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Living cells are highly complex systems comprising a multitude of elements that are engaged in the many convoluted processes observed during the cell cycle. However, not all elements and processes are essential for cell survival and reproduction under steady-state environmental conditions. To distinguish between essential from expendable cell components and thus define the ‘minimal cell’ and the corresponding ‘minimal genome’, we postulate that the synthesis of all cell elements can be represented as a finite set of binary operators, and within this framework we show that cell elements that depend on their previous existence to be synthesized are those that are essential for cell survival. An algorithm to distinguish essential cell elements is presented and demonstrated within an interactome. Data and functions implementing the algorithm are given as supporting information. We expect that this algorithmic approach will lead to the determination of the complete interactome of the minimal cell, which could then be experimentally validated. The assumptions behind this hypothesis as well as its consequences for experimental and theoretical biology are discussed. [ABSTRACT FROM AUTHOR]

The capacity of Acinetobacter baumannii to persist and cause infections depends on its interaction with abiotic and biotic surfaces, including those found on medical devices and host mucosal surfaces. However, the extracellular stimuli affecting these interactions are poorly understood. Based on our previous observations, we hypothesized that mucin, a glycoprotein secreted by lung epithelial cells, particularly during respiratory infections, significantly alters A. baumannii’s physiology and its interaction with the surrounding environment. Biofilm, virulence and growth assays showed that mucin enhances the interaction of A. baumannii ATCC 19606T with abiotic and biotic surfaces and its cytolytic activity against epithelial cells while serving as a nutrient source. The global effect of mucin on the physiology and virulence of this pathogen is supported by RNA-Seq data showing that its presence in a low nutrient medium results in the differential transcription of 427 predicted protein-coding genes. The reduced expression of ion acquisition genes and the increased transcription of genes coding for energy production together with the detection of mucin degradation indicate that this host glycoprotein is a nutrient source. The increased expression of genes coding for adherence and biofilm biogenesis on abiotic and biotic surfaces, the degradation of phenylacetic acid and the production of an active type VI secretion system further supports the role mucin plays in virulence. Taken together, our observations indicate that A. baumannii recognizes mucin as an environmental signal, which triggers a response cascade that allows this pathogen to acquire critical nutrients and promotes host-pathogen interactions that play a role in the pathogenesis of bacterial infections. [ABSTRACT FROM AUTHOR]

The 2-methylcitric acid cycle (2-MCC) is a common route of propionate catabolism in microorganisms. In Salmonella enterica, the prpBCDE operon encodes most of the 2-MCC enzymes. In other organisms, e.g., Shewanella oneidensis MR-1, two genes, acnD and prpF replace prpD, which encodes 2-methylcitrate dehydratase. We showed that together, S. oneidensis AcnD and PrpF (SoAcnD, SoPrpF) compensated for the absence of PrpD in a S. enterica prpD strain. We also showed that SoAcnD had 2-methylcitrate dehydratase activity and that PrpF has aconitate isomerase activity. Here we report in vitro evidence that the product of the SoAcnD reaction is an isomer of 2-methyl-cis-aconitate (2-MCA], the product of the SePrpD reaction. We show that the SoPrpF protein isomerizes the product of the AcnD reaction into the PrpD product (2-MCA], a known substrate of the housekeeping aconitase (AcnB]. Given that SoPrpF is an isomerase, that SoAcnD is a dehydratase, and the results from in vivo and in vitro experiments reported here, it is likely that 4-methylaconitate is the product of the AcnD enzyme. Results from in vivo studies using a S. enterica prpD strain show that SoPrpF variants with substitutions of residues K73 or C107 failed to support growth with propionate as the sole source of carbon and energy. High-resolution (1.22 Å) three-dimensional crystal structures of PrpFK73E in complex with trans-aconitate or malonate provide insights into the mechanism of catalysis of the wild-type protein. [ABSTRACT FROM AUTHOR]

The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes. [ABSTRACT FROM AUTHOR]

Our study aimed to produce the commercially promising platform chemical 3-hydroxypropionic acid (3-HP) via the propionyl-CoA pathway in genetically engineered Escherichia coli. Recombinant E. coli Ec-P overexpressing propionyl-CoA dehydrogenase (PACD, encoded by the pacd gene from Candida rugosa) under the T7 promoter produced 1.33 mM of 3-HP in a shake flask culture supplemented with 0.5% propionate. When propionate CoA-transferase (PCT, encoded by the pct gene from Megasphaera elsdenii) and 3-hydroxypropionyl-CoA dehydratase (HPCD, encoded by the hpcd gene from Chloroflexus aurantiacus) were expressed along with PACD, the 3-HP titer of the resulting E. coli Ec-PPH strain was improved by 6-fold. The effect of the cultivation conditions on the 3-HP yield from propionate in the Ec-PPH strain was also investigated. When cultured at 30°C with 1% glucose in addition to propionate, 3-HP production by Ec-PPH increased 2-fold and 12-fold compared to the cultivation at 37°C (4.23 mM) or without glucose (0.68 mM). Deletion of the ygfH gene encoding propionyl-CoA: succinate CoA-transferase from Ec-PPH (resulting in the strain Ec-△Y-PPH) led to increase of 3-HP production in shake flask experiments (15.04 mM), whereas the strain Ec-△Y-PPH with deletion of the prpC gene (encoding methylcitrate synthase in the methylcitrate cycle) produced 17.76 mM of 3-HP. The strain Ec-△Y-△P-PPH with both ygfH and prpC genes deleted produced 24.14 mM of 3-HP, thus showing an 18-fold increase in the 3-HP titer in compare to the strain Ec-P. [ABSTRACT FROM AUTHOR]

Two separate citrate synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric citrate synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both citrate synthase and 2-methyl citrate synthase activities. 2-Methyl citrate synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric citrate synthases and 2-methyl citrate synthase. [ABSTRACT FROM AUTHOR]