scholarly journals Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

2014 ◽  
Vol 81 (1) ◽  
pp. 320-331 ◽  
Author(s):  
Onur Ercan ◽  
Michiel Wels ◽  
Eddy J. Smid ◽  
Michiel Kleerebezem
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Growth Rates ◽  
Low Carbon ◽  
Content Type ◽  
Growth State ◽  
Mixed Acid ◽  
Metabolic Adaptations ◽  
Zero Growth ◽  
Growth Adaptation

ABSTRACTThis paper describes the molecular and metabolic adaptations ofLactococcus lactisduring the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY inL. lactisKF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation ofL. lactisto conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation.

scholarly journals Dynamics in Copy Numbers of Five Plasmids of a DairyLactococcus lactisStrain under Dairy-Related Conditions Including Near-Zero Growth Rates

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Oscar van Mastrigt ◽  
Marcel M. A. N. Lommers ◽  
Yorick C. de Vries ◽  
Tjakko Abee ◽  
Eddy J. Smid
Keyword(s):  
Nutrient Limitation ◽  
Lactococcus Lactis ◽  
Growth Rates ◽  
Copy Number ◽  
Plasmid Copy ◽  
Content Type ◽  
Copy Numbers ◽  
Wide Range ◽  
Zero Growth ◽  
The Impact

ABSTRACTLactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strainLactococcus lactisFM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h−1to 0.6 h−1), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids.IMPORTANCELactococcus lactisis extensively used in starter cultures for dairy fermentations. Important traits for the growth and survival ofL. lactisin dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers ofL. lactisat near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations.

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

2015 ◽  
Vol 81 (17) ◽  
pp. 5662-5670 ◽  
Author(s):  
Onur Ercan ◽  
Markus M. M. Bisschops ◽  
Wout Overkamp ◽  
Thomas R. Jørgensen ◽  
Arthur F. Ram ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Rates ◽  
Microbial Growth ◽  
Current Knowledge ◽  
Product Formation ◽  
Natural Ecosystems ◽  
Transcriptional Responses ◽  
Content Type ◽  
Growth Physiology ◽  
Zero Growth

ABSTRACTThe 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 microorganismsLactobacillus plantarum,Lactococcus lactis,Bacillus subtilis,Saccharomyces cerevisiae, andAspergillus 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.

scholarly journals D-Methionine and D-Phenylalanine Improve Lactococcus lactis F44 Acid Resistance and Nisin Yield by Governing Cell Wall Remodeling

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Hao Wu ◽  
Ershu Xue ◽  
Ning Zhi ◽  
Qianqian Song ◽  
Kairen Tian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Lactococcus Lactis ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Cell Wall Synthesis ◽  
Nisin Production ◽  
Content Type ◽  
Nisin Yield

ABSTRACT Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactis. IMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis, and this study provides new ideas for the enhancement of nisin production.

scholarly journals Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

2016 ◽  
Vol 82 (15) ◽  
pp. 4570-4583 ◽  
Author(s):  
Corinna Rebnegger ◽  
Tim Vos ◽  
Alexandra B. Graf ◽  
Minoska Valli ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Growth Rates ◽  
Specific Growth ◽  
Energy Requirement ◽  
Maintenance Energy ◽  
Content Type ◽  
Chemostat Cultures ◽  
Zero Growth ◽  
Maintenance Energy Requirement ◽  
Specific Growth Rates

ABSTRACTThe yeastPichia pastorisis a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented forP. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value ofmSat near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than themSestimated from faster-growing chemostat cultures. This difference indicated thatP. pastorisreduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.IMPORTANCEThe yeastPichia pastorisnaturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grownP. pastorisin a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found thatP. pastorisdecreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

scholarly journals Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Huong Thi Pham ◽  
Wen Shi ◽  
Yuwei Xiang ◽  
Su Yi Foo ◽  
Manuel R. Plan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Free Amino Acids ◽  
Free Amino ◽  
Second Messenger ◽  
Cell Lysis ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Counter Ion

ABSTRACT The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic β-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis. Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity. IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in β-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis. Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

scholarly journals Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters, SerP1 and SerP2, of Lactococcus lactis

2014 ◽  
Vol 197 (5) ◽  
pp. 951-958 ◽  
Author(s):  
Elke E. E. Noens ◽  
Juke S. Lolkema
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Lactococcus Lactis ◽  
Main Function ◽  
Content Type ◽  
High Affinity ◽  
Sequence Identity ◽  
Peptidoglycan Synthesis ◽  
Affinity Constants

TheserP1andserP2genes found adjacently on the chromosome ofLactococcus lactisstrains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transportsl-serine,l-threonine, andl-cysteine with high affinity. Affinity constants (Km) are in the 20 to 40 μM range. SerP2 is adl-alanine/dl-serine/glycine transporter. The preferred substrate appears to bedl-alanine for which the affinities were found to be 38 and 20 μM for thedandlisomers, respectively. The common substratel-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the mainl-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substratesl-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxicd-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake ofd-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA ofEscherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth ofL. lactison protein/peptide-rich media.

scholarly journals Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

2015 ◽  
Vol 198 (3) ◽  
pp. 477-485 ◽  
Author(s):  
Faizah Fulyani ◽  
Gea K. Schuurman-Wolters ◽  
Dirk-Jan Slotboom ◽  
Bert Poolman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Lactococcus Lactis ◽  
Abc Transporter ◽  
Essential Amino Acid ◽  
Transmembrane Domain ◽  
Affinity Constant ◽  
Remarkable Feature ◽  
Content Type

ABSTRACTThe GlnPQ transporter fromLactococcus lactishas the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate.IMPORTANCEGlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, includingL. lactisand several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine.

scholarly journals Draft Genome Sequences of Three Amino Acid-Secreting Lactococcus lactis Strains

2020 ◽  
Vol 9 (16) ◽  
Author(s):  
Jhonatan A. Hernandez-Valdes ◽  
Anne de Jong ◽  
Jan Kok ◽  
Oscar P. Kuipers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Lactococcus Lactis ◽  
Food Industry ◽  
Draft Genome ◽  
Bacterial Strains ◽  
Genome Sequences ◽  
Content Type ◽  
Acid Secreting

Three Lactococcus lactis strains with the ability to secrete various amino acids (leucine, isoleucine, methionine, valine, glutamic acid, and histidine) were sequenced in order to identify the mechanisms involved in the secretion. Amino acids contribute to flavor formation; therefore, bacterial strains with this ability are relevant for the food industry.

scholarly journals Quantitative physiology and aroma formation of a dairy Lactococcus lactis at near-zero growth rates

2018 ◽  
Vol 73 ◽  
pp. 216-226 ◽  
Author(s):  
Oscar van Mastrigt ◽  
Tjakko Abee ◽  
Søren K. Lillevang ◽  
Eddy J. Smid
Keyword(s):  
Lactococcus Lactis ◽  
Growth Rates ◽  
Zero Growth ◽  
Quantitative Physiology

scholarly journals Quantitative Physiology of Non-Energy-Limited Retentostat Cultures of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Yaya Liu ◽  
Anissa el Masoudi ◽  
Jack T. Pronk ◽  
Walter M. van Gulik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rates ◽  
Phosphate Limitation ◽  
High Yield ◽  
Content Type ◽  
Fermentation Processes ◽  
Yield Production ◽  
Zero Growth ◽  
Limited Supply ◽  
High Yield Production

ABSTRACT So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h−1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals. IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

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