Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.     

Effect of sludge age on methanogenic and glycogen accumulating organisms in an aerobic granular sludge process fed with methanol and acetate

The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35°C was investigated. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT values (51 days), γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed [so called metabolism of glycogen-accumulating organisms (GAO)]. When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35°C. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two GAO types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes.     

Cloning and sequencing of rhesus monkey pepsinogen A cDNA

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.     

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.     

Isolation of a cDNA representing the Fanconi anemia complementation group E gene

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.     

Anaerobic homolactate fermentation with Saccharomyces cerevisiae results in depletion of ATP and impaired metabolic activity

Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae . ATP-dependent export of the lactate anion and/or proton, resulting in net zero ATP formation, is suspected as the underlying cause. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S. cerevisiae , aerobic carbon-limited chemostats were performed and subjected to anaerobic perturbations in the presence of high glucose concentrations. Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol. Finally, the anticipated increase in maximal velocity ( V _max) of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.     

Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.     

Heat-labile enterotoxin of Escherichia coli. Characterization of different crystal forms

Heat-labile enterotoxin (LT) was obtained in large quantities (several-gram amounts) and great purity from Escherichia coli C600 carrying the LT-coding multicopy plasmid EWD299. By growing this strain on a medium that allows high cell densities in the early stationary phase, we increased the net LT production per milliliter by a factor of 200, compared to natural porcine enterotoxigenic E. coli. Adsorption and redesorption on Controlled-Pore Glass usually resulted in a 50-100-fold purification of LT in one simple step, which was followed by established purification procedures. LT shows a natural tendency to form large crystals, which, however, are disordered. After numerous trials, conditions were found which virtually eliminated the disorder. Much better crystals were obtained by employing CdCl2 and KF as coprecipitating agents. CdCl2 yielded crystals which did not contain A subunits as judged by electrophoresis of dissolved crystals. Adding KF results in beautifully shaped crystals which diffracted beyond 2 A and are suitable for a high resolution structure determination.