon and oxygen. Efficiencies of ATP synthesis (expressed as P/O ratios) were calculated from yields using the assumption that the energy required for cellular biosynthesis under anaerobic conditions applied to the aerobic cultures. P/O ratios were then compared with H+/O ratios of the harvested bacteria, 2. The maintenance requirement was lower for arithmetic cultures than for chemostat cultures, suggesting that dropwise addition of nutrient in chemostat culture might uncouple cellular growth from energy supply. 3. E.coli ML30 (which is lac-inducible) had the same maintenance requirement as its lac-constitutive derivative, ML3O8, . when it was grown on a non-inducing carbon source. The gratuitous synthesis and turnover of the lac enzymes therefore make negligible contributions to maintenance. 4. There were at least two classes of reaction responsible for maintenance one of which was independent of temperature while the other was strongly temperature-dependent. The latter had an activation energy similar to that of protein denaturation, suggesting that part of the maintenance requirement may be for macromolecular organisation. 5. Bacteria grown on glucose, glycerol, maltose, galactose or mannitol had P/O ratios of 2, while those grown on lactose, gluconate or glucose 6-phosphate had P/O ratios of 3. The phenotypes with the higher efficiency of energy conservation all transported their carbon sources- by a proton-symport mechanism. 6. In pulse-oxidant determinations of H+/O ratios, cells with a P/O ratio of 3 extruded more protons than those with P/O ratio of 2, This is an independent confirmation of the conclusion derived from growth yields. 7. Growth on lactose in batch culture or in the chemostat also gave a P/O ratio of 3. High P/O ratios are therefore not peculiar to arithmetic culture. 8. Bacteria which had high P/O ratios had high maintenance coefficients. It is speculated that the additional maintenance requirement may be spent for organisation of a more efficient electron transport chain. 9. A period of logarithmic growth on the proton-symported substrate was required for the extra coupling site to be expressed, since cells which had previously been grown on glycerol with P/O ratio of 2, grew on lactose with a P/O ratio of 2 under carbon limitation in arithmetic culture. 10. E.coli ML30 grew on gluconate with a P/O ratio of 3. Therefore, the lac enzymes themselves were not required for the higher growth efficiency. 11. When bacteria which had been grown on lactose were starved for 2 h, they lost the additional energy-coupling site as judged by H+/O ratios. The lac permease also decayed, indicating a possible relationship between the two processes. 12. Cultures of E.coli ML3O8 grown on either lactose or glycerol had active lac permease, but only the lactose phenotype had its H+/O ratio enhanced by adding non-metabolisable galactosides. Transport in cells growing on lactose can therefore affect energy conservation. 13. Addition of salt (NaCl, Na2SO4) to growth media reduced the P/O ratios of cultures but did not significantly change their H+/O ratios. Salt must therefore either decrease the efficiency of the ATPase machinery or have an un-coupling effect on the membrane. 14. Cultures grew on lactose/glucose mixtures with a P/O ratio of 3. Therefore when the additional coupling site was present, all carbon sources were metabolised through the same system with high efficiency. 15. The additional coupling site found in lactose/grown bacteria was not due to energy-conserving transhydrogenase activity nor to an alternative energy-conserving terminal oxidase. In the absence of other experimental data on the composition of the electron transport chain of this phenotype, models are presented to explain the results in terms of novel electron carriers or rearrangement within the membrane of preexisting electron carriers. 16. It is concluded that an additional site of energy conservation is synthesised by E.coli in response to transport of carbon sources which enter the cell via proton symport, This conclusion implies a relationship between active transport and electron transport which have hitherto been assumed to work independently of each other

Bod1, a novel kinetochore protein required for chromosome biorientation

We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule–kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles

Bilateral symmetry of linear streptomycete chromosomes

Here, we characterize an uncommon set of telomeres from Streptomyces rimosus ATCC 10970, the parental strain of a lineage of one of the earliest-discovered antibiotic producers. Following the closure of its genome sequence, we compared unusual telomeres from this organism with the other five classes of replicon ends found amongst streptomycetes. Closed replicons of streptomycete chromosomes were organized with respect to their phylogeny and physical orientation, which demonstrated that different telomeres were not associated with particular clades and are likely shared amongst different strains by plasmid-driven horizontal gene transfer. Furthermore, we identified a ~50 kb origin island with conserved synteny that is located at the core of all streptomycete chromosomes and forms an axis around which symmetrical chromosome inversions can take place. Despite this chromosomal bilateral symmetry, a bias in parS sites to the right of oriC is maintained across the family Streptomycetaceae and suggests that the formation of ParB/parS nucleoprotein complexes on the right replichore is a conserved feature in streptomycetes. Consequently, our studies reveal novel features of linear bacterial replicons that, through their manipulation, may lead to improvements in growth and productivity of this important industrial group of bacteria

Multitargeted anti-infective drugs : resilience to resistance in the antimicrobial resistance era

The standard drug discovery paradigm of single molecule - single biological target - single biological effect is perhaps particularly unsuitable for anti-infective drug discovery. This is due to the rapid evolution of resistance likely to be observed with single target drugs. Multi-targeted anti-infective drugs (MTAIDs) are likely to be superior due to their lower susceptibility to target-related resistance mechanisms. Strathclyde Minor Groove Binders (S-MGBs) are a class of compounds which have been developed by adopting the MTAID paradigm, and their effectiveness against a wide range of pathogenic organisms is discussed. The renaming of this class to Strathclyde Nucleic Acid Binders (SNABs) is also presented due to their likely targets including both DNA and RNA

ActDES- a Curated Actinobacterial Database for Evolutionary Studies

Actinobacteria is a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic and made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last 20 years, the large size, complexity and high G+C content of many actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large-scale comparative genomic and evolutionary studies. To enable greater understanding and exploitation of actinobacterial genomes, specialized genomic databases must be linked to high-quality genome sequences. Here, we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome re-annotation. Utilizing this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. This article contains data hosted by Microreact

Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2)

Background: Streptomycete bacteria are prolific producers of specialised metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalisation or sub-functionalisation. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. Results: RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialised metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. Conclusions: Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters

Long-term outcome of thyrotoxicosis in childhood and adolescence in the west of Scotland: the case for long-term antithyroid treatment and the importance of initial counselling

Background: Thyrotoxicosis is both rarer and more severe in children than in adults, rendering management difficult and often unsatisfactory. Objective: To ascertain outcome in a geographically defined area of Scotland between 1989 and 2014. Method: Retrospective case note review with follow-up questionnaire to family doctors for patients with Graves’ disease and Hashimoto’s thyroiditis. Results: Sixty-six patients (58 females:8 males) comprising 53 with Graves’ disease and 13 with Hashimoto’s thyroiditis were diagnosed at median 10.4 (2.9–15.8) years and followed up for 11.8 (2.6–30.2) years. Antithyroid drug (ATD) therapy was stopped electively in 35 patients after 4.5 (1.5–8.6) years, resulting in remission in 10/13 Hashimoto’s thyroiditis and 10/22 Graves’ disease. Side effects occurred in 12 patients receiving carbimazole, six of whom changed to propylthiouracil; no adverse events occurred in the latter patients. Second-line therapy was given to 37 patients (34 with Graves’ disease), comprising radioiodine (22) at 15.6 (9.3–24.4) years for relapse (6), poor control/adherence (14) or electively (2); and surgery (16) at 12 (6.4–21.3) years for relapse (4), poor control/adherence (5) and electively (7). Adherence problems with thyroxine replacement were reported in 10/33 patients in adulthood. Conclusions: Hashimoto’s thyroiditis should be distinguished from Graves’ disease at diagnosis since the prognosis for remission is better. Remission rates for Graves’ disease are low (10/53 patients), time to remission variable and adherence with both ATD and thyroxine replacement often problematic. We recommend (a) the giving of long-term ATD rather than a fixed course of treatment in GD and (b) meticulous and realistic counselling of families from the time of diagnosis onwards

Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland

Multi-locus sequencing typing (MLST) is widely used to monitor the phylogeny of microbial outbreaks. However, several strains of vancomycin-resistant Enterococcus faecium (VREfm) with a missing MLST locus (pstS) have recently emerged in Australia, with a few cases also reported in England. Here, we identified similarly distinct strains circulating in two neighbouring hospitals in Scotland. Whole genome sequencing of five VREfm strains isolated from these hospitals identified four pstS-null strains in both hospitals, while the fifth was multi-locus sequence type (ST) 262, which is the first documented in the UK. All five Scottish isolates had an insertion in the tetM gene, which is associated with increased susceptibility to tetracyclines, providing no other tetracycline-resistant gene is present. Such an insertion, which encompasses a dfrG gene and two currently uncharacterised genes, was additionally identified in all tested vanA-type pstS-null VREfm strains (5 English and 68 Australian). Phylogenetic comparison with other VREfm genomes indicates that the four pstS-null Scottish isolates sequenced in this study are more closely related to pstS-null strains from Australia rather than the English pstS-null isolates. Given how rapidly such pstS-null strains have expanded in Australia, the emergence of this clone in Scotland raises concerns for a potential outbreak

In-vivo antimalarial activity of the endophytic actinobacterium, Streptomyces SUK 10

Endophytic bacteria, such as Streptomyces, have the potential to act as a source for novel bioactive molecules with medicinal properties. The present study was aimed at assessing the antimalarial activity of crude extract isolated from various strains of actinobacteria living endophytically in some Malaysian medicinal plants. Using the four day suppression test method on male ICR strain mice, compounds produced from three strains of Streptomyces (SUK8, SUK10, and SUK27) were tested in vivo against Plasmodium berghei PZZ1/100 in an antimalarial screen using crude extracts at four different concentrations. One of these extracts, isolated from Streptomyces SUK10 obtained from the bark of Shorea ovalis tree, showed inhibition of the test organism and was further tested against P. berghei-infected mice for antimalarial activity at different concentrations. There was a positive relationship between the survival of the infected mouse group treated with 50 μg/kg body weight (bw) of ethyl acetate-SUK10 crude extract and the ability to inhibit the parasites growth. The parasite inhibition percentage for this group showed that 50% of the mice survived for more than 90 days after infection with the parasite. The nucleotide sequence and phylogenetic tree suggested that Streptomyces SUK10 may constitute a new species within the Streptomyces genus. As part of the drug discovery process, these promising finding may contribute to the medicinal and pharmaceutical field for malarial treatment

Expanding Primary Metabolism Helps Generate the Metabolic Robustness To Facilitate Antibiotic Biosynthesis in Streptomyces

Abstract The expansion of the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces bacteria are prolific producers of bioactive specialized metabolites that have adaptive functions in nature and have found extensive utility in human medicine. Whilst the biosynthesis of these specialized metabolites is directed by dedicated biosynthetic gene clusters, little attention has been focussed on how these organisms have evolved robustness into their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis during the complex metabolic transitions from vegetative growth to specialized metabolite production and sporulation. Here we examine genetic redundancy in Actinobacteria and show that specialised metabolite producing bacterial families exhibit gene family expansion in primary metabolism. Focussing on a gene duplication event we show that the two pyruvate kinases in the genome of S. coelicolor arose by an ancient duplication event and that each have evolved altered enzymatic kinetics, with Pyk1 having a 20-fold higher Kcat than Pyk2 (4703 sec-1 compared to 215 sec-1 respectively) yet both are constitutively expressed. The pyruvate kinase mutants were also found to be compromised in terms of fitness when compared to wild-type Streptomyces. These data suggest that expanding gene familes can help maintain cell functionality during metabolic perturbation such as nutrient limitation and/or specialized metabolite production. Importance The rise of antimicrobial resistant infections has prompted a resurgence in interest in understanding the production of specialized metabolites by Streptomyces such as antibiotics. The presence of multiple genes encoding the same enzymatic function is an aspect of Streptomyces biology that has received little attention, however understanding how the metabolic expansion influences these organisms can help enhance production of clinically useful molecules. Here we show that expanding the number of pyruvate kinases enables metabolic adaptation, increases strain fitness and represents an excellent target for metabolic engineering of industrial specialized metabolite producing bacteria and the activation of cryptic specialized metabolites