The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

The epithelial cell cytoskeleton and intracellular trafficking. II. Intestinal epithelial cell exosomes: perspectives on their structure and function

Intestinal epithelial cells (IEC) are located at the strategic interface between the external environment and the most extensive lymphoid compartment in the body. Besides their central role in the absorption of nutrients, they also provide sample information to the immune system on soluble or particulate antigens present in the intestinal lumen. Like professional antigen-presenting cells, IEC have recently been shown to secrete 30- to 90-nm diameter vesicles named exosomes from their apical and basolateral surfaces. These vesicles carry molecules that are implicated in adhesion and antigen presentation, such as major histocompatibility complex (MHC) class I molecules, MHC class II molecules, CD63, CD26/dipeptidyl-peptidase IV, tetraspan proteins, and A33 antigen. IEC exosomes therefore, constitute a link by which IEC may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.     

Oxygen Consumption by Desulfovibrio Strains with and without Polyglucose

The kinetics of oxygen reduction by Desulfovibrio salexigens Mast1 and the role of polyglucose in this activity were examined and compared with those of strains of D. desulfuricans and D. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O₂ min⁻¹ mg of protein⁻¹) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. In D. desulfuricans CSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 μM oxygen. In contrast to D. gigas and D. salexigens strains, the D. desulfuricans strains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O₂ min⁻¹ mg of protein⁻¹. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells of D. desulfuricans CSN and D. desulfuricans ATCC 27774, but inactivation was less pronounced with cells of D. salexigens Mast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth of D. salexigens Mast1 could be demonstrated neither in batch nor in continuous culture.     

Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.     

Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens.

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.     

A spatial model to jointly analyze self-reported survey data of COVID-19 symptoms and official COVID-19 incidence data

This work presents a joint spatial modeling framework to improve estimation of the spatial distribution of the latent COVID-19 incidence in Belgium, based on test-confirmed COVID-19 cases and crowd-sourced symptoms data as reported in a large-scale online survey. Correction is envisioned for stochastic dependence between the survey's response rate and spatial COVID-19 incidence, commonly known as preferential sampling, but not found significant. Results show that an online survey can provide valuable auxiliary data to optimize spatial COVID-19 incidence estimation based on confirmed cases in situations with limited testing capacity. Furthermore, it is shown that an online survey on COVID-19 symptoms with a sufficiently large sample size per spatial entity is capable of pinpointing the same locations that appear as test-confirmed clusters, approximately 1 week earlier. We conclude that a large-scale online study provides an inexpensive and flexible method to collect timely information of an epidemic during its early phase, which can be used by policy makers in an early phase of an epidemic and in conjunction with other monitoring systems.     

Activity of oxyntomodulin on gastric acid secretion induced by histamine or a meal in the rat

Conscious rats with chronic gastric fistula were trained for drinking a 14-ml milk meal. The activity of an intestinal hormone, oxyntomodulin (OXM), was studied in this model and compared to that observed when histamine was the stimulus. Under histamine (0.25 mg·kg⁻¹·h⁻¹) stimulation, OXM at doses (60–120 pmol·kg⁻¹·h⁻¹) that induced physiological circulating levels inhibited gastric acid secretion up to 50%. Under meal stimulation, OXM reduced up to 29% acid secretion at doses (1–1.5 nmol·kg⁻¹·h⁻¹) inducing supraphysiological levels. We conclude that at physiological concentrations OXM cannot counteract the complex processes triggered by a meal. OXM would be a component of enterogastrone, a combination of several intestinal hormones acting in synergy. The OXM action is related to pathways recognizing the C-terminal 19–37 moiety of the molecule.     

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation,metabolism, and survival in vivo

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.     

Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal

Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed.     

Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES)

Blepharophimosis, ptosis, epicanthus inversus syndrome type I (BPES; OMIM 110100) is an autosomal dominant disorder affecting craniofacial development and ovarian function. We have identified a patient with BPES who carried a de novo reciprocal translocation [46, XX,t(3;21)(q23;q22.1)]. Fluorescence in situ hybridization analysis at band 3q23 using probes derived from BAC 175G20 (Research Genetics), PACs 108L15 and 169C10 (RPCI1), and cosmids AC174D4, AC68D3, AC44F5, and AC125C5 (Lawrence Livermore National Laboratory) was performed. The patient's breakpoint was found to lie within the overlapping region of the BAC and PACs but centromeric to all the cosmids. However, a 10.5-kb BamHI-digested fragment, common to the BAC and PAC clones, was shown to cross the breakpoint. The results have placed our patient's breakpoint proximal to that of the previously reported patient [46,XY,t(3;4)(q23;p15.2)] and within a 10.5-kb interval. This is the second patient in which a breakpoint was refined by molecular cytogenetics. Our findings emphasize the significance of this region for BPES.