Characterization and whole genome sequencing of closely related multidrug-resistant Salmonella enterica serovar Heidelberg isolates from imported poultry meat in the Netherlands.

Multidrug-resistant Salmonella enterica serovar Heidelberg isolates are frequently recovered in the Netherlands from poultry meat imported from South America. Our aim was to retrospectively assess the characteristics of the antimicrobial determinants, gene content and the clonal relatedness of 122 unique S. Heidelberg isolates from chicken meat from Brazil (n = 119) and Argentina (n = 3) that were imported between 2010 and 2015. These isolates were subjected to antimicrobial susceptibility testing, PCR and Illumina HiSeq2500 whole genome sequencing. Draft genomes were assembled to assess the gene content, and the phylogenetic relationships between isolates were determined using single nucleotide polymorphisms. Ciprofloxacin-resistance was identified in 98.4% of the isolates and 83.7% isolates showed resistance to the extended-spectrum cephalosporins cefotaxime and ceftazidime (83.6% and 82.8% respectively). Of the latter, 97.1% exhibited an AmpC phenotype and contained blaCMY-2, whereas the remaining three isolates contained an extended spectrum beta-lactamase. Of the 99 extended-spectrum cephalosporins-resistant isolates harboring CMY-2 plasmids, 56.6% contained the incompatibility group I1 replicon. Phylogenetic cluster analysis showed that all isolates from Brazil clustered together, with 49% occurring in clusters larger than 5 isolates that revealed intra-cluster similarities based on geographical location and/or resistance profiles. The remaining isolates were classified in smaller clusters or as singletons, highlighting the large diversity of S. Heidelberg in the poultry chain in Brazil that was revealed by this study. Considering the potential public health risk associated with multidrug-resistant S. Heidelberg in imported poultry, collaborative whole genome sequencing-based surveillance is needed to monitor the spread, pathogenic properties and epidemiological distribution of these isolates.

Mixed and diverse metabolic and gene-expression regulation of the glycolytic and fermentative pathways in response to a HXK2 deletion in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the HXK2 gene, which encodes the glycolytic enzyme hexokinase II, is involved in the regulatory mechanism known as 'glucose repression'. Its deletion leads to fully respiratory growth at high glucose concentrations where the wild type ferments profusely. Here we describe that deletion of the HXK2 gene resulted in a 75% reduction in fermentative capacity. Using regulation analysis we found that the fluxes through most glycolytic and fermentative enzymes were regulated cooperatively by changes in their capacities (Vmax) and by changes in the way they interacted with the rest of the metabolism. Glucose transport and phosphofructokinase were regulated purely at the metabolic level. The reduction of fermentative capacity in the mutant was accompanied by a remarkable resilience of the remaining capacity to nutrient starvation. After starvation, the fermentative capacity of the hxk2Delta mutant was similar to that of the wild type. Based on our results and previous reports, we suggest an inverse correlation between glucose repression and the resilience of fermentative capacity towards nutrient starvation. Only a limited number of glycolytic enzyme activities changed upon starvation of the hxk2Delta mutant and we discuss to what extent this could explain the stability of the fermentative capacity.

Unraveling the complexity of flux regulation: a new method demonstrated for nutrient starvation in Saccharomyces cerevisiae.

An important question is to what extent metabolic fluxes are regulated by gene expression or by metabolic regulation. There are two distinct aspects to this question: (i) the local regulation of the fluxes through the individual steps in the pathway and (ii) the influence of such local regulation on the pathway's flux. We developed regulation analysis so as to address the former aspect for all steps in a pathway. We demonstrate the method for the issue of how Saccharomyces cerevisiae regulates the fluxes through its individual glycolytic and fermentative enzymes when confronted with nutrient starvation. Regulation was dissected quantitatively into (i) changes in maximum enzyme activity (Vmax, called hierarchical regulation) and (ii) changes in the interaction of the enzyme with the rest of metabolism (called metabolic regulation). Within a single pathway, the regulation of the fluxes through individual steps varied from fully hierarchical to exclusively metabolic. Existing paradigms of flux regulation (such as single- and multisite modulation and exclusively metabolic regulation) were tested for a complete pathway and falsified for a major pathway in an important model organism. We propose a subtler mechanism of flux regulation, with different roles for different enzymes, i.e., "leader," "follower," or "conservative," the latter attempting to hold back the change in flux. This study makes this subtlety, so typical for biological systems, tractable experimentally and invites reformulation of the questions concerning the drives and constraints governing metabolic flux regulation.

Training of yeast cell dynamics.

In both industrial fermenters and in their natural habitats, microorganisms often experience an inhomogeneous and fluctuating environment. In this paper we mimicked one aspect of this nonideal behaviour by imposing a low and oscillating extracellular glucose concentration on nonoscillating suspensions of yeast cells. The extracellular dynamics changed the intracellular dynamics--which was monitored through NADH fluorescence--from steady to equally dynamic; the latter followed the extracellular dynamics at the frequency of glucose pulsing. Interestingly, the amplitude of the oscillation of the NADH fluorescence increased with time. This increase in amplitude was sensitive to inhibition of protein synthesis, and was due to a change in the cells rather than in the medium; the cell population was 'trained' to respond to the extracellular dynamics. To examine the mechanism behind this 'training', we subjected the cells to a low and constant extracellular glucose concentration. Seventy-five minutes of adaptation to a low and constant glucose concentration induced the same increase of the amplitude of the forced NADH oscillations as did the train of glucose pulses. Furthermore, 75 min of adaptation to a low (oscillating or continuous) glucose concentration decreased the K(M) of the glucose transporter from 26 mm to 3.5 mm. When subsequently the apparent K(M) was increased by addition of maltose, the amplitude of the forced oscillations dropped to its original value. This demonstrated that the increased affinity of glucose transport was essential for the training of the cells' dynamics.

Hierarchical and metabolic regulation of glucose influx in starved Saccharomyces cerevisiae.

A novel method dissecting the regulation of a cellular function into direct metabolic regulation and hierarchical (e.g., gene-expression) regulation is applied to yeast starved for nitrogen or carbon. Upon nitrogen starvation glucose influx is down-regulated hierarchically. Upon carbon starvation it is down-regulated both metabolically and hierarchically. The method is expounded in terms of its implications for diverse types of regulation. It is also fine-tuned for cases where isoenzymes catalyze the flux through a single metabolic step.

Loss of fermentative capacity in baker's yeast can partly be explained by reduced glucose uptake capacity.

Initial attempts to increase fermentative capacity of baker's yeast focussed on the overproduction of single enzymes, which proved to be insufficient. Nowadays many components of the system are monitored simultaneously in a search for a correlation with fermentative capacity. However, this strategy has not yet proven fruitful either. Here we investigate an element previously neglected, the glucose transporter, and find that a loss of glucose transport capacity correlates with a decrease of fermentative capacity during nutrient starvation. However the correlation is not unique, suggesting that the loss of fermentative capacity cannot be attributed to an inactivation of glucose transport alone. Our results suggest the necessity to use a detailed kinetic model as an underlying working hypothesis and to use Metabolic Control Analysis to examine the pathway's control properties.

DNA supercoiling by gyrase is linked to nucleoid compaction.

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 microm cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.

DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase.

DNA of prokaryotes is in a nonequilibrium structural state, characterized as 'active' DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell 34, 105-113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem. 266, 865-877]. Of the two regulatory mechanisms via which homeostasis is conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).